Production process of peony composite small molecule peptide and application thereof in reducing blood sugar

Abstract

This invention discloses a production process for peony compound small molecule peptides and their application in lowering blood sugar, relating to the field of peptide production technology. The process includes the following steps: obtaining bovine bone, peony seed cake, and ginseng, and pretreating them separately, followed by sequential enzymatic hydrolysis in a first stage, a second stage, and a third stage to obtain a natural compound small molecule peptide base; adding ginsenoside-rich fractions and functional peptides prepared by solid-phase synthesis to the natural compound small molecule peptide base, mixing thoroughly, and drying to obtain the peony compound small molecule peptide product. This invention, through the synergistic utilization of bovine bone, peony seed cake, and ginseng, combined with three-stage enzymatic hydrolysis and graded ultrafiltration, not only improves the effective utilization of small molecule peptides and ginseng active ingredients in the compound system, and enhances the component stability and application compatibility of the final product, but also gives the obtained peony compound small molecule peptide product good comprehensive effects in terms of component quality, safety, and auxiliary blood sugar lowering application.

Description

Technical Field

[0001] This invention relates to the field of polypeptide production technology, specifically to a production process for peony compound small molecule peptides and their application in lowering blood sugar. Background Technology

[0002] Peony is a traditional and distinctive plant resource in my country. Besides its ornamental and medicinal value, its seeds, seed oil, and byproducts also have high development potential. Peony seed cake, a byproduct of peony seed oil extraction, contains a certain amount of plant protein, polyphenols, and other nutrients. Meanwhile, bovine bones are rich in collagen, which, after appropriate hydrolysis, can be used to obtain small-molecule collagen peptides, which are widely recognized for their easy digestibility, absorption, and high nutritional value. Ginseng, as a commonly used food and medicine ingredient, contains ginsenosides, polysaccharides, and various bioactive components, and has high application value in regulating metabolism and improving sub-health conditions.

[0003] Therefore, the synergistic development of peony-derived proteins, bovine bone collagen, and ginseng active ingredients to prepare compound small molecule peptide products with the potential to assist in lowering blood sugar has good application prospects and industrialization value. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a production process for peony compound small molecule peptides and their application in lowering blood sugar. To achieve the above objective, this invention provides the following technical solution:

[0005] In a first aspect, the present invention provides a production process for peony complex small molecule peptides, the method comprising the following steps:

[0006] S1. Obtain bovine bone, peony seed cake and ginseng, and pre-treat bovine bone, peony seed cake and ginseng respectively to obtain bovine bone collagen slurry, peony protein enriched phase, ginsenoside enriched part and ginseng slurry to be enzymatically hydrolyzed;

[0007] S2. The bovine bone collagen slurry is subjected to the first stage of enzymatic hydrolysis, and then subjected to 10kDa ultrafiltration and 3kDa ultrafiltration fractionation in sequence. The 3kDa permeate is collected as the first small molecule peptide component P1, and the 3kDa membrane retentate is combined with the 10kDa membrane retentate as the second stage of enzymatic hydrolysis raw material R1.

[0008] S3. Perform the second stage of enzymatic hydrolysis: Mix the truncated phase R1 with the peony protein enrichment phase, adjust the pH of the system to 7.2–7.8, and add 0.20%–0.40% neutral protease and 0.01%–0.06% elastin based on the total dry weight of the enzymatically hydrolyzable protein in the second stage reaction system at 40–46℃. After adding the enzymes, perform a stepwise ultra-high pressure assisted enzymatic hydrolysis treatment on the system. After the ultra-high pressure assisted enzymatic hydrolysis treatment is completed, continue enzymatic hydrolysis at atmospheric pressure for 55–65 min. Then, cool the system to 25–35℃ and adjust the pH to 5.2–6.0. ​​Add 0.005%–0.05% Carboxypeptidase Y based on the total dry weight of the enzymatically hydrolyzable protein in the second stage reaction system, and continue enzymatic hydrolysis at atmospheric pressure for 40–50 min. After the second stage of enzymatic hydrolysis is completed, perform 3 kDa ultrafiltration, collect the permeate as the second small molecule peptide component P2, and retain the truncated phase R2.

[0009] S4. Add the ginseng pulp to be enzymatically hydrolyzed to the truncated phase R2 for the third enzymatic hydrolysis, and then perform 1kDa ultrafiltration fractionation to collect the permeate as the third small molecule peptide component P3.

[0010] S5. Combine the first small molecule peptide component P1, the second small molecule peptide component P2 and the third small molecule peptide component P3, and after desalting and concentration, obtain a natural complex small molecule peptide base.

[0011] S6. Under conditions of 20-35℃, the ginsenoside-rich fraction and the functional peptide prepared by solid-phase synthesis are added to the natural complex small molecule peptide base, mixed and dried to obtain the peony complex small molecule peptide product.

[0012] Preferably, S2 specifically includes: adjusting the bovine bone collagen slurry to pH 2.8–3.2, adding 0.15%–0.45% acidic fungal protease based on the dry weight of bovine bone collagen at 40–44°C, and enzymatically hydrolyzing for 40–60 min; after the first enzymatic hydrolysis, adjusting the pH to 6.5–7.0 and incubating at 75–90°C for 5–15 min to inactivate the enzyme, followed by 10 kDa ultrafiltration, collecting the permeate, further performing 3 kDa ultrafiltration on the 10 kDa ultrafiltration permeate, collecting the permeate as the first small molecule peptide component P1, and combining the 3 kDa membrane retentate with the 10 kDa membrane retentate as the raw material R1 for the second enzymatic hydrolysis.

[0013] Preferably, S4 specifically includes: adding the ginseng slurry to be enzymatically hydrolyzed to the truncated phase R2; first adjusting the system to pH 6.3–6.8; then, at 37–42°C, adding 0.05%–0.12% proline-specific endonuclease based on the total dry weight of the enzymatically hydrolyzable proteins in the third-stage reaction system, and hydrolyzing for 30–40 min; then adjusting the system to pH 6.8–7.2; adding 0.10%–0.25% flavor protease at 45–50°C, and continuing hydrolysis for 30–40 min; after the third-stage hydrolysis is completed, performing 1 kDa ultrafiltration and collecting the permeate as the third small molecule peptide component P3.

[0014] Preferably, the preprocessing includes:

[0015] Fresh bovine bones are selected, washed, and crushed into 5-20mm particles. They are then rinsed in hot water at 85-95℃ for 10-20 minutes. Next, a mixed solution of 0.8%-2.0% citric acid and 0.2%-0.8% sodium citrate is used at a material-to-liquid ratio of 1:4-1:8, and treated at 20-35℃ for 4-10 hours. After washing until the pH reaches 5.5-6.5, the mixture is further refined using a wet colloid mill to obtain bovine bone collagen slurry. The bovine bone collagen slurry undergoes ultra-high pressure pretreatment at a pressure of 120-220MPa for 5-12 minutes.

[0016] Peony seed cake was pulverized through a 40-100 mesh and washed 1-2 times with 50%-75% edible ethanol at 35-50℃. After ethanol recovery, the cake was resuspended in water, and 0.1%-0.5% cellulase and 0.05%-0.3% pectinase were added. The cake was then subjected to cell wall disruption at 45-52℃ and pH 4.5-5.5 for 0.5-1.5 hours. Subsequently, the pH was adjusted to 8.5-9.5 for alkali extraction of protein for 30-90 minutes. The supernatant was collected by centrifugation at 3000-8000 rpm for 5-20 minutes. The pH was then adjusted to 4.2-4.8 for isoelectric precipitation. The precipitate was collected and redissolved at a pH of 6.5-7.5 to obtain a peony protein-enriched phase.

[0017] Ginseng is pulverized to 40-80 mesh and extracted 1-3 times with 55%-75% ethanol at 50-70℃ and a material-to-liquid ratio of 1:8-1:20. The extracts are combined, concentrated under reduced pressure, and enriched with macroporous resin to obtain the ginsenoside-rich fraction. The extracted residue is deethanolinated, resuspended in water, and then treated with 0.05%-0.2% α-amylase and 0.1%-0.4% cellulase at 50-58℃ for 0.5-1.5 hours to obtain the ginseng pulp to be enzymatically hydrolyzed.

[0018] Preferably, the pressure-assisted enzymatic hydrolysis treatment in step S3 is a stepped pressure treatment, with the following conditions: first, treatment at 80-120 MPa for 4-8 minutes, and then treatment at 120-160 MPa for 2-5 minutes.

[0019] Preferably, the proportions of each substance by weight are as follows:

[0020] 225-255 portions of beef bones;

[0021] 340-420 parts of peony seed cake meal;

[0022] 15-20 portions of ginseng;

[0023] 4-6 portions of functional peptides.

[0024] Preferably, the functional peptide is an oligopeptide prepared by solid-phase synthesis, and its amino acid sequence is IPWQHRAFNKR.

[0025] Preferably, in step S6, the ginsenoside-enriched fraction is first compounded with phospholipids and phytosterols, and then subjected to ethanol injection-high pressure homogenization to obtain ginsenoside phospholipid nanodispersions, which are then added to the natural complex small molecule peptide base.

[0026] Preferably, the weight ratio of the ginsenoside enriched fraction, total phospholipids and phytosterols is 1:(6-15):(0.5-4); the average Z-particle size of the nano-dispersion is 80-220 nm, the polydispersity index (PDI) is ≤0.35, and the encapsulation efficiency is 70%-92%.

[0027] Secondly, the present invention provides the application of products produced using the above-described peony complex small molecule peptide production process in the preparation of compositions for assisting in lowering blood sugar.

[0028] The present invention differs from the prior art in that it achieves the following technical effects:

[0029] This invention utilizes bovine bone, peony seed cake, and ginseng synergistically, combined with three-stage enzymatic hydrolysis, graded ultrafiltration, and optional ginsenoside nano-dispersion / microcapsule encapsulation. This not only improves the effective utilization of small molecule peptides and ginseng active ingredients in the composite system but also enhances the component stability and application suitability of the final product. Experimental results show that the peony composite small molecule peptides of this application exhibit an improving trend in α-glucosidase inhibition and glucose uptake-related indicators in HepG2 cells. Improvements are also observed in fasting blood glucose, HbA1c, and OGTT AUC. Therefore, the peony composite small molecule peptide product obtained in this application has good comprehensive effects in terms of component quality, safety, and auxiliary hypoglycemic application, demonstrating significant health benefits and broad market application prospects. Attached Figure Description

[0030] Figure 1 The image shows the third-party testing results for a representative batch A of the experimental products.

[0031] Figure 2 This is a graph showing the third-party inspection results of a representative finished product batch B in the experimental example.

[0032] Figure 3 This is a comparison chart of the α-glucosidase inhibition rates in each group of Experiment Example 2.

[0033] Figure 4 This is a comparison chart of OGTT_AUC in each group of mice in Experiment 3. Detailed Implementation

[0034] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0035] Example 1 This embodiment provides a production process for peony complex small molecule peptides. By weight, the process comprises 225 parts bovine bone, 340 parts peony seed cake, 15 parts ginseng, and 4 parts functional peptides. The functional peptides are oligopeptides prepared using a solid-phase synthesis method, and their amino acid sequence is IPWQHRAFNKR. The specific steps include:

[0036] S1, Raw material pretreatment

[0037] (1) Pretreatment of bovine bones:

[0038] Fresh bovine bones were selected, washed, and crushed into 5mm particles. They were then rinsed in 85℃ hot water for 10 minutes. Next, a mixed solution containing 0.8% citric acid and 0.2% sodium citrate was used at a material-to-liquid ratio of 1:4 and treated at 20℃ for 4 hours. After washing until the pH reached 5.5, the solution was further refined by wet colloid milling to obtain bovine bone collagen slurry. Subsequently, the bovine bone collagen slurry underwent ultra-high pressure pretreatment at a pressure of 120MPa for 5 minutes.

[0039] (2) Pretreatment of peony seed cake:

[0040] Peony seed cake was crushed and passed through a 40-mesh sieve. It was then rinsed once with 50% edible ethanol at 35°C. After recovering the ethanol, the cake was resuspended in water, and 0.1% cellulase and 0.05% pectinase were added. The cake was then subjected to cell wall disruption at 45°C and pH 4.5 for 0.5 hours. Subsequently, the pH was adjusted to 8.5 for alkali extraction of protein for 30 minutes. The supernatant was collected by centrifugation at 3000 rpm for 5 minutes. The pH was then adjusted to 4.2 for isoelectric precipitation. The precipitate was collected and reconstituted at pH 6.5 to obtain the peony protein-enriched phase.

[0041] (3) Ginseng pretreatment:

[0042] Ginseng was pulverized to 40 mesh and extracted once with 55% ethanol at 50°C and a material-to-liquid ratio of 1:8. The extracts were combined, and the ethanol was recovered under reduced pressure and concentrated to a relative density of 1.05 (60°C). The extracts were then enriched using an AB-8 or D101 macroporous adsorption resin column. Before use, the macroporous resin was soaked in 95% ethanol for 8 hours, followed by pretreatment with 2 column volumes of 95% ethanol and 3 column volumes of purified water until the eluent had no alcohol odor. The sample loading flow rate was controlled at 1 BV / h. After loading, the sample was eluted with 2 BV of purified water to remove sugars and highly polar impurities, then eluted with 20% ethanol for 1 BV to remove weakly adsorbed impurities, and finally eluted with 60% ethanol for 4 BV. This eluent was collected. The ethanol was recovered under reduced pressure and the eluent was concentrated or dried to obtain the ginsenoside-enriched fraction. Among them, one or more of ginsenosides Rg1, Re, and Rb1 were used as quality control indicators to detect the total saponin content of the enriched fraction before being used in subsequent batches. The extracted residue was deethanolinated, resuspended in water, and then treated with 0.05% α-amylase and 0.1% cellulase at 50℃ for 0.5 h to obtain the ginseng pulp to be enzymatically hydrolyzed.

[0043] S2, First stage of enzymatic hydrolysis:

[0044] The bovine bone collagen slurry was adjusted to pH 2.8, and 0.15% acidic fungal protease was added at 40℃ based on the dry weight of bovine bone collagen for 40 min of enzymatic hydrolysis. After the first stage of enzymatic hydrolysis, the pH was adjusted to 6.5 and incubated at 75℃ for 5 min to inactivate the enzyme. Subsequently, 10 kDa ultrafiltration was performed, and the permeate was collected. The 10 kDa ultrafiltration permeate was then further subjected to 3 kDa ultrafiltration, and the permeate was collected as the first small molecule peptide component P1. The 3 kDa membrane retentate and the 10 kDa membrane retentate were combined as the raw material R1 for the second stage of enzymatic hydrolysis.

[0045] S3, Second stage of enzymatic hydrolysis:

[0046] The truncated phase R1 was mixed with the peony protein enrichment phase, and the pH of the system was adjusted to 7.2. At 40°C, 0.20% neutral protease and 0.01% elastase were added based on the total dry weight of the enzymatically hydrolyzable protein in the second reaction system. After adding the enzymes, the system was subjected to a stepwise ultra-high pressure-assisted enzymatic hydrolysis treatment, specifically: first, treatment at 80 MPa for 4 min, then treatment at 120 MPa for 2 min. After the ultra-high pressure-assisted enzymatic hydrolysis treatment, enzymatic hydrolysis was continued at atmospheric pressure for 55 min. Subsequently, the system was cooled to 25°C and the pH was adjusted to 5.2. 0.005% carboxypeptidase Y was added based on the total dry weight of the enzymatically hydrolyzable protein in the second reaction system, and enzymatic hydrolysis was continued at atmospheric pressure for 40 min. After the second enzymatic hydrolysis was completed, 3 kDa ultrafiltration was performed, and the permeate was collected as the second small molecule peptide component P2, while the truncated phase R2 was retained. The enzyme preparation is added based on the total dry weight of the enzymatically hydrolyzable protein in the reaction system. The total dry weight of the enzymatically hydrolyzable protein is the sum of the protein mass of each protein component in the reaction system after dry matter conversion. The protein content is determined using the Kjeldahl method and converted according to the corresponding nitrogen-protein conversion factor. The amount of enzyme added is calculated as a percentage of the enzyme preparation mass to the total dry weight of the enzymatically hydrolyzable protein. Furthermore, the total dry weight of the enzymatically hydrolyzable protein includes at least the sum of the protein mass in the second enzymatic hydrolysis raw material R1 and the peony protein enrichment phase.

[0047] S4, Third stage of enzymatic hydrolysis:

[0048] Add the ginseng slurry to be enzymatically hydrolyzed to the retrieval phase R2. First, adjust the system to pH 6.3. At 37°C, add 0.05% proline-specific endonuclease based on the total dry weight of the enzymatically hydrolyzable protein in the third reaction stage, and hydrolyze for 30 min. Then, adjust the system to pH 6.8, and at 45°C, add 0.10% flavor protease based on the total dry weight of the enzymatically hydrolyzable protein in the third reaction stage, and continue hydrolysis for 30 min. After the third hydrolysis is completed, perform 1 kDa ultrafiltration, and collect the permeate as the third small molecule peptide component P3. The total dry weight of the enzymatically hydrolyzable protein includes at least the sum of the protein content in the retrieval phase R2 and the ginseng slurry to be enzymatically hydrolyzed.

[0049] S5. Preparation of composite peptide substrate:

[0050] The first small molecule peptide component P1, the second small molecule peptide component P2, and the third small molecule peptide component P3 were combined, and after desalting and concentration, a natural complex small molecule peptide base was obtained.

[0051] S6. Finished Product Preparation:

[0052] At 20°C, ginsenoside-rich fractions and functional peptides were added to a natural complex small molecule peptide base, mixed thoroughly, and dried to obtain a peony complex small molecule peptide product.

[0053] Example 2 This embodiment provides a production process for peony complex small molecule peptides. By weight, the process comprises 240 parts bovine bone, 380 parts peony seed cake, 17 parts ginseng, and 5 parts functional peptides. The functional peptides are oligopeptides prepared using a solid-phase synthesis method, and their amino acid sequence is IPWQHRAFNKR. The specific steps include:

[0054] S1. Raw material pretreatment:

[0055] (1) Pretreatment of bovine bones:

[0056] Fresh bovine bones were selected, washed, and crushed into 12mm particles. They were then rinsed in 90℃ hot water for 15 minutes. Next, a mixed solution containing 1.4% citric acid and 0.5% sodium citrate was used at a material-to-liquid ratio of 1:6 and treated at 28℃ for 7 hours. After washing until the pH reached 6.0, the solution was further refined by wet colloid milling to obtain bovine bone collagen slurry. Subsequently, the bovine bone collagen slurry underwent ultra-high pressure pretreatment at a pressure of 170MPa for 9 minutes.

[0057] (2) Pretreatment of peony seed cake:

[0058] Peony seed cake was crushed and passed through a 70-mesh sieve. It was then washed twice with 63% edible ethanol at 43°C. After recovering the ethanol, the cake was resuspended in water, and 0.3% cellulase and 0.175% pectinase were added. The cake was then subjected to cell wall disruption at 48°C and pH 5.0 for 1.0 h. Subsequently, the pH was adjusted to 9.0 for alkali extraction of protein for 60 min. The supernatant was collected by centrifugation at 5500 rpm for 13 min. The pH was then adjusted to 4.5 for isoelectric precipitation. The precipitate was collected and reconstituted at pH 7.0 to obtain the peony protein-enriched phase.

[0059] (3) Ginseng pretreatment:

[0060] Ginseng was pulverized to 60 mesh and extracted twice with 65% ethanol at 60°C and a material-to-liquid ratio of 1:14. The extracts were combined, and the ethanol was recovered under reduced pressure and concentrated to a relative density of 1.1 (60°C). The extracts were then enriched using an AB-8 or D101 macroporous adsorption resin column. Before use, the macroporous resin was soaked in 95% ethanol for 10 hours, followed by pretreatment with 2 column volumes of 95% ethanol and 4 column volumes of purified water until the eluent had no alcohol odor. The sample loading flow rate was controlled at 2 BV / h. After loading, the sample was eluted with 3 BV of purified water to remove sugars and highly polar impurities, then eluted with 25% ethanol for 2 BV to remove weakly adsorbed impurities, and finally eluted with 65% ethanol for 5 BV. This eluent was collected. The ethanol was recovered under reduced pressure and the eluent was concentrated or dried to obtain the ginsenoside-enriched fraction. After extraction, the residue was de-alcoholized and resuspended in water. Then, it was treated with 0.12% α-amylase and 0.25% cellulase at 54℃ for 1.0 h to obtain the ginseng pulp to be enzymatically hydrolyzed.

[0061] S2, First stage of enzymatic hydrolysis:

[0062] The bovine bone collagen slurry was adjusted to pH 3.0, and 0.30% acidic fungal protease was added at 42℃ (based on the dry weight of bovine bone collagen) for 50 min of enzymatic hydrolysis. After the first stage of enzymatic hydrolysis, the pH was adjusted to 6.75 and the mixture was incubated at 83℃ for 10 min to inactivate the enzyme. Subsequently, 10 kDa ultrafiltration was performed, and the permeate was collected. The 10 kDa ultrafiltration permeate was then further subjected to 3 kDa ultrafiltration, and the permeate was collected as the first small molecule peptide component P1. The 3 kDa membrane retentate was combined with the 10 kDa membrane retentate and used as the raw material R1 for the second stage of enzymatic hydrolysis.

[0063] S3, Second stage of enzymatic hydrolysis:

[0064] The truncated phase R1 was mixed with the peony protein enrichment phase, and the pH of the system was adjusted to 7.5. At 43°C, 0.30% neutral protease and 0.03% elastolate were added based on the total dry weight of the enzymatically hydrolyzable protein in the second-stage reaction system. After adding the enzymes, the system was subjected to a stepwise ultra-high pressure-assisted enzymatic hydrolysis treatment, specifically: first, treatment at 100 MPa for 6 min, then treatment at 140 MPa for 3 min. After the ultra-high pressure-assisted enzymatic hydrolysis treatment, enzymatic hydrolysis was continued at atmospheric pressure for 60 min. Subsequently, the system was cooled to 30°C and the pH was adjusted to 5.6. 0.02% Carboxypeptidase Y was added based on the total dry weight of the enzymatically hydrolyzable protein in the second-stage reaction system, and enzymatic hydrolysis was continued at atmospheric pressure for 45 min. After the second-stage enzymatic hydrolysis was completed, 3 kDa ultrafiltration was performed, and the permeate was collected as the second small molecule peptide component P2, while the truncated phase R2 was retained. The total dry weight of the enzymatically hydrolyzable protein includes at least the sum of the protein content in the second enzymatic hydrolysis feedstock R1 and the protein enrichment phase of peony protein.

[0065] S4, Third stage of enzymatic hydrolysis:

[0066] Add the ginseng slurry to be enzymatically hydrolyzed to the retrieval phase R2. First, adjust the system to pH 6.55. At 39°C, add 0.08% proline-specific endonuclease based on the total dry weight of the enzymatically hydrolyzable protein in the third reaction phase, and hydrolyze for 35 min. Then, adjust the system to pH 7.0. At 47°C, add 0.17% flavor protease based on the total dry weight of the enzymatically hydrolyzable protein in the third reaction phase, and continue hydrolysis for 35 min. After the third hydrolysis is completed, perform 1 kDa ultrafiltration and collect the permeate as the third small molecule peptide component P3. The total dry weight of the enzymatically hydrolyzable protein includes at least the sum of the protein content in the retrieval phase R2 and the ginseng slurry to be enzymatically hydrolyzed.

[0067] S5. Preparation of composite peptide substrate:

[0068] The first small molecule peptide component P1, the second small molecule peptide component P2, and the third small molecule peptide component P3 were combined, and after desalting and concentration, a natural complex small molecule peptide base was obtained.

[0069] S6. Finished Product Preparation:

[0070] At 27°C, ginsenoside-rich fractions and functional peptides were added to a natural complex small molecule peptide base, mixed thoroughly, and dried to obtain a peony complex small molecule peptide product.

[0071] Example 3 This embodiment provides a production process for peony complex small molecule peptides. By weight, the process comprises 255 parts bovine bone, 420 parts peony seed cake, 20 parts ginseng, and 6 parts functional peptides. The functional peptides are oligopeptides prepared using a solid-phase synthesis method, and their amino acid sequence is IPWQHRAFNKR. The specific steps include:

[0072] S1. Raw material pretreatment:

[0073] (1) Pretreatment of bovine bones:

[0074] Fresh bovine bones were selected, washed, and crushed into 20mm particles. They were then rinsed in 95℃ hot water for 20 minutes. Next, a mixed solution containing 2.0% citric acid and 0.8% sodium citrate was used at a material-to-liquid ratio of 1:8 and treated at 35℃ for 10 hours. After washing until the pH reached 6.5, the solution was further refined by wet colloid milling to obtain bovine bone collagen slurry. Subsequently, the bovine bone collagen slurry underwent ultra-high pressure pretreatment at 220MPa for 12 minutes.

[0075] (2) Pretreatment of peony seed cake:

[0076] Peony seed cake was crushed and passed through a 100-mesh sieve. It was then washed twice with 75% edible ethanol at 50°C. After recovering the ethanol, the cake was resuspended in water, and 0.5% cellulase and 0.3% pectinase were added. The cake was then subjected to cell wall disruption at 52°C and pH 5.5 for 1.5 hours. Subsequently, the pH was adjusted to 9.5 for alkali extraction of protein for 90 minutes. The supernatant was collected by centrifugation at 8000 rpm for 20 minutes. The pH was then adjusted to 4.8 for isoelectric precipitation. The precipitate was collected and reconstituted at pH 7.5 to obtain the peony protein-enriched phase.

[0077] (3) Ginseng pretreatment:

[0078] Ginseng was pulverized to 80 mesh and extracted three times with 75% ethanol at 70°C and a material-to-liquid ratio of 1:20. The extracts were combined, and the ethanol was recovered under reduced pressure and concentrated to a relative density of 1.15 (60°C). The extracts were then enriched using an AB-8 or D101 macroporous adsorption resin column. Before use, the macroporous resin was soaked in 95% ethanol for 12 hours, followed by pretreatment with 3 column volumes of 95% ethanol and 5 column volumes of purified water until the eluent had no alcohol odor. The sample loading flow rate was controlled at 2 BV / h. After loading, the sample was eluted with 4 BV of purified water to remove sugars and highly polar impurities, then eluted with 30% ethanol for 3 BV to remove weakly adsorbed impurities, and finally eluted with 75% ethanol for 6 BV. This eluent was collected. The ethanol was recovered under reduced pressure and the eluent was concentrated or dried to obtain the ginsenoside-enriched fraction. After extraction, the residue was de-alcoholized and resuspended in water. Then, it was treated with 0.2% α-amylase and 0.4% cellulase at 58℃ for 1.5 h to obtain the ginseng pulp to be enzymatically hydrolyzed.

[0079] S2, First stage of enzymatic hydrolysis:

[0080] The bovine bone collagen slurry was adjusted to pH 3.2, and 0.45% acidic fungal protease was added at 44℃ based on the dry weight of bovine bone collagen for 60 min of enzymatic hydrolysis. After the first stage of enzymatic hydrolysis, the pH was adjusted to 7.0 and incubated at 90℃ for 15 min to inactivate the enzyme. Subsequently, 10 kDa ultrafiltration was performed, and the permeate was collected. The 10 kDa ultrafiltration permeate was then further subjected to 3 kDa ultrafiltration, and the permeate was collected as the first small molecule peptide component P1. The 3 kDa membrane retentate and the 10 kDa membrane retentate were combined as the raw material R1 for the second stage of enzymatic hydrolysis.

[0081] S3, Second stage of enzymatic hydrolysis:

[0082] The retrieval phase R1 was mixed with the peony protein enrichment phase, and the pH of the system was adjusted to 7.8. At 46°C, 0.40% neutral protease and 0.06% elastolate were added based on the total dry weight of the enzymatically hydrolyzable proteins in the second-stage reaction system. After adding the enzymes, the system was subjected to a stepwise ultra-high pressure-assisted enzymatic hydrolysis treatment, specifically: first, treatment at 120 MPa for 8 min, then treatment at 160 MPa for 5 min. After the ultra-high pressure-assisted enzymatic hydrolysis treatment, enzymatic hydrolysis was continued at atmospheric pressure for 65 min. Subsequently, the system was cooled to 35°C, and the pH was adjusted to 6.0. 0.05% Carboxypeptidase Y was added based on the total dry weight of the enzymatically hydrolyzable proteins in the second-stage reaction system, and enzymatic hydrolysis was continued at atmospheric pressure for 50 min. After the second-stage enzymatic hydrolysis, 3 kDa ultrafiltration was performed, and the permeate was collected as the second small molecule peptide component P2, while the retrieval phase R2 was retained. The total dry weight of the enzymatically hydrolyzable protein includes at least the sum of the protein content in the second enzymatic hydrolysis feedstock R1 and the protein enrichment phase of peony protein.

[0083] S4, Third stage of enzymatic hydrolysis:

[0084] Add the ginseng slurry to be enzymatically hydrolyzed to the retrieval phase R2. First, adjust the system to pH 6.8. At 42℃, add 0.12% proline-specific endonuclease based on the total dry weight of the enzymatically hydrolyzable protein in the third reaction stage, and hydrolyze for 40 min. Then, adjust the system to pH 7.2. At 50℃, add 0.25% flavor protease based on the total dry weight of the enzymatically hydrolyzable protein in the third reaction stage, and continue hydrolysis for 40 min. After the third hydrolysis is completed, perform 1 kDa ultrafiltration and collect the permeate as the third small molecule peptide component P3. The total dry weight of the enzymatically hydrolyzable protein includes at least the sum of the protein content in the retrieval phase R2 and the ginseng slurry to be enzymatically hydrolyzed.

[0085] S5. Preparation of composite peptide substrate:

[0086] The first small molecule peptide component P1, the second small molecule peptide component P2, and the third small molecule peptide component P3 were combined, and after desalting and concentration, a natural complex small molecule peptide base was obtained.

[0087] S6. Finished Product Preparation:

[0088] At 35°C, ginsenoside-rich fractions and functional peptides were added to a natural complex small molecule peptide base, mixed thoroughly, and dried to obtain a peony complex small molecule peptide product.

[0089] Example 4 This embodiment optimizes step S6 based on the natural complex small molecule peptide base obtained in Example 2. First, the ginsenoside-rich fraction is prepared into a ginsenoside phospholipid nanodispersion, which is then compounded with the natural complex small molecule peptide base and functional peptides to obtain the peony complex small molecule peptide product. By weight, the raw materials are: 240 parts bovine bone, 380 parts peony seed cake, 17 parts ginseng, and 5 parts functional peptides. The functional peptides are oligopeptides prepared by solid-phase synthesis, with the amino acid sequence IPWQHRAFNKR.

[0090] Steps S1 to S5 can be performed according to the method of Example 2 to obtain the natural complex small molecule peptide base. The difference is that step S6 in this example is as follows:

[0091] S6. Preparation and acquisition of ginsenoside phospholipid nanodispersions:

[0092] (1) Preparation of ginsenoside phospholipid nanodispersions:

[0093] The ginsenoside-rich fraction, total phospholipids, and phytosterols obtained in step S1 were mixed in a weight ratio of 1:10:2. Specifically, the ginsenoside-rich fraction, total phospholipids, and phytosterols were added to ethanol to prepare an organic phase, wherein the total solids concentration of the organic phase was controlled at 6 wt%; purified water was used as the aqueous phase, and the temperature of the aqueous phase was controlled at 45℃. Under stirring conditions of 600 rpm, the organic phase was slowly injected into the aqueous phase at a rate of 2 mL / min. After the injection was completed, stirring was continued for 20 min to obtain the colostrum dispersion system.

[0094] The colostrum dispersion system was then homogenized using a high-pressure homogenizer at a pressure of 80 MPa, repeating the homogenization process three times. The system temperature was controlled to not exceed 45°C during homogenization. After homogenization, ethanol was removed under reduced pressure to obtain ginsenoside phospholipid nanodispersions.

[0095] (2) Performance testing of nano-dispersions:

[0096] The obtained ginsenoside phospholipid nanodispersions were analyzed: the average particle size (Z-axis) was 138 nm, and the PDI was 0.21, determined by dynamic light scattering; the encapsulation efficiency was 85.4%, determined by ultrafiltration centrifugation combined with high-performance liquid chromatography. The encapsulation efficiency (%) was calculated as follows: (Total ginsenoside content - Free ginsenoside content) / Total ginsenoside content × 100%.

[0097] (3) Combining with natural complex small molecule peptide bases and functional peptides:

[0098] At 28°C, the above-mentioned ginsenoside phospholipid nanodispersion was added to the natural composite small molecule peptide base obtained in Example 2, and stirred for 15 min. Then, the functional peptide was added, and stirring was continued for 20 min to make the system uniformly mixed and form a composite formulation liquid.

[0099] (4) The product is obtained by drying:

[0100] The compound ingredient liquid is dried at low temperature under vacuum conditions, with the drying temperature controlled below 40°C. After drying until the moisture content meets the product requirements, it is pulverized and sieved to obtain the peony compound small molecule peptide product.

[0101] In the product obtained in this embodiment, ginsenosides are constructed into a nano-dispersion system through phospholipids and phytosterols, and then compounded with natural complex small molecule peptide base and functional peptides. This is beneficial to improve the dispersion stability of ginsenosides in the complex peptide system and helps to improve its subsequent utilization effect.

[0102] Example 5 This embodiment further optimizes step S6 based on embodiment 4. Specifically, it first prepares ginsenoside phospholipid nanodispersions, then combines them with a natural complex small molecule peptide substrate and functional peptides to form a composite core material. Subsequently, sodium alginate and carboxymethyl chitosan are used as composite wall materials, and Ca2+ is added as the composite core material. 2+ The composite microcapsule powder was obtained by iontophoresis encapsulation using a crosslinking agent and then dried. The raw materials, by weight, were: 240 parts bovine bone, 380 parts peony seed cake, 17 parts ginseng, and 5 parts functional peptides. Steps S1 to S5 could be performed according to the method in Example 2 to obtain a natural composite small molecule peptide base.

[0103] Step S6 is as follows:

[0104] S6. Preparation of composite microcapsule powder:

[0105] (1) Preparation of ginsenoside phospholipid nanodispersions:

[0106] First, ginsenoside phospholipid nanodispersions were prepared according to the method in Example 4. Specifically, ginsenoside-rich fractions, total phospholipids, and phytosterols were mixed at a weight ratio of 1:10:2. Ethanol was used as the organic phase solvent, and the total solids concentration of the organic phase was controlled at 6 wt%. Purified water was used as the aqueous phase, and the aqueous phase temperature was controlled at 45°C. Under 600 rpm, the organic phase was injected into the aqueous phase at a rate of 2 mL / min, and stirring was continued for 20 min to form a primary emulsion system. The system was then homogenized three times under 80 MPa pressure, and ethanol was removed under reduced pressure to obtain the ginsenoside phospholipid nanodispersions. The Z-average particle size of the prepared nanodispersions was measured to be 145 nm, the PDI was 0.24, and the encapsulation efficiency was 86.1%.

[0107] (2) Formation of composite core material:

[0108] At 28°C, the ginsenoside phospholipid nanodispersion was added to a natural composite small molecule peptide substrate and stirred for 15 min; then functional peptides were added and stirring was continued for 20 min to obtain a uniformly dispersed composite core material system.

[0109] (3) Preparation of composite wall material solution:

[0110] Sodium alginate solution and carboxymethyl chitosan solution were prepared separately and mixed at a mass ratio of 5:1 to form a composite wall material. Specifically, a composite wall material solution with a total concentration of 2.4 wt% was prepared, in which sodium alginate accounted for 2.0 wt% and carboxymethyl chitosan accounted for 0.4 wt%. After mixing the two, the mixture was stirred at room temperature for 30 minutes to ensure that the wall material system was homogeneous and stable.

[0111] (4) Ion gelation embedding:

[0112] The composite core material was slowly added to the composite wall material solution to ensure thorough mixing, with the mass ratio of the composite core material to the composite wall material controlled at 1:3. After uniform mixing, a 2.0 wt% calcium chloride solution was added to the system under stirring as a Ca2+ solution. 2+ The cross-linking agent was used for ion gelation embedding for 20 minutes to obtain the embedding slurry.

[0113] (5) Spray drying:

[0114] The above-mentioned encapsulation slurry was spray-dried under the following conditions: inlet air temperature 150℃ and outlet air temperature 78℃. After drying, the powder was collected to obtain peony composite small molecule peptide composite microcapsule powder.

[0115] (6) Performance testing of composite microcapsule powder:

[0116] The obtained composite microcapsule powder was analyzed based on total ginsenosides. The results are as follows: encapsulation rate was 82.7%; D50 was 36 μm; and water content was 3.9%. D50 was determined using laser particle size analysis, and water content was determined using the 105℃ constant weight method.

[0117] Furthermore, an in vitro simulated release experiment was conducted on the composite microcapsule powder: under simulated gastric juice pH 1.2 conditions, the cumulative release rate of total ginsenosides was 21.3% after 30 minutes; under simulated intestinal juice pH 6.8 conditions, the cumulative release rate of total ginsenosides was 72.5% after 120 minutes.

[0118] The above results indicate that the composite microcapsule powder prepared in this embodiment has high encapsulation efficiency, suitable particle size distribution and low water content. At the same time, the composite microcapsule releases slowly under simulated gastric juice conditions but quickly under simulated intestinal juice conditions, indicating that it has a good protective effect on ginsenosides and helps to improve their intestinal release characteristics.

[0119] Comparative Example 1 Except for the following changes in step S3, this comparative example follows the same procedure as Example 2.

[0120] In step S3, after mixing the chopped phase R1 with the peony protein enrichment phase, the pH of the system was adjusted to 7.5. At 43°C, 0.30% neutral protease was added based on the total dry weight of the enzymatically hydrolyzable proteins in the second reaction system for enzymatic hydrolysis. After the same stepwise ultra-high pressure assisted treatment as in Example 2, enzymatic hydrolysis was continued for 60 min at atmospheric pressure. Subsequently, the system was cooled to 30°C, the pH was adjusted to 5.6, and 0.02% Carboxypeptidase Y was added based on the total dry weight of the enzymatically hydrolyzable proteins in the second reaction system, and enzymatic hydrolysis was continued for 45 min. After the second enzymatic hydrolysis was completed, 3 kDa ultrafiltration was performed, and the permeate was collected as the second small molecule peptide component P2, while the chopped phase R2 was retained. The remaining steps were the same as in Example 2, yielding the product of Comparative Example 1.

[0121] Comparative Example 2 Except for the following changes in step S3, this comparative example follows the same procedure as Example 2.

[0122] In step S3, after mixing the chopped phase R1 with the peony protein enrichment phase, the pH of the system was adjusted to 7.5. At 43°C, 0.30% neutral protease was added based solely on the total dry weight of the enzymatically digestible proteins in the second reaction system. After adding the enzyme, the system underwent the same stepwise ultra-high pressure-assisted enzymatic hydrolysis treatment as in Example 2: first, treatment at 100 MPa for 6 min, then at 140 MPa for 3 min. After the ultra-high pressure-assisted enzymatic hydrolysis, enzymatic hydrolysis continued at atmospheric pressure for 60 min. The system was then cooled to 30°C and the pH was adjusted to 5.6. Carboxypeptidase Y was not added, and the system was incubated for another 45 min under the same conditions. After the second enzymatic hydrolysis, 3 kDa ultrafiltration was performed, and the permeate was collected as the second small molecule peptide component P2, while the chopped phase R2 was retained. The remaining steps were the same as in Example 2, yielding the product of Comparative Example 2.

[0123] Comparative Example 3 Except for the following changes in step S3, this comparative example follows the same procedure as Example 2.

[0124] In step S3, after mixing the chopped phase R1 with the peony protein enrichment phase, the pH of the system was adjusted to 7.5. At 43°C, 0.30% neutral protease and 0.035% papain were added based on the total dry weight of the enzymatically hydrolyzable proteins in the second reaction system. After adding the enzymes, the same stepwise ultra-high pressure assisted treatment as in Example 2 was performed, and enzymatic hydrolysis continued for 60 min at atmospheric pressure. Subsequently, the system was adjusted to pH 7.0 and temperature 45°C, and 0.175% flavor protease was added based on the total dry weight of the enzymatically hydrolyzable proteins in the second reaction system, and enzymatic hydrolysis continued for 45 min. After the second enzymatic hydrolysis was completed, 3 kDa ultrafiltration was performed, and the permeate was collected as the second small molecule peptide component P2, while the chopped phase R2 was retained. The remaining steps were the same as in Example 2, yielding the product of Comparative Example 3.

[0125] Comparative Example 4 Except for the following changes in step S3, this comparative example follows the same procedure as Example 2.

[0126] In step S3, after mixing the chopped phase R1 with the peony protein enrichment phase, the pH of the system was adjusted to 7.5. At 43°C, 0.30% neutral protease and 0.03% elastolate were added based on the total dry weight of the enzymatically hydrolyzable proteins in the second-stage reaction system. However, instead of performing stepwise ultra-high pressure assisted enzymatic hydrolysis, enzymatic hydrolysis was directly performed at atmospheric pressure for 60 min. Subsequently, the system was cooled to 30°C, the pH was adjusted to 5.6, and 0.0275% Carboxypeptidase Y was added based on the total dry weight of the enzymatically hydrolyzable proteins in the second-stage reaction system. Enzymatic hydrolysis continued for 45 min. After the second-stage enzymatic hydrolysis was completed, 3 kDa ultrafiltration was performed, and the permeate was collected as the second small molecule peptide component P2, while the chopped phase R2 was retained. The remaining steps were the same as in Example 2, yielding the product of Comparative Example 4.

[0127] Comparative Example 5 Except for the following changes in step S2, this comparative example follows the same procedure as Example 2.

[0128] In step S2, the bovine bone collagen slurry was adjusted to pH 2.0, and 0.30% pepsin was added at 37°C (based on the dry weight of bovine bone collagen) for 90 min of enzymatic hydrolysis. After the first stage of enzymatic hydrolysis, the pH was adjusted to 7.0 and incubated at 95°C for 10 min to inactivate the enzyme. Subsequently, 10 kDa ultrafiltration was performed, followed by 3 kDa ultrafiltration. The 3 kDa permeate was collected as the first small molecule peptide component P1. The 3 kDa membrane retentate and the 10 kDa membrane retentate were combined as the raw material R1 for the second stage of enzymatic hydrolysis. The remaining steps were the same as in Example 2, yielding the product of Comparative Example 5.

[0129] Comparative Example 6 Except for the following changes in step S4, this comparative example follows the same procedure as Example 2.

[0130] In step S4, after adding the ginseng slurry to be enzymatically hydrolyzed to the truncated phase R2, the system was uniformly adjusted to pH 7.0. At 50°C, 0.20% papain was added based on the total dry weight of the enzymatically hydrolyzable proteins in the third-stage reaction system, and enzymatic hydrolysis was performed for 70 minutes at a time. After the third-stage enzymatic hydrolysis was completed, 1 kDa ultrafiltration was performed, and the permeate was collected as the third small molecule peptide component P3. The remaining steps were the same as in Example 2, yielding the product of Comparative Example 6.

[0131] Comparative Example 7 Except for the following changes to the preprocessing method in step S1, the comparative example is performed in accordance with Example 2.

[0132] In step S1: the bovine bones are simply washed, crushed into 12.5mm particles, rinsed with 90℃ hot water for 15 minutes, and then directly subjected to wet colloid milling without citric acid / sodium citrate demineralization or ultra-high pressure pretreatment to obtain bovine bone slurry; the peony seed cake is pulverized through 70 mesh, then without ethanol rinsing or cellulase / pectinase cell wall breaking treatment, but directly adjusted to pH 9.0 for alkali extraction of protein for 60 minutes, centrifuged to collect the supernatant, adjusted to pH 4.5 for isoelectric precipitation, and reconstituted to obtain the peony protein phase; the ginseng is pulverized and extracted twice with 65% ethanol at 60℃ and a material-to-liquid ratio of 1:14, but the extract is concentrated under reduced pressure without enrichment with macroporous resin and is directly used as the ginseng extract; the extracted residue is deethanoluted, resuspended in water, and without α-amylase and cellulase treatment, is directly used as the slurry for the subsequent third enzymatic hydrolysis.

[0133] The remaining steps are the same as in Example 2, resulting in Comparative Example 7 product.

[0134] Comparative Example 8 Except for the following changes in the raw material ratio, this comparative example follows the same procedure as Example 1. By weight, the following ingredients were used: 210 parts bovine bone, 320 parts peony seed cake, 12 parts ginseng, and 3 parts functional peptides.

[0135] The remaining steps are the same as in Example 1, resulting in Comparative Example 8 product.

[0136] Comparative Example 9 Except for the following changes in the raw material ratio, this comparative example follows the same procedure as Example 3.

[0137] By weight, take: 270 parts of beef bone, 450 parts of peony seed cake, 24 parts of ginseng, and 7 parts of functional peptides.

[0138] The remaining steps are the same as in Example 3, resulting in Comparative Example 9 product.

[0139] Comparative Example 10 Except for the removal of ginseng and its related processes, this comparative example follows the same procedure as Example 2.

[0140] By weight, only 240 parts of beef bone, 380 parts of peony seed cake, and 5 parts of functional peptides are used, without adding ginseng.

[0141] In step S1, ginseng extraction and processing are not performed; in step S4, the ginseng slurry to be enzymatically hydrolyzed is not added, and the third enzymatic hydrolysis step is omitted; in step S5, only the first small molecule peptide component P1 and the second small molecule peptide component P2 are combined, and after desalting and concentration, a composite peptide base is obtained; in step S6, the ginsenoside-enriched fraction is not added, only the functional peptide is added and then dried. Comparative Example 10 product is obtained.

[0142] Comparative Example 11 Except for the removal of peony seed cake and related processes, this comparative example follows the same procedure as Example 2.

[0143] By weight, only 240 parts of beef bone, 17 parts of ginseng, and 5 parts of functional peptides are used, without adding peony seed cake.

[0144] In step S1, no pretreatment of peony seed cake is performed; in step S3, only the second enzymatic hydrolysis raw material R1 is treated, and no peony protein enrichment phase is added. The remaining second enzymatic hydrolysis conditions are the same as in Example 2; the remaining steps are the same as in Example 2, and the product of Comparative Example 11 is obtained.

[0145] Comparative Example 12 Except for the deletion of the functional peptide and its related processes, the comparative example was carried out in accordance with Example 2.

[0146] By weight, only 240 parts of beef bone, 380 parts of peony seed cake, and 17 parts of ginseng are used, without the addition of functional peptides.

[0147] Steps S1 to S5 are the same as in Example 2; in step S6, only the ginsenoside-enriched fraction is added to the natural complex small molecule peptide base, mixed well, and then vacuum-dried at low temperature to obtain the product of Comparative Example 12.

[0148] Comparative Example 13 Except for the following changes in step S6, this comparative example follows the same procedure as Example 4.

[0149] In step S6, instead of combining the ginsenoside-enriched fraction with phospholipids and phytosterols, and without performing ethanol injection-high pressure homogenization, the ginsenoside-enriched fraction is directly added to the natural complex small molecule peptide base, followed by the addition of functional peptides. After mixing, the mixture is vacuum-dried at low temperature to obtain the product of Comparative Example 13.

[0150] Comparative Example 14 Except for the following changes in step S6, this comparative example follows the same procedure as Example 4.

[0151] In step S6, the ginsenoside-rich fraction is compounded with total phospholipids at a weight ratio of 1:10, but no phytosterols are added. Subsequently, an ethanol injection-high pressure homogenization process is used to obtain a dispersion system, which is then added to a natural complex small molecule peptide base. Functional peptides are then added, mixed, and vacuum low-temperature drying is performed to obtain the product of Comparative Example 14.

[0152] Comparative Example 15 Except for the following changes in step S6, this comparative example follows the same procedure as Example 5.

[0153] In step S6, ginsenoside phospholipid nanodispersions are first prepared according to the method of Example 4, and then mixed with natural complex small molecule peptide substrates and functional peptides to form a composite core material. However, sodium alginate / carboxymethyl chitosan composite wall material is not used for ion gelation embedding, and Ca is not added. 2+ Instead of crosslinking, the composite core material was directly spray-dried to obtain the product of Comparative Example 15.

[0154] Comparative Example 16 Except for the following changes in step S6, this comparative example follows the same procedure as Example 5.

[0155] In step S6, after mixing the ginsenoside phospholipid nanodispersion with the natural composite small molecule peptide matrix and functional peptides to form a composite core material, only sodium alginate is used as a single wall material for encapsulation, and Ca... 2+ The product was used as a crosslinking agent for ion gelation and then spray-dried to obtain product 16 of Comparative Example; without the addition of carboxymethyl chitosan.

[0156] Third-party quality inspection of representative finished products in Experiment Example 1

[0157] A representative batch A of finished products prepared according to the process of Example 2 of this invention, and another batch B of finished products prepared using the same process but in a different batch, were selected and commissioned to different third-party testing institutions for quality testing. Batch A was sent to SGS-CSTC Standards Technical Services Co., Ltd. Dalian Branch for testing, and the test report number was DLF25-0025100-01; batch B was sent to Jilin Provincial Institute of Product Quality Supervision and Inspection for testing, and the test report number was WTSP2502092. The testing items included sensory evaluation, moisture content, protein (on a dry basis), peptide content (on a dry basis), total ginsenosides, lead, and microbiological indicators.

[0158] The test results showed that batch A had a protein content (dry basis) of 99.2%, a peptide content (dry basis) of 91.0%, a moisture content of 4.26%, a total ginsenoside content of 0.37%, and no detectable lead. The total bacterial count, coliform bacteria, mold, Salmonella, and Staphylococcus aureus all met the requirements of Q / JLTG0035S-2025. Batch B had a protein content (dry basis) of 96.9%, a peptide content (dry basis) of 92.7%, a moisture content of 2.86%, a total ginsenoside content of 0.11%, and no detectable lead. The microbiological items also met the requirements of Q / JLTG0035S-2025. This indicates that the representative product obtained by this invention has high protein and peptide content, a certain amount of ginsenosides, and good product safety.

[0159]

[0160] Experiment Example 2: In vitro assisted hypoglycemic activity experiment

[0161] Take the samples obtained in Examples 1-5 and Comparative Examples 1-16, pulverize them, and pass them through an 80-mesh sieve. Weigh an appropriate amount of each group of samples, prepare a 20 mg / mL sample solution with deionized water, shake and dissolve at 37°C for 30 min, centrifuge at 10000 rpm for 10 min, and take the supernatant for later use.

[0162] The degree of hydrolysis (DH) was determined using the OPA method; the proportion of <1kDa peptides was characterized by the percentage of 1kDa ultrafiltration-permeable peptides in the total peptides of the sample supernatant; α-glucosidase inhibitory activity was determined using the PNPG method, with a final sample concentration of 1.0 mg / mL; and the glucose uptake enhancement rate was detected using the HepG2 insulin resistance model. HepG2 cells were routinely cultured in high-glucose DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Logarithmic growth phase cells were seeded into 96-well plates at a density of 8 × 10⁶ cells per well. 3 ~1×10 4 Cells were cultured for 24 hours, then the culture medium was discarded, and the cells were simultaneously cultured in serum-free medium for 12 hours; subsequently, a solution containing 1×10⁻⁶ cells was added. -6 Insulin resistance model was established by culturing in medium containing 100 μmol / L insulin for 24 h. After model establishment, sample solution was added to each drug-treated group to bring the final sample concentration to 0.5 mg / mL, and cultured for another 24 h. Equal volumes of medium were added to the normal control wells and the model control wells. The supernatant was then discarded, and the cells were washed once with glucose-free medium. Glucose-free medium containing 100 μmol / L 2-NBDG was added, and the cells were incubated at 37°C for 30 min. After washing, the fluorescence intensity was measured using a fluorescence microplate reader (excitation wavelength 485 nm, emission wavelength 535 nm). The glucose uptake enhancement rate was calculated for each group, using the model group as a baseline. Before formal testing, the CCK-8 assay was used to verify that the selected sample concentration had no significant effect on cell viability.

[0163] Each group of samples was prepared and tested independently three times, and the results are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used for inter-group comparisons, and Tukey's method was used for post-hoc multiple comparisons. A p-value < 0.05 was considered statistically significant. This experiment was mainly used to compare the differences in in vitro glucose-lowering activities among the various examples and comparative examples.

[0164] The experimental results are shown in Table 2.

[0165] Table 2. In vitro screening results of each embodiment and comparative example.

[0166] Conclusions: As shown in Table 2, under in vitro screening conditions, Example 2 was superior to Comparative Examples 1-6 in terms of α-glucosidase inhibition rate and glucose uptake enhancement rate. This indicates that the use of neutral protease, elase, and carboxypeptidase Y in the second stage, combined with stepwise ultra-high pressure assisted treatment and the enzymatic hydrolysis conditions in the first and third stages, is beneficial to improving the in vitro hypoglycemic activity of the samples. The results of Comparative Examples 7-9 show that there is a certain correlation between the current pretreatment method and formulation range and the in vitro activity level. The results of Comparative Examples 10-12 show that when any component of ginseng, peony seed cake, or functional peptides is removed, the activity of the samples decreases to varying degrees. The decrease is relatively large after removing ginseng, suggesting that ginseng-related components are an important part of the overall activity. Examples 4 and 5 further improved the in vitro activity indicators, with Example 5 showing the best overall performance. Meanwhile, although Comparative Examples 13-16 showed some improvement compared to Example 2, they were still lower than Example 5 overall, indicating that optimizing the ginsenoside delivery form in S6 helps to further improve the in vitro utilization performance of the final product.

[0167] Experiment Example 3: Mouse-assisted hypoglycemic experiment

[0168] SPF-grade male C57BL / 6J mice, 6 weeks old and weighing 18–22 g, were selected and acclimatized for one week before the experiment. The rearing conditions were: temperature 22±2℃, relative humidity 50%–60%, 12-hour light-dark cycle, and free access to food and water. Except for the normal control group, all other groups of mice were fed a high-fat diet for 4 weeks; subsequently, they were intraperitoneally injected with streptozotocin (STZ) for 5 consecutive days. STZ was freshly prepared with 0.1 mol / L citrate-sodium citrate buffer (pH 4.5) at a dose of 35 mg / kg. Fasting blood glucose was measured 7 days after the last injection; mice with a fasting blood glucose level ≥11.1 mmol / L were considered successfully modeled. Successfully modeled mice were randomly divided into a model group, a positive control group, an example group, and a comparative group, with 6 mice in each group. The normal and model groups were administered an equal volume of purified water by gavage, the positive control group was administered acarbose 30 mg / kg by gavage, and each example group and comparative group was administered the corresponding sample at 500 mg / kg by gavage. The gavage volume was uniformly 10 mL / kg, once daily for 6 consecutive weeks. Fasting blood glucose, HbA1c, and OGTT AUC were measured after the administration period.

[0169] The oral glucose tolerance test (OGTT) was conducted before the last administration. Mice in each group were fasted for 12 hours and then given free access to water before being administered glucose solution by gavage at a dose of 2.0 g / kg body weight. Tail venous blood was collected before glucose administration (0 min) and at 30 min, 60 min, and 120 min after administration, and blood glucose levels were measured using a glucometer. Glucose tolerance was evaluated using the area under the blood glucose-time curve (AUC), calculated using the trapezoidal method: OGTT AUC = (G0 + G30) × 30 / 2 + (G30 + G60) × 30 / 2 + (G60 + G120) × 60 / 2, where G0, G30, G60, and G120 represent blood glucose values ​​at each time point in mmol / L, and AUC is expressed in mmol·min / L.

[0170] Data from each group are expressed as mean ± standard deviation (mean ± SD), with n = 6 for each group. One-way ANOVA was used for inter-group comparisons, and Tukey's method was used for post-hoc multiple comparisons. A p-value < 0.05 was considered statistically significant. This experiment was primarily used to verify the differences in in vivo adjuvant blood glucose lowering effects among the various embodiments and comparative examples. Experimental results are shown in Table 3.

[0171] Table 3. Results of assisted blood glucose reduction in mice in each group (mean±SD, n=6)

[0172] Conclusions: As shown in Table 3, compared with the model group, the fasting blood glucose, HbA1c, and OGTT AUC of each example group and most comparative groups showed a decreasing trend, indicating that each sample exhibited varying degrees of in vivo auxiliary hypoglycemic effect under the experimental conditions. Among them, Example 5 showed the greatest overall improvement, followed by Example 4, and Example 2 was better than Example 3 and Example 1, suggesting that further introducing ginsenoside phospholipid nano-dispersion and composite microencapsulation treatment on the basic three-stage enzymatic hydrolysis scheme helps to improve the in vivo performance of the samples. Comparative Examples 1-6 were generally lower than Example 2, indicating that the key enzyme system in the second stage, ultra-high pressure auxiliary treatment, and the enzymatic hydrolysis conditions in the first and third stages are correlated with the in vivo effect of the final product; Comparative Examples 7-9 further suggest that the integrity of pretreatment and the range of formulation also affect the in vivo performance. The results of Comparative Examples 10-12 show that ginseng, peony seed cake, and functional peptides all participated in constructing the overall effect, with the decrease being more significant after removing ginseng. Although Comparative Examples 13-16 performed better than some of the front-end process destructive comparative examples, they were still lower than Example 5 overall, indicating that optimizing the delivery structure in S6 helps to further improve the in vivo adjuvant hypoglycemic effect.

[0173] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A production process for peony complex small molecule peptides, characterized in that, The process includes the following steps: S1. Obtain bovine bone, peony seed cake and ginseng, and pre-treat bovine bone, peony seed cake and ginseng respectively to obtain bovine bone collagen slurry, peony protein enriched phase, ginsenoside enriched part and ginseng slurry to be enzymatically hydrolyzed; S2. The bovine bone collagen slurry is subjected to the first stage of enzymatic hydrolysis, and then subjected to 10kDa ultrafiltration and 3kDa ultrafiltration fractionation in sequence. The 3kDa permeate is collected as the first small molecule peptide component P1, and the 3kDa membrane retentate is combined with the 10kDa membrane retentate as the second stage of enzymatic hydrolysis raw material R1. S3. Perform the second stage of enzymatic hydrolysis: Mix the truncated phase R1 with the peony protein enrichment phase, adjust the pH of the system to 7.2–7.8, and add 0.20%–0.40% neutral protease and 0.01%–0.06% elastin based on the total dry weight of the enzymatically hydrolyzable protein in the second stage reaction system at 40–46°C. After adding the enzymes, perform a stepwise ultra-high pressure assisted enzymatic hydrolysis treatment on the system. After the ultra-high pressure assisted enzymatic hydrolysis treatment is completed, continue enzymatic hydrolysis at atmospheric pressure for 55–65 min. Then, cool the system to 25–35°C and adjust the pH to 5.2–6.

0. ​​Add 0.005%–0.05% Carboxypeptidase Y based on the total dry weight of the enzymatically hydrolyzable protein in the second stage reaction system, and continue enzymatic hydrolysis at atmospheric pressure for 40–50 min. After the second stage of enzymatic hydrolysis is completed, perform 3 kDa ultrafiltration, collect the permeate as the second small molecule peptide component P2, and retain the truncated phase R2. S4. Add the ginseng pulp to be enzymatically hydrolyzed to the truncated phase R2 for the third enzymatic hydrolysis, and then perform ultrafiltration fractionation at 1 kDa. Collect the permeate as the third small molecule peptide component P3. S5. Combine the first small molecule peptide component P1, the second small molecule peptide component P2 and the third small molecule peptide component P3, and after desalting and concentration, obtain a natural complex small molecule peptide base. S6. Under conditions of 20-35℃, the ginsenoside enrichment fraction and the functional peptide prepared by solid-phase synthesis are added to the natural complex small molecule peptide base, mixed and dried to obtain the peony complex small molecule peptide product. Specifically, S2 includes: adjusting the bovine bone collagen slurry to pH 2.8–3.2, adding 0.15%–0.45% acidic fungal protease based on the dry weight of bovine bone collagen at 40–44°C, and enzymatically hydrolyzing for 40–60 min; after the first enzymatic hydrolysis, adjusting the pH to 6.5–7.0 and incubating at 75–90°C for 5–15 min to inactivate the enzyme, followed by 10 kDa ultrafiltration, collecting the permeate, further performing 3 kDa ultrafiltration on the 10 kDa ultrafiltration permeate, collecting the permeate as the first small molecule peptide component P1, and combining the 3 kDa membrane retentate with the 10 kDa membrane retentate as the raw material R1 for the second enzymatic hydrolysis; S4 specifically includes: adding the ginseng slurry to be enzymatically hydrolyzed to the truncated phase R2; first adjusting the system to pH 6.3–6.8; then, at 37–42°C, adding 0.05%–0.12% proline-specific endonuclease based on the total dry weight of the enzymatically hydrolyzable proteins in the third-stage reaction system, and hydrolyzing for 30–40 min; then adjusting the system to pH 6.8–7.2; adding 0.10%–0.25% flavor protease at 45–50°C, and continuing hydrolysis for 30–40 min; after the third-stage hydrolysis is completed, performing 1 kDa ultrafiltration and collecting the permeate as the third small molecule peptide component P3; The proportions of each substance by weight are as follows: 225-255 parts of bovine bone; 340-420 parts of peony seed cake; 15-20 parts of ginseng; and 4-6 parts of functional peptides. The functional peptides are oligopeptides prepared by solid-phase synthesis, and their amino acid sequence is IPWQHRAFNKR. In step S6, the ginsenoside-enriched fraction is first compounded with phospholipids and phytosterols, and then subjected to ethanol injection-high pressure homogenization to obtain ginsenoside phospholipid nanodispersions, which are then added to the natural complex small molecule peptide base.

2. The production process of peony compound small molecule peptides as described in claim 1, characterized in that, The preprocessing includes: Fresh bovine bones are selected, washed, and crushed into 5-20 mm particles. They are then rinsed in hot water at 85-95℃ for 10-20 minutes. Next, a mixed solution of 0.8%-2.0% citric acid and 0.2%-0.8% sodium citrate is used at a material-to-liquid ratio of 1:4-1:8, and treated at 20-35℃ for 4-10 hours. After washing until the pH reaches 5.5-6.5, the mixture is further refined using a wet colloid mill to obtain bovine bone collagen slurry. The bovine bone collagen slurry is then subjected to ultra-high pressure pretreatment at a pressure of 120-220 MPa for 5-12 minutes. Peony seed cake was pulverized through a 40-100 mesh and washed 1-2 times with 50%-75% edible ethanol at 35-50℃. After ethanol recovery, the cake was resuspended in water, and 0.1%-0.5% cellulase and 0.05%-0.3% pectinase were added. The cake was then subjected to cell wall disruption at 45-52℃ and pH 4.5-5.5 for 0.5-1.5 hours. Subsequently, the pH was adjusted to 8.5-9.5 for alkali extraction of protein for 30-90 minutes. The supernatant was collected by centrifugation at 3000-8000 rpm for 5-20 minutes. The pH was then adjusted to 4.2-4.8 for isoelectric precipitation. The precipitate was collected and redissolved at a pH of 6.5-7.5 to obtain a peony protein-enriched phase. Ginseng is pulverized to 40-80 mesh and extracted 1-3 times with 55%-75% ethanol at 50-70℃ and a material-to-liquid ratio of 1:8-1:

20. The extracts are combined, concentrated under reduced pressure, and enriched with macroporous resin to obtain the ginsenoside-rich fraction. The extracted residue is deethanolinated, resuspended in water, and then treated with 0.05%-0.2% α-amylase and 0.1%-0.4% cellulase at 50-58℃ for 0.5-1.5 hours to obtain the ginseng pulp to be enzymatically hydrolyzed.

3. The production process of peony complex small molecule peptides as described in claim 2, characterized in that, The pressure-assisted enzymatic hydrolysis treatment in step S3 is a stepped pressure treatment, with the following conditions: first, treatment at 80-120 MPa for 4-8 minutes, and then treatment at 120-160 MPa for 2-5 minutes.

4. The production process of peony complex small molecule peptides as described in claim 1, characterized in that, The weight ratio of the ginsenoside enriched fraction, total phospholipids and phytosterols is 1:6-15:0.5-4; the average Z-particle size of the nano-dispersion is 80-220 nm, the polydispersity index (PDI) is ≤0.35, and the encapsulation efficiency is 70%-92%.

5. The use of a product manufactured using the production process of peony complex small molecule peptides according to any one of claims 1-4 in the preparation of a composition for assisting in lowering blood sugar.

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