Ginseng peptide with anti-aging activity as well as preparation method and application thereof

Abstract

The invention particularly relates to a ginseng peptide with anti-aging activity and a preparation method and application thereof, and belongs to the technical field of bioactive peptides. The method comprises the following steps: by taking ginseng fibrous roots, ginseng leaves and other by-products as raw materials, carrying out weak acid ultrasonic pretreatment, loading natural anti-aging active components, carrying out low-temperature high-pressure homogenization and airflow crushing synergistic treatment, carrying out double-stage composite enzymolysis, clarifying with an alternating electric field ceramic membrane, carrying out five-stage series ultrafiltration classification, carrying out four-stage filter element sterilization, freeze-drying and the like in sequence. The high-purity ginseng peptide is prepared. The total nitrogen content of the prepared ginseng peptide is larger than or equal to 14.8%, the proportion of a target peptide fragment is larger than or equal to 97.2%, the yield is larger than or equal to 18.5%, and the ginseng peptide has good antioxidant and anti-aging activity and can be used for development of functional food and skin care products. The method is low in cost, high in resource utilization rate and green and efficient in process, and the product purity and activity are remarkably superior to those of a traditional method.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioactive peptides, and specifically relates to a ginseng peptide with anti-aging activity and a preparation method and application thereof. BACKGROUND

[0002] With the intensification of population aging trend and the improvement of health awareness, the global anti-aging market demand continues to grow. Skin aging is a complex physiological process, which is closely related to oxidative stress, mitochondrial function decline, collagen loss and other factors. Therefore, the development of natural active ingredients that can effectively scavenge free radicals, protect cell function and promote skin health has become a research hotspot in the field of functional foods and cosmetics.

[0003] Ginseng is a traditional and valuable Chinese medicinal material, and its main active ingredient ginsenoside has been widely recognized for its antioxidant and anti-aging effects. However, ginseng peptides are small molecular peptide segments derived from the enzymatic hydrolysis of ginseng proteins. Compared with ginsenosides, ginseng peptides have the advantages of small molecular weight, easier absorption by the human body, high bioavailability, and low antigenicity. Studies have shown that ginseng peptides with a specific molecular weight range (such as 300-1800 daltons) have good skin permeability, can stimulate collagen production and inhibit matrix metalloproteinase activity, thereby delaying skin aging.

[0004] Currently, the raw materials for preparing ginseng peptides are mainly concentrated in the main roots of ginseng, which is costly and limits its industrial development. In fact, a large amount of rootlets, ginseng leaves, stems and other by-products are produced during the processing of ginseng, which contain rich protein resources but are often discarded or used at low value, resulting in resource waste. How to efficiently utilize these ginseng by-products to prepare high-activity ginseng peptides is the key to realizing resource value-added and cost reduction.

[0005] In terms of preparation process, the traditional production method of ginseng peptides has many limitations. First, high-temperature water extraction or strong acid and alkali treatment is often used in the pretreatment stage, which can easily lead to the hydrolysis and inactivation of heat-sensitive active ingredients (such as ginsenosides) and the denaturation of peptide chains. Second, single protease is often used in the enzymatic hydrolysis process, which has low enzymatic efficiency, wide molecular weight distribution of products, and low proportion of target small molecular peptides, affecting the activity and uniformity of the final product. Third, in the subsequent separation and purification stage, conventional separation techniques such as alcohol precipitation and centrifugation are difficult to accurately separate peptides with specific molecular weights, and membrane separation process is prone to blockage due to impurities, resulting in low efficiency. Finally, spray drying is commonly used in the drying stage, which may cause damage to the stereostructure of the peptides due to the instantaneous high temperature, affecting the activity.

[0006] In addition, the existing anti-aging product ingredients are relatively single, and simple physical mixing between different active ingredients cannot achieve synergistic effect. Organic combination of natural active ingredients with different anti-aging mechanisms (such as resveratrol targeting mitochondria, procyanidin targeting free radicals, etc.) with ginseng peptides in the early stage of preparation is expected to improve the comprehensive anti-aging effect of the product from multiple pathways and multiple targets, but there is no related technical report.

[0007] Therefore, there is an urgent need in the art for an innovative method capable of efficiently and low-cost utilizing ginseng by-products to prepare ginseng peptides with high purity, high activity and synergistic anti-aging effect by green and mild processing technology. SUMMARY

[0008] In view of this, the present application provides a ginseng peptide with anti-aging activity and a preparation method and application thereof.

[0009] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0010] In a first aspect, the present application discloses a preparation method of a ginseng peptide with anti-aging activity, comprising the following steps:

[0011] (1) ultrasonic pretreatment: the raw material is placed in weakly acidic deionized water, and ultrasonic pretreatment is carried out at 28-58℃, 280-780W and 28-48kHz for 18-48min;

[0012] Among them, the weakly acidic deionized water is adjusted to pH 4.2-5.0 by malic acid, and the weakly acidic environment can avoid the hydrolysis of ginsenosides and the denaturation of peptide segments, and the ultrasonic cavitation effect can efficiently destroy the cell wall structure of ginseng, thereby improving the dissolution efficiency of active ingredients in the subsequent soaking process;

[0013] (2) natural anti-aging active ingredient loading: after draining, 0.6-1.3% of the natural anti-aging active ingredient is added to the raw material in a warm water at a solid-liquid ratio of 1:20-30 at 58-78℃ for 8-14h;

[0014] (3) low-temperature high-pressure homogenizer and jet mill treatment: the soaked material is put into a low-temperature high-pressure homogenizer, and homogenized at 1150-1550bar, 38-45℃ and a flow rate of 12-24L / h, and then subjected to pulverization treatment by a jet mill to form a uniform material liquid;

[0015] Among them, low-temperature homogenization can avoid thermal denaturation of ginseng peptides, and jet milling can further destroy the intercellular matrix by high-speed airflow impact, increase the enzyme contact area, and improve the subsequent enzymolysis efficiency;

[0016] (4) enzymolysis: after the uniform material liquid is sealed, it is cooked at 98-118℃ for 2.8-4.8h, cooled to 44±2℃, adjusted to pH 6.6-6.8, and then 1.5-4.5% of the raw material mass of composite enzyme I is added for enzymolysis, and then 0.7-1.8% of the raw material mass of composite enzyme II is added for enzymolysis, to obtain an enzymolysis liquid;

[0017] Here, long-time cooking at low temperature can make the large molecular proteins in ginseng denature preliminarily, so as to facilitate the exposure of the enzyme cutting site, and at the same time, avoid the carbonization or structural damage of active ingredients caused by high temperature; the composite enzyme I enzymolysis can preliminarily cut the peptide bond of large molecular proteins to generate medium molecular peptide segments; the composite enzyme III enzymolysis can further degrade the medium molecular peptide segments into small molecular peptides, and the α-galactosidase can specifically degrade the saponin glycosidic bond impurities in ginseng to improve the purity and antioxidant activity of ginseng peptides; the composite enzyme I enzymolysis time is 0.8-1.8h, and the composite enzyme III enzymolysis time is 2.5-3.5h;

[0018] (5) After centrifugation of the enzymolysis liquid, the supernatant is absorbed, the supernatant is clarified by ceramic membrane, concentrated by membrane, and then fractionated by an ultrafiltration membrane with a molecular weight cutoff of 300-1800Da; sterilized by a four-stage filter cartridge, freeze-dried, and then aseptically packaged.

[0019] Here, the centrifugal speed is 2500-5500rpm for 15-35min, and high-speed centrifugation can effectively separate the unenzymolyzed coarse fibers from the supernatant, reduce the impurity blockage in the subsequent membrane filtration process, and improve the membrane flux.

[0020] Specifically, the natural anti-aging active ingredients in step (1) are puerarin, resveratrol, procyanidine, and vitamin E in a mass ratio of 3:2:1:0.9. The preliminary loading of the anti-aging ingredients is realized synchronously in the warm water soaking process, and the vitamin E in the composite solution can protect the active groups of ginseng peptides from oxidation, and puerarin and resveratrol can synergistically enhance the mitochondrial protection ability of ginseng peptides. Procyanidine can scavenge free radicals on the skin surface and synergistically play an antioxidant role with ginseng peptides.

[0021] Specifically, the specific steps of the jet mill treatment in step (2) include: treating for 2.5-6.5min at 14-24kw, 3200-5200rpm, and a feeding rate of 6-12kg / h, so that the particle size of the material is reduced to 4-8μm to form a uniform material liquid.

[0022] Specifically, the composite enzyme I in step (3) is a ginseng special protease, a neutral protease, and a ficin in a mass ratio of (1.5-3.5):(0.5-1.5):(0.6-1.8), and the enzyme activity of each is ≥1.5×10 5 U / g.

[0023] Specifically, the complex enzyme I I in step (3) is serine protease, pepsin and alpha-galactosidase with a mass ratio of 1: (1.5-2.5): (0.3-0.5), and the enzyme activity is all greater than or equal to 2.5 x 10 5 U / g.

[0024] The step-by-step enzymolysis of the complex enzyme I and the complex enzyme II can avoid uneven distribution of the peptide segment caused by single enzymolysis, and the enzymolysis temperature is controlled at 46±2℃, which is highly matched with the optimum temperature (43-49℃) of the two kinds of complex enzymes, thereby significantly improving the enzymolysis efficiency and the generation rate of the target peptide segment.

[0025] Specifically, an alternating electric field is applied in the ceramic membrane clarification process in step (4), the electric field strength is 7-12V / cm, and the pulse frequency is 28-58Hz. The alternating electric field can form a charge barrier on the membrane surface to inhibit the adsorption of impurities such as proteins and polysaccharides, thereby improving the membrane flux and the clarification efficiency.

[0026] Specifically, the pore size of the ceramic membrane in step (4) is 0.15-0.25μm, the membrane filtration pressure is 0.18-0.48MPa, and the temperature is controlled at 40-60℃.

[0027] In some embodiments, the ultrafiltration fractionation adopts five-stage series ultrafiltration membranes (polyvinylidene fluoride hollow fiber ultrafiltration membranes with molecular weight cutoffs of 1800Da, 1500Da, 1200Da, 800Da and 300Da), the feed liquid flow rate is 0.7-1.2m / s, and the target peptide segments of 300-1800Da (the ginseng peptides in this molecular weight range are easily absorbed by the human body, and have the optimal antioxidant activity and skin permeability) are accurately collected. Among them, the five-stage series ultrafiltration membranes adopt polyvinylidene fluoride material, which is resistant to acid and alkali and has strong oxidation resistance, so that chemical reaction between the membrane material and the ginseng peptides can be avoided, and the gradient cutoff design can accurately separate peptide segments with different molecular weights, effectively remove free amino acids with a molecular weight of <300Da and macromolecular impurities with a molecular weight of >1800Da, and improve the proportion of the target peptide segments.

[0028] In some embodiments, the four-stage filter element is sterilized (passes through polyether sulfone filter elements with pore sizes of 0.5μm, 0.25μm, 0.12μm and 0.08μm in sequence), freeze-dried (vacuum degree 0.09-0.13mbar, temperature -42 to -52℃), and then packaged aseptically; freeze-drying can maximize the retention of active ingredients of ginseng peptides, avoid the damage to the structure of the peptide segments caused by high temperature in spray drying, and improve the product stability and shelf life.

[0029] In some embodiments, the raw material adopts by-products such as ginseng rootlets and ginseng leaves.

[0030] In the second aspect, the present application discloses a ginseng peptide with anti-aging activity prepared by the preparation method.

[0031] Among them, the total nitrogen content of ginseng peptides is ≥14.8%, the proportion of target peptides is ≥97.2%, and the yield of ginseng peptides is ≥18.5%.

[0032] Thirdly, this invention discloses the application of the aforementioned ginseng peptides in the preparation of anti-aging functional foods and skin care products. The functional foods include oral liquids and compressed candies, and the skin care products include serums, face creams, and face masks.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] This invention uses ginseng rootlets and leaves as byproducts, which, compared to the traditional method using ginseng rootlets, not only reduces raw material costs by 30%–40% but also achieves full utilization of ginseng resources, effectively avoiding environmental problems caused by byproduct waste. In the pretreatment stage, an innovative synergistic process system of "weakly acidic ultrasound-low temperature high-pressure homogenization-airflow pulverization" is constructed: the weakly acidic environment protects ginsenosides from hydrolysis, low-temperature high-pressure homogenization helps avoid thermal denaturation of active peptides, and airflow pulverization significantly improves subsequent enzymatic hydrolysis efficiency. Compared to traditional single ultrasound or homogenization pretreatment methods, this synergistic process increases the yield of target peptides by 18%–23%. In the purification stage, a combination of "alternating electric field ceramic membrane clarification + five-stage tandem ultrafiltration" technology is used. The introduction of the alternating electric field effectively reduces membrane fouling, and the five-stage ultrafiltration achieves precise retention of target peptides. Compared to the traditional "single ceramic membrane + three-stage ultrafiltration" process, this method increases the proportion of target peptides in the product by 6%–9%, ​​significantly improving product purity. Attached Figure Description

[0035] Figure 1 Example 3 shows the scavenging rate of ginseng peptides against DPPH free radicals.

[0036] Figure 2 Example 3 shows the scavenging rate of ginseng peptides against superoxide anion free radicals;

[0037] Figure 3 Example 3 shows the scavenging rate of ginseng peptides against hydroxyl radicals. Detailed Implementation

[0038] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0039] Example 1: A method for preparing ginseng peptides with anti-aging activity

[0040] (1) Ultrasonic pretreatment: Ginseng rootlets or leaves and other by-products were placed in weakly acidic deionized water (pH 4.2, adjusted with malic acid) and ultrasonically pretreated for 18 min at 28℃, 280W and 28kHz.

[0041] (2) Anti-aging ingredient loading: After draining, add 0.6% of the natural anti-aging active ingredients by weight of the raw material to 58℃ warm water at a material-to-liquid ratio of 1:20 and soak for 8 hours (mass ratio of puerarin, resveratrol, proanthocyanidins and vitamin E 3:2:1:0.9);

[0042] (3) Low-temperature high-pressure homogenizer and air jet mill treatment: The soaked material is put into a low-temperature high-pressure homogenizer and homogenized at 1150 bar, 38℃ and 12L / h flow rate; then it is pulverized by an air jet mill (power 14kw, pulverizing speed 3200rpm, feeding rate 6kg / h, processing time 2.5min) to control the particle size of the material to 4-6μm and form a uniform material liquid;

[0043] (4) Enzymatic hydrolysis: After sealing the homogenized liquid material, it was cooked at 98℃ for 2.8h, cooled to 44℃, and the pH was adjusted to 6.8 (using potassium bicarbonate). Then, 1.5% of the substrate mass of compound enzyme I was added for 0.8h of enzymatic hydrolysis, followed by 0.7% of the substrate mass of compound enzyme II for 2.5h of enzymatic hydrolysis. Among them, compound enzyme I consisted of ginseng-specific protease, neutral protease, and fig protease in a mass ratio of 1.5:0.5:0.6, all with enzyme activities greater than 1.5×10⁻⁶. 5 U / g; Complex enzyme II consists of serine protease, pepsin, and α-galactosidase in a mass ratio of 1:1.5:0.3, with enzyme activities all greater than 2.5 × 10⁻⁶ U / g; 5 U / g; after enzymatic hydrolysis, centrifuge at 2500 rpm for 15 min and collect the supernatant;

[0044] (5) The supernatant was clarified and concentrated by a ceramic membrane with a pore size of 0.15 μm and a membrane filtration pressure of 0.18 MPa (an alternating electric field was applied at 40°C with an electric field strength of 7 V / cm and a pulse frequency of 28 Hz). Then, it was graded by ultrafiltration membranes with molecular weight cutoffs of 1800 Da, 1500 Da, 1200 Da, 800 Da and 300 Da. The feed flow rate during ultrafiltration was 0.7 m / s. The ultrafiltration membrane was a polyvinylidene fluoride hollow fiber ultrafiltration membrane. After ultrafiltration, it was sterilized by polyethersulfone filter cartridges with pore sizes of 0.5 μm, 0.25 μm, 0.12 μm and 0.08 μm. It was then freeze-dried at a vacuum of 0.09 mbar and a temperature of -42°C and aseptically packaged.

[0045] Example 2: A method for preparing ginseng peptides with anti-aging activity

[0046] (1) Ultrasonic pretreatment: Ginseng rootlets or leaves and other by-products were placed in weakly acidic deionized water (pH 5.0, adjusted with malic acid) and ultrasonically pretreated for 48 min at 58℃, 780W and 48kHz.

[0047] (2) Anti-aging ingredient loading: After draining, add 1.3% of the natural anti-aging active ingredients by weight of the raw material to warm water at 78℃ at a material-to-liquid ratio of 1:30 and soak for 14 hours (mass ratio of puerarin, resveratrol, proanthocyanidins and vitamin E 3:2:1:0.9);

[0048] (3) Low-temperature high-pressure homogenizer and air jet mill treatment: The soaked material is put into a low-temperature high-pressure homogenizer and homogenized at 1550 bar, 45℃ and 24 L / h flow rate; then it is pulverized by an air jet mill (power 24kw, pulverizing speed 5200rpm, feeding rate 12kg / h, processing time 6.5min) to control the particle size of the material to 6-8μm and form a uniform material liquid;

[0049] (4) Enzymatic hydrolysis: After sealing the homogenized liquid material, it was cooked at 118℃ for 4.8h, cooled to 48℃, and the pH was adjusted to 8.8 (using potassium bicarbonate). Then, 4.5% of the substrate mass of compound enzyme I was added for 1.8h of enzymatic hydrolysis, followed by 1.8% of the substrate mass of compound enzyme II for 3.5h of enzymatic hydrolysis. Compound enzyme I consisted of ginseng-specific protease, neutral protease, and fig protease in a mass ratio of 3.5:1.5:1.8, all with enzyme activities greater than 1.5×10⁻⁶. 5 U / g; Complex enzyme II consists of serine protease, pepsin, and α-galactosidase in a mass ratio of 1:2.5:0.5, with enzyme activities all greater than 2.5 × 10⁻⁶ U / g; 5 U / g; after enzymatic hydrolysis, centrifuge at 2500 rpm for 15 min and collect the supernatant;

[0050] (5) The supernatant was clarified and concentrated by a ceramic membrane with a pore size of 0.25 μm and a membrane filtration pressure of 0.48 MPa (clarification was carried out at 60°C with an alternating electric field strength of 12 V / cm and a pulse frequency of 58 Hz), and then graded by ultrafiltration membranes with molecular weight cutoffs of 1800 Da, 1500 Da, 1200 Da, 800 Da and 300 Da. The feed flow rate during ultrafiltration was 1.2 m / s, and the ultrafiltration membrane was a polyvinylidene fluoride hollow fiber ultrafiltration membrane. After ultrafiltration, the supernatant was sterilized by polyethersulfone filter cartridges with diameters of 0.5 μm, 0.25 μm, 0.12 μm and 0.08 μm, and then freeze-dried at a vacuum of 0.13 mbar and a temperature of -52°C, and aseptically packaged.

[0051] Example 3: A method for preparing ginseng peptides with anti-aging activity

[0052] (1) Ultrasonic pretreatment: Ginseng rootlets or leaves and other by-products were placed in weakly acidic deionized water (pH 4.6, adjusted with malic acid) and ultrasonically pretreated for 33 min at 43℃, 530W and 38kHz.

[0053] (2) Anti-aging ingredient loading: After draining, add 0.95% of the natural anti-aging active ingredients by weight of the raw material to 68℃ warm water at a material-to-liquid ratio of 1:25 and soak for 11 hours (mass ratio of puerarin, resveratrol, proanthocyanidins and vitamin E 3:2:1:0.9).

[0054] (3) Low-temperature high-pressure homogenizer and air jet mill treatment: The soaked material is put into a low-temperature high-pressure homogenizer and homogenized at 1350 bar, 41℃ and 18L / h flow rate; then it is pulverized by an air jet mill (power of 19kw, pulverizing speed of 4200rpm, feeding rate of 9kg / h and processing time of 4.5min) to control the particle size of the material to 5-7μm and form a uniform material liquid;

[0055] (4) Enzymatic hydrolysis: After sealing the homogenized liquid, it was cooked at 108℃ for 3.8h, cooled to 46℃, and the pH was adjusted to 7.8 (using potassium bicarbonate). Then, 3.0% of the substrate mass of compound enzyme I was added for 1.3h of enzymatic hydrolysis, followed by 1.25% of the substrate mass of compound enzyme II for 3.0h of enzymatic hydrolysis. Compound enzyme I consisted of ginseng-specific protease, neutral protease, and fig protease in a mass ratio of 2.5:1.0:1.2, all with enzyme activities greater than 1.5×10⁻⁶. 5 U / g; Complex enzyme II consists of serine protease, pepsin, and α-galactosidase in a mass ratio of 1:2.0:0.4, with enzyme activities all greater than 2.5 × 10⁻⁶ U / g; 5 U / g; after enzymatic hydrolysis, centrifuge at 2500 rpm for 15 min and collect the supernatant;

[0056] (5) The supernatant was clarified and concentrated by a ceramic membrane with a pore size of 0.20 μm and a membrane filtration pressure of 0.33 MPa (an alternating electric field was applied at 50°C with an electric field strength of 9.5 V / cm and a pulse frequency of 43 Hz). Then, it was graded by ultrafiltration membranes with molecular weight cutoffs of 1800 Da, 1500 Da, 1200 Da, 800 Da and 300 Da. The feed flow rate during ultrafiltration was 0.95 m / s. The ultrafiltration membrane was a polyvinylidene fluoride hollow fiber ultrafiltration membrane. After ultrafiltration, it was sterilized by polyethersulfone filter cartridges with diameters of 0.5 μm, 0.25 μm, 0.12 μm and 0.08 μm. It was then freeze-dried at a vacuum of 0.11 mbar and a temperature of -47°C and aseptically packaged.

[0057] Comparative Example 1

[0058] Compared with Example 3, the difference is that the amount of compound enzyme I is 5.5% of the substrate mass, and the remaining steps and parameters are the same as in Example 3.

[0059] Comparative Example 2

[0060] Compared with Example 3, the difference is that only the enzymatic hydrolysis of compound enzyme I is retained, and compound enzyme II is omitted. The remaining steps and parameters are the same as in Example 3.

[0061] Comparative Example 3

[0062] Compared with Example 3, the difference is that the amount of compound enzyme ⅠⅠ is 2.5% of the substrate mass, and the remaining steps and parameters are the same as in Example 3.

[0063] Comparative Example 4

[0064] Compared with Example 3, the difference is that the enzymatic hydrolysis time of compound enzyme ⅠⅠ is extended to 5 hours, while the remaining steps and parameters are the same as in Example 3.

[0065] Comparative Example 5

[0066] Compared with Example 3, the difference is that the ultrasonic pretreatment time is extended to 1.5 hours, while the remaining steps and parameters are the same as in Example 3.

[0067] Comparative Example 6

[0068] Compared with Example 3, the difference is that the ultrasonic pretreatment time is extended to 1.5 hours and the enzymatic hydrolysis time of compound enzyme ⅠⅠ is extended to 5 hours. The remaining steps and parameters are the same as in Example 3.

[0069] Comparative Example 7

[0070] Compared with Example 3, the difference is that the airflow pulverization process is omitted, and only the low temperature and high pressure homogenization process is retained. The remaining steps and parameters are the same as those in Example 3.

[0071] Comparative Example 8

[0072] Compared with Example 3, the difference is that no alternating electric field was applied during the ceramic membrane clarification process, while the remaining steps and parameters were the same as in Example 3.

[0073] Comparative Example 9

[0074] Compared with Example 3, the difference is that the addition of natural anti-aging active ingredients is omitted, while the remaining steps and parameters are the same as in Example 3.

[0075] Comparative Example 10

[0076] Compared with Example 3, the difference is that this comparative example only uses ultrasonic pretreatment, neutral protease hydrolysis, ordinary ceramic membrane clarification, and three-stage ultrafiltration; the remaining steps and parameters are the same as in Example 3. The specific steps are as follows:

[0077] (1) Ultrasonic pretreatment: Ginseng rootlets or leaves and other by-products were placed in weakly acidic deionized water (pH 4.6, adjusted with malic acid) and ultrasonically pretreated for 33 min at 43℃, 530W and 38kHz.

[0078] (2) After draining, add 3.0% (by weight of raw material) of neutral protease to warm water at 45℃ at a material-to-liquid ratio of 1:25 and hydrolyze for 3.0 h. The enzyme activity is greater than 1.5 × 10⁻⁶. 5 U / g; after enzymatic hydrolysis, centrifuge at 2500 rpm for 15 min and collect the supernatant;

[0079] (3) After the supernatant is clarified and concentrated by a ceramic membrane with a pore size of 0.20 μm and a membrane filtration pressure of 0.33 MPa, it is further classified by ultrafiltration membranes with molecular weight cutoffs of 1800 Da, 800 Da and 300 Da. The feed flow rate during ultrafiltration is 0.95 m / s, and the ultrafiltration membrane is a polyvinylidene fluoride hollow fiber ultrafiltration membrane. After ultrafiltration, it is freeze-dried at a vacuum of 0.11 mbar and a temperature of -47 °C and then aseptically packaged.

[0080] Comparative Example 11

[0081] Compared with Example 3, the difference is that the pH of the weakly acidic deionized water in step (1) is 7.8, and the rest of the steps and parameters are the same as in Example 3.

[0082] Comparative Example 12

[0083] Compared with Example 3, the difference is that the pH of the weakly acidic deionized water in step (1) is 3.8, and the rest of the steps and parameters are the same as in Example 3.

[0084] Comparative Example 13

[0085] Compared with Example 3, the difference is that the ultrafiltration grading in step (5) adopts the traditional three-stage ultrafiltration (molecular weight cutoff of 1800 Da, 800 Da, and 300 Da), while the other steps and parameters are the same as in Example 3.

[0086] Example 1: Detection of Key Indicators of Ginseng Peptides

[0087] The ginseng peptides prepared in Examples 1-3 and Comparative Examples 1-9 were tested for the following indicators.

[0088] Total nitrogen content: The Kjeldahl method was used to determine the total nitrogen content of the sample by heating and digesting the sample with concentrated sulfuric acid and a catalyst to convert organic nitrogen into ammonium salt. After distillation, absorption and titration, the total nitrogen content of the sample was calculated.

[0089] Peptide proportion: High performance liquid chromatography (HPLC) was used to separate and determine peptides with molecular weight exclusion chromatography based on the retention time of peptides with different molecular weights, namely 500-2000 Da (total target peptides), 1000-2000 Da, and 500-1000 Da, according to the principle of molecular weight exclusion chromatography. The proportion of each peptide range in the total peptides was calculated.

[0090] Ginseng peptide yield: Accurately weigh the raw materials of ginseng fibrous roots, ginseng leaves and other by-products (m1, unit: g), collect the ginseng peptide powder after final freeze-drying and weigh it (m2, unit: g), and calculate the yield according to the following formula: Yield (%) = (m2 / m1) × 100%.

[0091] Saponin retention rate = (A / B) × 100%, where B is the saponin content of the raw material and A is the saponin content after treatment.

[0092] Table 1. Results of key indicators of ginseng peptides

[0093]

[0094] Table 2. Results of key indicators of ginseng peptides

[0095]

[0096] As shown in Table 1, the total nitrogen content of Examples 1-3 was ≥14.4%, with Example 3 reaching 14.9%, significantly higher than the comparative example (maximum 14.2%). This indicates that the synergistic process of stepwise enzymatic hydrolysis with compound enzyme, clarification with pulsed electric field ceramic membrane, and air jet milling effectively improved the protein purity of ginseng peptides. The proportion of target peptides: The proportion of total target peptides (<2000 Da) in Examples 1-3 was ≥96.6%, with Example 3 reaching 97.1%. The proportion of the comparative example decreased due to the absence of key processes (such as compound enzyme II and air jet milling), proving that the combination of four-stage ultrafiltration and air jet milling can accurately enrich highly active peptides. The yield: Example 3 had the highest yield (18.4%), while the yields of Comparative Example 2 (without compound enzyme II) and Comparative Example 6 (dual parameter deviation) were lower. This indicates that the matching of stepwise enzymatic hydrolysis parameters and pretreatment processes is crucial to the yield; excessive enzyme or excessively long treatment time can lead to the generation of ineffective byproducts, reducing the yield.

[0097] As shown in Table 2, all comparative examples showed significantly lower performance than Example 3 in key indicators, fully demonstrating the necessity and synergistic advantages of each step in the process of this invention. Comparative Example 10 was significantly inferior to Example 3 in terms of total nitrogen content, target peptide ratio, yield, and saponin retention rate, with the target peptide ratio being 14.6 percentage points lower and the yield 6.8 percentage points lower. This result clearly indicates that the complete process system of "synergistic pretreatment-stepwise enzymatic hydrolysis-electric field membrane separation" adopted in this invention can systematically improve product purity and production efficiency, effectively solving the problems of low efficiency and insufficient product purity in traditional processes. The total nitrogen content, target peptide ratio, and saponin retention rate of Comparative Example 11 were all lower than those of Example 3, especially the saponin retention rate, which was only 68.5%, far lower than the level of the example. This indicates that saponins are prone to hydrolysis under alkaline conditions, further verifying the rationality of controlling the reaction system under non-alkaline conditions in this invention. Comparative Example 12 showed that excessive acidity in the system inhibited enzyme activity, resulting in a product yield decrease to 12.5%. This demonstrates, from the opposite perspective, the crucial role of the optimal weakly acidic environment (pH 4.2–5.0) selected in this invention—effectively protecting active ingredients such as saponins while ensuring efficient enzymatic hydrolysis. The target peptide proportion in Comparative Example 13 was 5.3 percentage points lower than in Example 3, indicating that the five-stage tandem ultrafiltration, with its finer molecular weight cutoff gradient, can achieve precise enrichment of target peptides from 300-1800 Da. The separation effect is significantly better than the traditional three-stage ultrafiltration process, highlighting the technological advancement of this invention in the separation and purification stage.

[0098] Example 2: Determination of the free radical scavenging ability of ginseng peptides

[0099] DPPH free radical scavenging rate:

[0100] Add 1.5 mg of ginseng peptide sample to 1.5 mL of 0.1 mmol / L DPPH (95% ethanol), incubate at 25 °C for 30 min, and measure the absorbance at 517 nm. Using VC solution as a control, calculate the clearance rate according to the following formula:

[0101] Clearance rate (%) = [1 - (A1 - A2) / A0] × 100%;

[0102] Wherein, A0 is the absorbance of 1.5 mL distilled water + 1.5 mL DPPH solution, A1 is the absorbance of 1.5 mL ginseng peptide solution + 1.5 mL DPPH solution, and A2 is the absorbance of 1.5 mL ginseng peptide solution + 1.5 mL 95% ethanol.

[0103] Superoxide anion radical scavenging rate:

[0104] Following the instructions of the superoxide anion radical scavenging kit, ginseng peptide samples and reagents of different concentrations were added. After incubating in a 37°C water bath for 40 min, a colorimetric reagent was added, and the absorbance was measured at 550 nm after 10 min. Calculation formula:

[0105] Clearance rate (%) = [(A2-A1) / A2] × 100%;

[0106] Where A1 is the absorbance of the test tube and A2 is the absorbance of the control tube.

[0107] Hydroxyl radical scavenging rate:

[0108] Using the Fenton reaction system, 50 μL of ginseng peptide samples at different concentrations (5, 10, and 20 mg / mL) were taken, reagents were added according to the kit instructions, and the reaction was carried out at 37℃ for 20 min. After centrifugation at 8000 r / min for 5 min, the absorbance of the clear liquid was measured at 510 nm. Calculation formula:

[0109] Clearance rate (%) = [1 - (A1 - A2) / A0] × 100%;

[0110] Where A0 is the absorbance of the blank tube, A1 is the absorbance of the test tube, and A2 is the absorbance of the control tube.

[0111] The results are as follows Figures 1-3 As the concentration increased, the scavenging rate of ginseng peptides against DPPH free radicals gradually increased, demonstrating a good linear dependence on this free radical. Ginseng peptides also showed good scavenging effects against superoxide anion free radicals, achieving nearly 100% scavenging at a total concentration of 20 mg / mL. Furthermore, ginseng peptides showed good scavenging effects against hydroxyl free radicals, with the highest scavenging rate at a total concentration of 30 mg / mL.

[0112] Example 3: Detection of the anti-glycation efficacy of ginseng peptides

[0113] Using Comparative Example 9 as a control, the anti-glycation activity was verified using a fructose-bovine serum albumin (BSA) model:

[0114] System preparation: Mix 2 mL of 300 mg / mL fructose solution + 2 mL of ginseng peptide solution (Examples 1-3 / Comparative Example 9) + 2 mL of 30 mg / mL BSA solution (dissolved in 50 mmol / L pH 7.4 phosphate buffer), with 0.1 and 1.5 mg / mL aminoguanidine (AG) as positive controls and phosphate buffer as a blank control, and incubate in a 50°C water bath for 24 h;

[0115] Fructose amine content determination: Take 0.2 mL of reaction solution + 0.8 mL of 0.3 mmol / L nitrotetrazole blue reagent, add 2 mL of 100 mmol / L pH 10.1 carbonate buffer, react at room temperature for 30 min, and then measure the OD value at 530 nm. Calculation formula:

[0116] Fructose amine inhibition rate (%) = [(F0-F1) / F0] × 100%;

[0117] Where F0 is the OD value without ginseng peptides, and F1 is the OD value with ginseng peptides.

[0118] Table 3. Results of fructosamine inhibition rate of ginseng peptides

[0119] Total concentration 2.5 mg / mL inhibition rate (%) Example 1 46 Example 2 48 Example 3 45 Comparative Example 9 32

[0120] The results are shown in Table 3. In Examples 1-3, the fructosamine inhibition rate was significantly higher than that in Comparative Example 9 due to the addition of natural anti-aging ingredients, indicating that the synergistic effect of natural anti-aging ingredients and ginseng peptides can significantly enhance antioxidant activity.

[0121] In summary, the ginseng peptides prepared by this invention not only have excellent physicochemical properties, but also possess significant anti-aging and anti-glycation activities, and can be widely used in the fields of functional foods and skin care products.

[0122] The above description is only a part of the embodiments of the present invention and does not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made using the present invention specification, or directly / indirectly applied to other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing a ginseng peptide having an anti-aging activity, characterized by, It comprises the following steps: (1) natural anti-aging active ingredient loading: the raw material is treated by ultrasonic wave, drained, and then soaked in warm water at 58-78℃ with the addition of 0.6-1.3% of the natural anti-aging active ingredient based on the mass of the raw material at a ratio of 1:20-30; (2) low-temperature high-pressure homogenizer and air flow pulverizer treatment: the soaked material is put into a low-temperature high-pressure homogenizer and treated at 1150-1550bar, 38-45℃ and a flow rate of 12-24L / h, and then treated by an air flow pulverizer to form a uniform material liquid; (3) enzyme hydrolysis: the uniform material liquid is sealed and cooked at 98-118℃ for 2.8-4.8h, cooled to 44±2℃, adjusted to pH 6.6-6.8, and then sequentially added with 1.5-4.5% of a composite enzyme I based on the mass of the raw material and 0.7-1.8% of a composite enzyme II based on the mass of the raw material for enzyme hydrolysis to obtain an enzyme hydrolysate; (4) centrifugation of the enzyme hydrolysate, absorption of the supernatant, clarification of the supernatant by ceramic membrane, membrane concentration, fractionation by an ultrafiltration membrane with a molecular weight cutoff of 300-1800Da, sterilization by a four-stage filter, freeze-drying, and aseptic packaging.

2. The production method according to claim 1, characterized by, The natural anti-aging active ingredient in step (1) is puerarin, resveratrol, procyanidin, and vitamin E at a mass ratio of 3:2:1:0.

9.

3. The preparation method according to claim 1, characterized in that, The specific steps of the air flow pulverizer treatment in step (2) include: treatment at 14-24kw, 3200-5200rpm, and a feeding rate of 6-12kg / h for 2.5-6.5min to reduce the particle size of the material to 4-8μm to form a uniform material liquid.

4. The method of claim 1, wherein, The complex enzyme I in step (3) is panax special protease, neutral protease and ficin with mass ratio of (1.5-3.5):(0.5-1.5):(0.6-1.8), and enzyme activity is all ≥1.5×10 5 U / g.

5. The preparation method according to claim 1, characterized in that, The complex enzyme I I in step (3) is serine protease, pepsin and α-galactosidase with mass ratio of 1: (1.5-2.5) : (0.3-0.5) and enzyme activity of all ≥2.5×10 5 U / g. The complex enzyme I I in step (3) is serine protease, pepsin and α-galactosidase with mass ratio of 1: (1.5-2.5) : (0.3-0.5) and enzyme activity of all ≥2.5×10 5 U / g.

6. The method of claim 1, wherein, In the ceramic membrane clarification process in step (4), an alternating electric field is applied with an electric field strength of 7-12V / cm and a pulse frequency of 28-58Hz.

7. The production method according to claim 6, wherein The pore size of the ceramic membrane in step (4) is 0.15-0.25μm, the membrane filtration pressure is 0.18-0.48MPa, and the temperature is 40-60℃.

8. The method of claim 1, wherein, The ultrasonic wave treatment in step (1) is a pretreatment of the raw material in weakly acidic deionized water at 28-58℃, 280-780W, and 28-48kHz for 18-48min.

9. The ginseng peptide prepared by the method of any one of claims 1-8, wherein the ginseng peptide is characterized by, The total nitrogen content is ≥14.8%, the target peptide segment accounts for ≥97.2%, and the ginseng peptide yield is ≥18.5%.

10. The use of the ginseng peptide of claim 9 in the preparation of anti-aging functional food and skin care products, characterized in that, The functional food includes oral liquids and tablet candies, and the skin care products include serums, creams, and masks.

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