Preparation method of peony and ginseng peptide for enhancing immunity and tonifying qi and blood

Abstract

The invention provides a preparation method of peony and ginseng peptide for enhancing immunity and tonifying qi and blood, and belongs to the field of nutritious food. The composition comprises a peony peptide extract and ginseng polypeptide, the peony peptide extract is prepared by the following steps: soaking peony seeds in an elicitor solution, and fermenting with rhizopus oligosporus; and separating and purifying to obtain the peony peptide extract. Fresh peony seeds are soaked in the elicitor, biological stress is simulated to activate the defensive reaction of the peony seeds, and protein accumulation is induced; the rhizopus oligosporus is fermented for biotransformation, protein degradation is promoted, and active substances are possibly generated. And carrying out subsequent extraction to obtain the peony peptide extract. The extract and ginseng polypeptide are mixed in proportion, bioactive components from different sources are integrated, small molecule peptide is easy to absorb, and the immune regulation function is enhanced through the multi-target synergistic effect. The whole process is based on plant physiological response and biotransformation, and the preparation efficiency and quality of the peptide are optimized, so that the final composition has the effects of enhancing immunity and tonifying qi and blood.

Description

Technical Field

[0001] This invention belongs to the field of nutritional foods, and in particular relates to a method for preparing peony ginseng peptides that enhance immunity and replenish qi and blood. Background Technology

[0002] As an important component of modern health management, the development of health supplements has always been closely linked to humanity's pursuit of a higher quality of life. Among the many products claiming to promote health, the function of enhancing immunity has attracted particular attention. The immune system is a complex network that protects the body from pathogens and maintains homeostasis; its balanced and robust function is the core foundation of health. In exploring ways to support the immune system through external supplementation, peptides, with their unique biological activity and mechanisms, have gradually become a focus of in-depth research in both the scientific and industrial communities.

[0003] Peptides are short-chain molecules composed of amino acids linked by peptide bonds, with molecular weights between those of amino acids and proteins. This structural feature gives them unique advantages: compared to large protein molecules, peptides are more easily absorbed and utilized by the human body; compared to single amino acids, they possess specific spatial structures and biological information, enabling them to participate in more precise physiological regulation. In the field of immune regulation, some bioactive peptides have shown the ability to bind to specific receptors on the surface of immune cells, acting like a precise key to activate or regulate intracellular signaling pathways. For example, some enzymatically hydrolyzed peptides derived from milk proteins, soy proteins, or marine fish proteins have been found to gently regulate the activity of immune cells such as macrophages and natural killer cells, promote the secretion of certain cytokines with anti-inflammatory or immune-enhancing effects, and help maintain the balance of the immune response, avoiding the negative effects of overactivation. This characteristic of "regulation" rather than simple "stimulation" is considered its value, because ideal immune support should focus on restoring and maintaining optimal immune homeostasis, rather than indiscriminately stimulating the immune response.

[0004] The potential mechanisms by which peptides act on the immune system are multidimensional. Besides directly interacting with immune cells, some studies indicate that certain peptide fragments indirectly influence immunity by supporting gut health. The gut is the largest immune organ in the human body, and its mucosal immune system function is crucial. Some peptides may help maintain the integrity of the intestinal mucosal barrier or have beneficial effects on the gut microbiota, thus laying the foundation for overall immune health. Furthermore, during periods of high physical exertion or sustained mental stress, the body's amino acid requirements may change. Supplementation with certain peptides may help optimize protein metabolism, providing necessary nutritional support for vital functions such as the immune system and mitigating temporary fluctuations in immune function caused by stress.

[0005] In the broad spectrum of immune enhancement, peptides are not isolated players. Other components, with more extensive research and validation, also play important roles. For example, polysaccharides, especially β-glucan from edible fungi, have been widely studied for their regulatory effects on innate immunity. Vitamins and minerals are the cornerstones of a properly functioning immune system; adequate intake of vitamin C, vitamin D, and elements such as zinc and selenium is crucial for immune function.

[0006] Currently, research on peptide-based health supplements is deepening. The research trend lies not only in discovering new active peptides, but also in using proteomics, bioinformatics, and other methods to more precisely elucidate their targets and molecular mechanisms; in utilizing advanced delivery technologies to improve their bioavailability; and in defining specific populations and application scenarios through more refined clinical studies. Summary of the Invention

[0007] The purpose of this invention is to provide a peony ginseng peptide that enhances immunity and replenishes qi and blood, comprising peony peptide extract and ginseng polypeptide; The mass ratio of the peony peptide extract to the ginseng polypeptide is 7-8:2-3; The peony peptide extract is obtained by soaking fresh peony seeds in an elicitor solution, followed by solid-state aerobic fermentation with Rhizopus oligosporus and sterilization. Fermented peony seeds are obtained by pressing the fermented peony seeds for oil. Fermented peony seed meal is obtained by crushing, alkali dissolving and acid precipitation, and enzymatic hydrolysis of the fermented peony seed meal. The enzymatic hydrolysate is then separated and purified to obtain the peony peptide extract.

[0008] Preferably, the exciton solution contains at least an exciton with a cyclopentenone structure, and the concentration of the exciton is 50-200 μM; the soaking time of the exciton solution is at least 6 hours.

[0009] Preferably, the specific steps of the solid-state aerobic fermentation of Rhizopus oligosporus are as follows: S1, Rhizopus oligosporus was activated and cultured to prepare a spore suspension with a concentration of 10. 6 -10 7 spores / mL; S2, the spore suspension is sprayed onto the surface of peony seeds for inoculation, and the surface of the peony seeds is coated with a carbon source; S3, control the initial pH of fermentation to ≤7, the temperature to 29±1℃, maintain oxygen supply, and ferment for at least 72 hours under an ambient humidity of ≥80%; after fermentation, sterilize and inactivate enzymes at high temperature to obtain fermented peony seeds.

[0010] In step S2 The inoculation amount of the spore suspension is at least 5 wt% of the dry weight of the peony seeds; the amount of carbon source added is at least 2 wt% of the dry weight of the peony seeds.

[0011] Preferably, the alkali dissolution and acid precipitation involves stirring the crushed fermented peony seed meal with an alkaline solution with a pH of 8.0-9.0 under heating to form a hot alkali mixture; after centrifugation of the hot alkali mixture, the supernatant is collected, and an acidic solution is added to the supernatant to adjust the pH to ≤4.5; then centrifugation is performed again, and the precipitate is collected; after washing the precipitate, it is redispersed in water and the pH is adjusted to neutral or alkaline to obtain a crude protein solution.

[0012] Preferably, the enzymatic hydrolysis involves first hydrolyzing the crude protein solution obtained after alkali dissolution and acid precipitation of fermented peony seeds with alkaline protease, and then performing a second hydrolysis using flavor protease to obtain the hydrolysate. The first enzymatic hydrolysis should be performed at a temperature of 40-55℃ and a pH of 8.0-9.0, for at least 3 hours, and the amount added should be at least 2% of the weight of the fermented peony seed meal. The second enzymatic hydrolysis is performed at a temperature of 40-55℃ and a pH of 6.0-7.5 for at least 3 hours, with an addition amount of at least 2% of the fermented peony seed meal mass.

[0013] Preferably, the enzymatic hydrolysate separation and purification involves inactivating the enzymatic hydrolysate, cooling it, centrifuging it, and collecting the supernatant; the supernatant is fractionated using an ultrafiltration membrane to obtain the fraction with a molecular weight of 2 kDa or less; after concentration, it is freeze-dried to obtain the peony peptide extract.

[0014] Preferably, the fresh peony seeds are first washed before soaking in the stimulating solution, and then soaked in an ethanol aqueous solution, followed by soaking in a disinfectant and rinsing with water for surface sterilization.

[0015] The present invention also provides the application of the aforementioned peony ginseng peptide, which enhances immunity and replenishes qi and blood, in the preparation of products that enhance immunity.

[0016] This invention relates to a substance prepared using a specific process, which is a compound of peony peptide extract and ginseng polypeptide in a specific ratio, and claims to support immune function. The principle and advantages of this method are rooted in the cross-application of modern plant physiology, fermentation engineering, and bio-enzymatic hydrolysis technology. Its core design concept is to directionally enrich and prepare polypeptide components with potential biological activity through the pretreatment and biotransformation of raw materials.

[0017] The starting point of this invention is "active peony seeds with germination potential." Only living seeds can initiate a complete defense response when sensing stress. Soaking the seeds in elicitors containing cyclopentenone structures (such as methyl jasmonic acid) is to simulate stress signals such as pathogen attack, thereby activating defense signaling pathways such as jasmonic acid within the seeds and inducing the synthesis and accumulation of more defense-related proteins. This is the material basis and prerequisite for all subsequent processes.

[0018] Rhizopus oligosporus grows and metabolizes under solid-state aerobic conditions. On the one hand, it secretes a variety of enzymes that degrade the cell walls of peony seeds, destroying structural barriers such as cellulose and pectin, making the proteins enriched in the cells easier to extract through subsequent alkali dissolution and acid precipitation processes. On the other hand, the small molecules produced during microbial metabolism can preliminarily modify the proteins, enhancing the activity of subsequent enzymatic hydrolysis products. At the same time, the fermentation environment can inhibit contamination by miscellaneous bacteria, ensuring the safety of the raw materials.

[0019] After fermentation, the seed meal undergoes alkali dissolution and acid precipitation to remove most non-protein impurities and initially enrich the target protein. A tandem enzymatic hydrolysis strategy of "alkaline protease → flavor protease" is then employed. First, endonucleases cleave large protein molecules into peptides, followed by exonucleases to further trim the ends, aiming to obtain a mixture of peptides with smaller molecular weights, easier absorption, and potentially lower bitterness. Finally, ultrafiltration (≤2kDa) is used to achieve molecular weight fractionation, ensuring the consistency of the final product composition.

[0020] This invention uses a compound of peony peptide extract and ginseng peptide in a mass ratio of 7-8:2-3. The immune mechanisms of the two active peptides complement each other: peony peptide is derived from plant defense-related proteins and can regulate the proliferation and differentiation of immune cells in the body; ginseng peptide is an active component of traditional food and medicine raw materials and can enhance the activity of immune cells and the secretion of cytokines. The two work synergistically to achieve an immune enhancement effect of "1+1>2".

[0021] This invention does not simply extract inherent components from raw materials, but creatively integrates a three-step biotechnology process: "plant activation - fungal fermentation - enzymatic purification". It actively intervenes in the metabolic state of raw materials, utilizing the organism's own response mechanisms and the transformation capabilities of microorganisms, aiming to increase the reserves of functional proteins in the raw materials and optimize their existing forms.

[0022] This invention, from activation pretreatment and solid-state fermentation with specific microbial strains to stepwise hydrolysis with dual enzymes and membrane separation purification, designs each step with a clear progressive objective and an inherent logical connection. For example, fermentation alters the protein's susceptibility to enzymatic digestion, which in turn directly determines the yield and properties of peptides, while membrane separation enables precise control over the molecular weight range.

[0023] This product, prepared using a novel process, is a complex of peony peptides and ginseng polypeptides designed to support the body's "Qi and Blood" balance from a modern biological perspective. Its small-molecule peptide components are easily absorbed, providing specific nutritional support to target cells. The traditional tonifying components derived from ginseng help mobilize bodily functions and promote energy metabolism, corresponding to the "Qi tonification" aspect. Meanwhile, the peony active peptides, enriched and transformed through a special process, may support intrinsic nourishing and regulatory functions, working synergistically with the ginseng peptides to address the "blood nourishment" aspect. The two are combined in a specific ratio, working synergistically on the body's energy metabolism and internal balance system, thereby supporting immune function while helping to maintain overall physiological coordination and vitality.

[0024] This invention utilizes cyclopentenone elicitors to soak fresh peony seeds, simulating biological stress to activate their defense response and induce protein accumulation, providing a rich substrate for subsequent peptide preparation. Solid-state aerobic fermentation with *Rhizopus oligosporus* under controlled temperature and humidity conditions promotes protein degradation and may generate bioactive substances. After fermentation, the oil is extracted, and the resulting meal is purified by alkali dissolution and acid precipitation. The protein is then efficiently generated through stepwise enzymatic hydrolysis with alkaline proteases and flavor proteases, followed by ultrafiltration purification to obtain a peony peptide extract with a molecular weight ≤2kDa. This extract is mixed with ginseng peptides in a specific ratio, integrating bioactive components from different sources. The small-molecule peptides are easily absorbed and enhance immunomodulatory function through multi-target synergistic effects. The entire process is based on plant physiological responses and biotransformation, optimizing the efficiency and quality of peptide preparation, resulting in a final composition with potential immune-enhancing effects. Detailed Implementation

[0025] To better understand the present invention, the present invention will be further described below with reference to specific serial numbers. The terminology used in the serial numbers is for describing specific embodiments and does not constitute a limitation on the scope of protection of the present invention.

[0026] In the specific implementation methods, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0027] In the specific implementation method, unless otherwise specified, percentages and % are assumed to be mass percentages; non-mass percentages will be specified.

[0028] The Rhizopus oligosporus used in this invention is ATCC 22959. The Bacillus subtilis used in this invention is ATCC 6633. The ginseng polypeptide used in this invention is a food-grade water-soluble ginseng polypeptide with a molecular weight ≤500 Da and a purity higher than 99%.

[0029] The peony seeds used in this invention are mature seeds of oil peony, identified as Paeonia suffruticosa Andr.

[0030] Example 1: Preparation of peony seeds fermented with methyl jasmonate-Rhizopus oligosporus Includes the following steps: S1 Peony Seed Treatment: Rinse whole peony seeds with clean water to remove impurities; then soak them in 70% ethanol for 60 seconds and pour them out; then soak them in 2wt% sodium hypochlorite solution for 15 minutes, gently shaking them during the process; finally, rinse them thoroughly three times with sterile water to remove any residual disinfectant; drain and set aside.

[0031] S2 strain activation: Inoculate *Rhizopus oligosporus* onto PDA (potato dextrose agar) slant medium and incubate at 28-30°C for 5 days until spores mature. Wash off the spores with sterile water to prepare a spore suspension, and adjust the concentration to 10 using a hemocytometer. 6 -10 7 Spores / mL.

[0032] S3 Peony Seed Activation Treatment: Soak peony seeds in an aqueous solution of methyl jasmonate (50 μM) (containing a small amount of Tween 80) for 6 hours, then remove and drain for later use.

[0033] S4 Solid-State Fermentation: Place the drained peony seeds into a sterilized shallow dish.

[0034] Inoculate the seeds with a suspension of Rhizopus spores at a rate of 5% of the dry weight of the peony seeds. Then, sprinkle wheat bran (2 wt% of the dry weight of the peony seeds) on the surface of the seeds; turn the seeds over once to ensure even adhesion.

[0035] Then, control the initial fermentation pH to ≤7 (if the pH does not meet the standard, use dilute HCl solution to adjust), the temperature to 29±1℃, maintain oxygen supply, and ferment for at least 72 hours with an ambient humidity ≥80%. During the 24 and 48 hours of fermentation, the material is gently turned over once under aseptic conditions to break up the clumps formed by the mycelium and promote internal oxygen exchange.

[0036] S5 sterilization post-treatment: After fermentation, steam at 121℃ for 20 minutes; then cool rapidly to obtain fermented peony seeds.

[0037] Example 2: Preparation of peony seeds fermented with methylcyclopentenolone-Rhizopus oligosporus The difference from Example 1 is that in step S3, the peony seed activation treatment uses a methylcyclopentenolone aqueous solution (50 μM concentration) instead of a methyl jasmonate aqueous solution (50 μM concentration).

[0038] Example 3: Preparation of peony seeds fermented by Rhizopus oligosporus The difference from Example 1 is that after the bacterial strain is activated in step S2, step S3, the peony seed activation treatment is skipped, and step S4, solid-state fermentation is carried out directly.

[0039] Example 4: Preparation of peony seeds fermented with methyl jasmonate-Bacillus subtilis The difference from Example 1 is that, Step S2, bacterial activation, is modified as follows: Bacillus subtilis is streaked onto LB agar plates and cultured at 37°C for 18 hours to obtain single colonies.

[0040] Pick a single colony and inoculate it into an Erlenmeyer flask containing LB liquid medium. Incubate at 37°C and 150 rpm with shaking for 16 hours until the late logarithmic growth phase (OD2). 600 (Approximately 1.0-1.5), to obtain seed liquid.

[0041] Inoculum adjustment: Centrifuge the seed culture to collect the bacterial cells, resuspend in sterile physiological saline, and adjust the bacterial concentration to approximately 10. 8 -10 9 Prepare a bacterial suspension at CFU / mL for later use.

[0042] Step S4, solid-state fermentation, is modified as follows: Inoculate the seeds with the bacterial suspension at a rate of 5% of their dry weight, spraying it evenly onto the seeds. Then, sprinkle wheat bran (2 wt% of the dry weight of the peony seeds) onto the surface of the seeds; turn the seeds over once to ensure even adhesion.

[0043] Then control the initial fermentation pH to ≤7 (if the pH does not meet the standard, use dilute HCl solution to adjust), the temperature to 36±1℃, maintain oxygen supply, and ferment for at least 60 hours under ambient humidity ≥80%; During the 12th and 24th hours of fermentation, the material is gently turned over once under aseptic conditions to dissipate heat and ensure even aeration.

[0044] Example 5: Preparation of peony seeds fermented by Bacillus subtilis The difference from Example 4 is that after the bacterial strain is activated in step S2, step S3, the peony seed activation treatment is skipped, and step S4, solid-state fermentation is carried out directly.

[0045] The protein content of fermented peony seeds and fresh peony seeds prepared in Examples 1-5 above was determined by the Kjeldahl method in GB5009.5-2016 "National Food Safety Standard - Determination of Protein in Food".

[0046] The part measured was the kernel of the peony seed after dehulling, and it had been degreased with petroleum ether.

[0047] The results are as follows: Example 1: Methyl jasmonate-Rhizopus oligosporus fermented peony seeds: 31.3% Example 2: Methylcyclopentenolone-Rhizopus oligosporus fermentation of peony seeds: 30.8% Example 3: Fermented peony seeds by Rhizopus oligosporus: 28.1% Example 4: Methyl jasmonate-Bacillus subtilis fermentation of peony seeds: 27.8% Example 5: Bacillus subtilis fermentation of peony seeds: 26.6% Fresh peony seeds: 24.6%.

[0048] To investigate the effect of elicitor concentration on protein content, peony seeds were soaked in aqueous solutions of methyl jasmonate (containing a small amount of Tween 80) at concentrations of 20 μM, 100 μM, 200 μM, and 400 μM, using the same preparation method as in Example 1. The protein content was then determined using the Kjeldahl method, and the results are as follows: Peony seeds fermented with Rhizopus oligosporus treated with methyl jasmonate at a concentration of 20 μM: 28.2% Peony seeds fermented with Rhizopus oligosporus treated with methyl jasmonate at a concentration of 100 μM: 31.6% Peony seeds fermented with Rhizopus oligosporus treated with methyl jasmonate at a concentration of 200 μM: 32.2% Peony seeds fermented with Rhizopus oligosporus treated with methyl jasmonate at a concentration of 400 μM: 26.1% The results show that too high an elicitor concentration can have an inhibitory effect, while too low a concentration will not reach the threshold and will not be effective.

[0049] Example 6 Extraction of peony ginseng peptides Includes the following steps: S1 Pretreatment and Alkali Dissolution and Acid Precipitation Extraction of Crude Protein: Peony seed meal obtained after pressing peony seeds for oil was passed through an 80-mesh sieve to obtain uniform meal powder.

[0050] Mix the meal powder with 0.1M sodium hydroxide solution at a ratio of 1:10 (w / v) and adjust the pH of the mixture to 8.5. Stir continuously in a 50°C constant temperature water bath for 60 minutes to fully dissolve the protein.

[0051] Centrifuge the above hot alkali mixture at 4°C and 8000×g for 20 minutes and collect the supernatant.

[0052] While stirring, slowly add 1.0M hydrochloric acid (HCl) solution dropwise to the supernatant to adjust the pH to 4.2.

[0053] At this point, the protein flocculates and precipitates. The solution is centrifuged again at 4°C and 8000×g for 15 minutes, the supernatant is discarded, and the precipitate (protein precipitate) is collected.

[0054] The precipitate was washed three times with pre-cooled deionized water to remove residual salts and acids. The precipitate was then redispersed in deionized water, and the pH was adjusted to 7.0 with dilute NaOH solution to obtain a crude protein solution.

[0055] S2 two-step enzymatic hydrolysis method for peptide preparation: Adjust the temperature of the crude protein solution to 50°C and the pH to 8.5.

[0056] Add alkaline protease at 3% of the initial dry weight of peony seed meal; continue enzymatic hydrolysis for 4 hours under these conditions while maintaining a constant pH.

[0057] Adjust the pH of the first enzymatic hydrolysate to 7.0 and maintain the temperature at 50°C.

[0058] Add flavor protease at 2.5% of the initial dry weight of peony seed meal; continue enzymatic hydrolysis for 3 hours under these conditions.

[0059] After enzymatic hydrolysis, the hydrolysate is rapidly heated to 90°C and held for 10 minutes to inactivate the protease, and then immediately cooled to room temperature in an ice bath.

[0060] Separation and purification of S3 enzyme hydrolysate: Centrifuge the cooled enzymatic hydrolysate at 4°C and 10000×g for 30 minutes and collect the clear supernatant.

[0061] A tangential flow ultrafiltration system was used, with polyethersulfone (PES) membrane packs having molecular weight cutoffs of 10 kDa, 5 kDa, and 2 kDa, respectively.

[0062] First, pass the supernatant through a 10kDa membrane to remove large protein molecules that have not been fully digested; then collect the permeate.

[0063] The permeate is then passed through a 5kDa membrane to collect peptide components with a molecular weight ≤5kDa (permeate).

[0064] Finally, the permeate is passed through a 2kDa membrane to collect peptide components with a molecular weight ≤2kDa.

[0065] The obtained peptide fraction solution of ≤2kDa was concentrated to a solid content of about 30% at below 50°C using a rotary evaporator or vacuum concentrator.

[0066] The concentrate was freeze-dried to obtain a white peony peptide extract powder.

[0067] The degree of hydrolysis and protein content of peony peptide extract were determined; the degree of hydrolysis was determined by the ninhydrin colorimetric method; the protein content was determined by the Kjeldahl method; the results are as follows: Peony seed meal was derived from the peony peptide extract of Example 1, with a degree of hydrolysis of 31.3% and a protein content of 95.5%.

[0068] Peony seed meal was derived from the peony peptide extract of Example 2, with a degree of hydrolysis of 30.6% and a protein content of 96.1%.

[0069] Peony seed meal was derived from the peony peptide extract of Example 3, with a degree of hydrolysis of 32.6% and a protein content of 95.4%.

[0070] Peony seed meal was derived from the peony peptide extract of Example 4, with a degree of hydrolysis of 31.8% and a protein content of 94.3%.

[0071] Peony seed meal was derived from the peony peptide extract of Example 5, with a degree of hydrolysis of 30.4% and a protein content of 94.7%.

[0072] Peony seed meal is derived from peony peptide extract from fresh peony seeds, with a degree of hydrolysis of 29.3% and a protein content of 93.6%.

[0073] The results show that the two-step enzymatic hydrolysis and ultrafiltration purification method used in this invention can yield peony peptide extract with high degree of hydrolysis and high purity.

[0074] Example 7 Preparation and performance testing of peptide compositions 1. Prepare peptide compositions according to the proportions in Table 1 for performance testing.

[0075] Table 1 Preparation of peptide compositions

[0076] The sources of peony peptide extracts in Table 1 refer to the various peony peptide extracts prepared in Example 6, with the source of peony seed meal continuing to follow the corresponding example number.

[0077] II. DPPH Antioxidant Scavenging Capacity Test: The antioxidant capacity of the peptides was determined using the DPPH method in GB / T 39100-2020, "Determination of Antioxidant Activity of Peptides - DPPH and ABTS Methods"; the results are shown in Table 2 below.

[0078] Table 2 DPPH free radical scavenging rate

[0079] As shown in Table 2, the comparison of items 1, 6, 9, and 10 reveals that the antioxidant capacity of peony peptide and ginseng polypeptide after mixing is not a weighted average of their individual components, but rather higher, proving that there is a certain synergistic effect between peony peptide and ginseng polypeptide. The comparison of items 1, 6, and 7 shows that this synergistic effect has a threshold; when the proportion of ginseng polypeptide exceeds a certain limit, it will decrease significantly.

[0080] III. Lymphocyte Proliferation Capacity Detection (MTT Method) BALB / c mouse spleen lymphocytes (sterilely isolated, RPMI-1640 medium containing 10% FBS and 1% penicillin antibiotics), adjusted to a cell concentration of 1×10⁻⁶. 6 The peptide composition was seeded at 100 μL / mL in 96-well plates. Each sequence of peptide compositions was dissolved in DMSO (final concentration ≤0.1%), diluted with culture medium to 400 μg / mL, and a blank control group (culture medium only) was included. 50 μL of each peptide composition was added to each well of the 96-well plates. The plates were incubated at 37℃ and 5% CO2 for 48 hours; then 20 μL of MTT (5 mg / mL) was added, and the plates were incubated for another 4 hours. After centrifugation and discarding the supernatant, 150 μL of DMSO was added, and the plates were shaken for 15 minutes (protected from light). The absorbance (OD value) of each well was measured at 570 nm using a microplate reader. A higher OD value indicates stronger lymphocyte proliferation capacity. The results are shown in Table 3 below.

[0081] IV. Macrophage Phagocytic Capacity Detection RAW264.7 macrophages (DMEM high-glucose medium, containing 10% FBS) were seeded in 96-well plates (5 × 10⁶ cells / well). 4 100 μL of each macrophage was added to each well after 12 hours of adhesion. Then, 50 μL of the peptide composition diluted to 400 μg / mL was added to each well. A blank control group was set up (only an equal volume of cells and culture medium was added). After adding the composition, the cells were cultured for another 24 hours. Then, 1 mL of 0.075% neutral red solution was added to each well, and the cells were cultured for another 2 hours. After the culture was completed, the cells were gently washed three times with PBS to remove any unphagocytosed neutral red. 1 mL of cell lysis buffer (ethanol:acetic acid = 1:1) was added to each well, and the cells were shaken at room temperature for 10 minutes to completely release the neutral red from the cells. The OD value of each well was measured at 540 nm using a microplate reader. A higher OD value indicates a stronger phagocytic capacity of the macrophages. The results are shown in Table 3 below.

[0082] V. Detection of interleukin-2 and interferon-γ secretion levels (ELISA method) BALB / c mouse spleen lymphocytes (sterilely isolated, RPMI-1640 medium containing 10% FBS and 1% penicillin antibiotics) were adjusted to a cell concentration of 1×10⁶ cells / mL and seeded into 24-well plates, 1 mL per well. The 24-well plates were incubated at 37℃ in a 5% CO₂ incubator for 2 h to allow cell adhesion. Peptide compositions for each sequence number were dissolved in DMSO (final concentration ≤0.1%), diluted with medium to 400 μg / mL, and a blank control group (medium only) was added to each well (50 μL). After adding the compositions, the cells were incubated for another 48 h, and the supernatant was collected. The ELISA kit was operated according to the instructions. The OD value of each well was measured at 450 nm using a microplate reader, and the secretion levels (pg / mL) of interleukin-2 and interferon-γ in the samples were calculated based on the standard curve. The results are shown in Table 3 below.

[0083] Table 3 Results of Immunological Performance Tests

[0084] The results in Table 3 show that: I. Differences in source treatment determine the potential of basic immune activity. The differences in immune activity stem primarily from the different treatment methods applied to peony seeds before fermentation, which directly affect the composition and function of the final polypeptides.

[0085] The choice of elicitor is crucial: treatment with methyl jasmonate (numbers 1 and 4) or methylcyclopentenolone (number 2) showed a significant advantage over samples without elicitors (numbers 3 and 5) across all immunological indicators. This is because methyl jasmonate, as a natural defense signaling molecule in plants, efficiently activates the jasmonic acid pathway, inducing seeds to synthesize more defense-related functional proteins (such as disease-associated proteins). These proteins, after enzymatic hydrolysis, may produce peptides with specific immunomodulatory activities. Although methylcyclopentenolone has a similar structure, its biological activity is weaker; therefore, the activity in Example 2 was slightly lower than in Example 1, but still significantly higher than the group without elicitors. Protein content assays have preliminarily confirmed this difference in the accumulation of substances at the source.

[0086] The fermentation strains produce different metabolites: Comparing strains 1 (Rhizopus oligosporus) and 4 (Bacillus subtilis), both using methyl jasmonate but different strains, the former exhibits significantly higher immunomodulatory activity than the latter. This difference likely stems not only from variations in protein content but also from the different metabolites and enzyme systems produced by the two microorganisms during fermentation. As a fungus, Rhizopus oligosporus's complex secondary metabolism may produce or modify more specific peptides or accessory components with immunomodulatory functions, while Bacillus subtilis has a different metabolic profile, leading to differences in the bioactivity of the final products.

[0087] II. The proportions of the composite components exhibit a clear synergistic effect window. The data in Table 3 clearly reveal the nonlinear synergistic effect of the combination of peony peptide and ginseng polypeptide, rather than a simple additive effect.

[0088] The optimal ratio range: Group 1 (peony peptide: ginseng polypeptide = 7:3) and Group 6 (8:2) showed the best performance across all immune indicators, significantly outperforming groups using peony peptide (Groups 8 and 10) or ginseng polypeptide (Group 9) alone. This indicates that at specific ratios, the two can produce synergistic effects through multi-target and multi-pathway interactions. For example, peony peptide may primarily activate a certain type of immune cell or factor, while ginseng polypeptide enhances another pathway; the combination of the two achieves a more comprehensive activation of the immune regulatory network.

[0089] Imbalanced proportions led to diminished effects: when the proportion of ginseng peptides was increased to 4 parts (number 7), all immunomodulatory activity indicators decreased. This confirms that there is an optimal threshold for synergistic effects. Excessive ginseng peptides may alter the overall absorption of the mixed peptides, receptor competition, or intracellular signaling balance, thereby interfering with optimal synergistic effects. This underscores the importance of precise proportion control in formulation.

[0090] III. Peony peptide extract alone already possesses core activities, but its efficacy is amplified when combined with other ingredients. The data show that even the untreated peony peptide (using item 8 of Example 3) exhibited significantly higher immunomodulatory activity when used alone than the control group, demonstrating that the basic process (fermentation and enzymatic hydrolysis) itself can produce immunomodulatory peptides. However, the peony peptide stimulated by methyl jasmonate (using item 10 of Example 1) showed even higher activity when used alone, further confirming the contribution of source stimulation treatment to enhancing product functionality. Regardless of the type of peony peptide, its immunomodulatory efficacy was further amplified when combined with appropriate amounts of ginseng polypeptides (items 1 and 6).

[0091] Any change in any step (such as changing the elicitor, altering the bacterial strain, or adjusting the ratio) will affect the final output of this chain.

[0092] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the present invention.

Claims

1. A peony ginseng peptide that enhances immunity and replenishes qi and blood, characterized in that, Including peony peptide extract and ginseng polypeptide; The mass ratio of the peony peptide extract to the ginseng polypeptide is 7-8:2-3; The peony peptide extract is obtained by soaking fresh peony seeds in an elicitor solution, followed by solid-state aerobic fermentation with Rhizopus oligosporus and sterilization. Fermented peony seeds are obtained by pressing the fermented peony seeds for oil. Fermented peony seed meal is obtained by crushing, alkali dissolving and acid precipitation, and enzymatic hydrolysis of the fermented peony seed meal. The enzymatic hydrolysate is then separated and purified to obtain the peony peptide extract.

2. The peony ginseng peptide for enhancing immunity and replenishing qi and blood according to claim 1, characterized in that, The exciton solution contains at least an exciton with a cyclopentenone structure, and the concentration of the exciton is 50-200 μM; the soaking time of the exciton solution is at least 6 h.

3. The peony ginseng peptide for enhancing immunity and replenishing qi and blood according to claim 1, characterized in that, The specific steps of the solid-state aerobic fermentation of Rhizopus oligosporus are as follows: S1, Rhizopus oligosporus was activated and cultured to prepare a spore suspension with a concentration of 10. 6 -10 7 spores / mL; S2, the spore suspension is sprayed onto the surface of peony seeds for inoculation, and the surface of the peony seeds is coated with a carbon source; S3, control the initial pH of fermentation to ≤7, the temperature to 29±1℃, maintain oxygen supply, and ferment for at least 72 hours under an ambient humidity of ≥80%; after fermentation, sterilize and inactivate enzymes at high temperature to obtain fermented peony seeds.

4. The peony ginseng peptide for enhancing immunity and replenishing qi and blood according to claim 3, characterized in that, In step S2 The inoculation amount of the spore suspension is at least 5 wt% of the dry weight of the peony seeds; the amount of carbon source added is at least 2 wt% of the dry weight of the peony seeds.

5. The peony ginseng peptide for enhancing immunity and replenishing qi and blood according to claim 1, characterized in that, The alkaline dissolution and acid precipitation process involves stirring the crushed fermented peony seed meal with an alkaline solution with a pH of 8.0-9.0 under heating conditions to form a hot alkaline mixture. After centrifuging the hot alkali mixture, take the supernatant and add acidic solution to adjust the pH to ≤4.5; Then centrifuge again and collect the precipitate; After precipitation and washing, the protein is redispersed in water and the pH is adjusted to neutral or alkaline to obtain a crude protein solution.

6. The peony ginseng peptide for enhancing immunity and replenishing qi and blood according to claim 1, characterized in that, The enzymatic hydrolysis involves first hydrolyzing the crude protein solution obtained after alkali dissolution and acid precipitation of fermented peony seeds with alkaline protease, and then performing a second enzymatic hydrolysis using flavor protease to obtain the hydrolysate. The first enzymatic hydrolysis should be performed at a temperature of 40-55℃ and a pH of 8.0-9.0, for at least 3 hours, and the amount added should be at least 2% of the weight of the fermented peony seed meal. The second enzymatic hydrolysis is performed at a temperature of 40-55℃ and a pH of 6.0-7.5 for at least 3 hours, with an addition amount of at least 2% of the fermented peony seed meal mass.

7. The peony ginseng peptide for enhancing immunity and replenishing qi and blood according to claim 1, characterized in that, The enzymatic hydrolysate separation and purification process involves inactivating the enzymatic hydrolysate, cooling it, centrifuging it, and collecting the supernatant. The supernatant is then fractionated using an ultrafiltration membrane to separate the fractions with a molecular weight of 2 kDa or less. After concentration, the fractions are freeze-dried to obtain the peony peptide extract.

8. The peony ginseng peptide for enhancing immunity and replenishing qi and blood according to claim 1, characterized in that, Before soaking in the stimulating solution, the fresh peony seeds are first washed, then soaked in an ethanol aqueous solution, then soaked in a disinfectant, and finally rinsed with water for surface sterilization.

9. The application of the peony ginseng peptides described in claims 1-8, which enhance immunity and replenish qi and blood, in the preparation of products that enhance immunity.

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