Preparation method of sturgeon peptide and application thereof in improving immunity and sexual function

Abstract

The application discloses a preparation method of sturgeon peptide and application of the sturgeon peptide in improving immunity and sexual function. The method comprises the following steps: S1, one or more of sturgeon cartilage, fish bone and fish skin are crushed after being thawed, and pure water is added according to a material-liquid ratio of 1:3-1:4 and is pumped into an enzyme hydrolysis tank; S2, the enzyme hydrolysis tank is heated to 85-90 DEG C under the condition of a closed low pressure of 0.06-0.08 MPa, and protein is denatured for 15-20 min; S3, the temperature is reduced to 60-65 DEG C, the pH is adjusted to 8.0-8.5, 2‰-3‰ alkaline protease of the raw material amount is added, and constant-temperature enzyme hydrolysis is carried out for 170-190 min; the temperature is increased to 85-90 DEG C after the enzyme hydrolysis is completed, and the enzyme is inactivated for 14-16 min; S4, decolorization is carried out; S5, filtration and concentration are carried out; S6, vacuum concentration is carried out; S7, sterilization and drying are carried out, and sturgeon peptide finished products are obtained. The sturgeon peptide obtained by the preparation method has small molecular weight and high active ingredients.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide preparation technology, specifically to a method for preparing sturgeon peptide and its application in improving immunity and sexual function. Background Technology

[0002] In recent years, problems such as weakened immunity, sub-health, and decreased male sexual function have become increasingly common. Many commercially available products suffer from drawbacks such as limited functionality, poor absorption, and significant side effects. Sturgeon-derived active peptides are rich in arginine, chondroitin sulfate, and small molecule peptides, possessing the potential to enhance immunity, tonify the kidneys and replenish essence, and combat inflammation and protect the prostate. However, existing preparation methods lack specificity, their molecular weight is uncontrollable, and their activity is insufficiently preserved.

[0003] Therefore, researchers have conducted studies on the preparation technology of low molecular weight peptides, such as the invention application with publication number 107653290A, which discloses a method for preparing sturgeon protein peptides to prepare low molecular weight sturgeon protein peptides. However, its preparation route can be improved or replaced: 1. It uses high temperature and high pressure steam explosion treatment to destroy protein structure, which will excessively shear collagen peptides and break active oligopeptides into free amino acids, thus destroying and rendering the active oligopeptides ineffective; in addition, high temperature and high pressure cause irreversible denaturation of proteins, making them prone to flocculation, precipitation, and difficult filtration after enzymatic hydrolysis, resulting in low yield and poor solubility; and it requires high pressure equipment, which requires large equipment investment and high safety requirements. 2. Under high temperature and pressure, if oxygen is not removed, the Maillard reaction will be violent, which will easily cause browning, scorching, and a strong fishy smell, resulting in poor product appearance and flavor. Therefore, a deodorization treatment step needs to be set in the subsequent steps. 3. The raw material is whole sturgeon, which is high in fat, making it prone to strong fishy smell, oxidation, and rancidity. It also contains more common muscle protein and less collagen, resulting in a low concentration of active protein. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing sturgeon peptides, aiming to propose a new method for preparing small molecule peptides.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing sturgeon peptide, comprising the following steps: S1. Raw material pretreatment: The raw material is one or more of sturgeon cartilage, fish bone, and fish skin. After thawing, it is minced and added to purified water at a material-to-liquid ratio of 1:3 to 1:4. The mixture is then pumped into an enzymatic hydrolysis tank. S2. Protein denaturation: The enzymatic hydrolysis vessel is heated to 85-90°C under a closed, low-pressure condition of 0.06-0.08 MPa and maintained for 15-20 minutes to fully denature the protein. S3. Enzymatic hydrolysis: Cool to 60-65℃, adjust pH to 8.0-8.5, add 2‰-3‰ alkaline protease to the raw material, and hydrolyze at a constant temperature for 170-190 min; after the enzymatic hydrolysis is completed, raise the temperature to 85-90℃ and maintain it for 14-16 min to inactivate the enzyme. S4. Decolorization: Add an appropriate amount of activated carbon to the enzymatic hydrolysate, and filter to remove the activated carbon after decolorization. S5. Filtration and Concentration: Remove large protein molecules and impurities, desalt, dehydrate, and remove free amino acids to obtain a concentrated solution; S6. Vacuum concentration: Concentrate the concentrate at 75-85℃ to 25%-35% under vacuum; S7. Sterilization and drying: After sterilization at 121-125℃ for 5-15 seconds, the sturgeon peptide product is obtained by spray drying.

[0006] Furthermore, after S5 microfiltration, the proportion of peptides with a relative molecular mass <10000Da in the filtrate is ≥95%.

[0007] Further, in S3, the temperature is lowered to 60-62℃, the pH is adjusted to 8.0-8.2, and 1.2‰-1.8‰ of alkaline protease is added. The mixture is then hydrolyzed at a constant temperature for 80-90 minutes. In situ, the temperature of the solution is raised to 63-65℃, the pH is adjusted to 8.3-8.5, and 1.0‰-1.2‰ of alkaline protease is added. The mixture is then hydrolyzed at a constant temperature for 90-100 minutes.

[0008] Further, in S4, the enzymatic hydrolysate is transferred to a decolorizing tank, and 0.5% of the raw material amount of powdered activated carbon is added to the enzymatic hydrolysate. The decolorization reaction is carried out at 55-65°C for 30-40 minutes.

[0009] Furthermore, in S5, ceramic membrane microfiltration is first used, controlling the pressure difference between the membrane inlet and outlet at 0.1–0.3 MPa. The filter is circulated and washed with water 2–4 times to remove large molecular proteins and impurities, obtaining filtrate. Then, nanofiltration membrane concentration is used, controlling the membrane inlet pressure at 1.3–1.5 MPa, the membrane outlet pressure at 1.1–1.3 MPa, and the feed temperature at 55–60°C, to desalinate, dehydrate, and remove free amino acids, obtaining concentrated solution.

[0010] Furthermore, a pH fine-tuning step is included before nanofiltration membrane concentration: the pH of the concentrate is adjusted to 5.5–6.5 using citric acid.

[0011] Furthermore, the process before S5 also includes a centrifugation step: centrifugation at 8000-10000 r / min for 10-15 min to remove fat, bone residue, and colloidal impurities.

[0012] Furthermore, a closed aseptic temporary storage step is added between sterilization and spray drying, where the liquid is temporarily stored in an aseptic environment at 4℃~10℃ for 5min~30min.

[0013] Furthermore, the protein content is ≥78.5g / 100g, the peptide content is ≥75.2g / 100g, the arginine content is ≥5.80g / 100g, and the chondroitin sulfate content is ≥11.1%; the relative molecular mass is less than 10000Da and greater than 98%, and the proportion of oligopeptides less than 1000Da is greater than 95%; its active short peptides include VLPEDGRP, ASYLDCIK, DGLGDVAFVK, APNHAVVSRK, DDTKCLVDLR, NSEPWAKDLK, GSNFQLNELQGK, AVKWCALSHHER, YLGADYIKAVSNLR, CSTSRLLEACTFHK, TVRWCAVNDHEASK, and QETIFENLPSKDER.

[0014] Furthermore, the present invention also discloses the application of sturgeon peptides in the preparation of products that enhance immunity, improve male sexual function, or improve prostatitis, wherein the products are health foods, functional foods, or dietary supplements.

[0015] By adopting the above technical solution, the present invention has the following advantages compared with the prior art: 1. This invention utilizes collagen from sturgeon cartilage / fish bones / fish skin to reduce the impact of muscle protein and fat in the raw materials on the product's fishy smell. Collagen exists in a dense triple helix structure with high stability due to hydrogen bonds and hydrophobic bonds. The sealed, low-pressure environment of this invention lowers the boiling point of water, allowing water molecules to more easily penetrate into the collagen. At the same temperature, water molecules move more vigorously and have stronger permeability. The low pressure weakens the hydrogen bonds, van der Waals forces, and hydrophobic interactions between collagen molecular chains, allowing the triple helix structure to unfold more fully and evenly, exposing more enzyme cleavage sites. The unfolded peptide chains have a larger contact area with proteases, improving enzymatic hydrolysis efficiency. 2. Furthermore, the low-pressure environment significantly reduces oxygen content, effectively inhibiting side reactions such as Maillard reaction, polyphenol oxidation, and amino acid oxidation. It also reduces local overheating and browning / scorching caused by rapid oxidation under normal pressure and high temperature. Moreover, the amines, sulfur-containing compounds, and low-carbon chain aldehydes naturally present in sturgeon raw materials are all small-molecule volatile fishy-smelling substances. Under normal pressure and high temperature conditions, due to the high external pressure, these substances are difficult to escape from the material. High temperature also easily promotes fat oxidation and protein decomposition, generating more odorous components and intensifying the fishy smell. This invention lowers the boiling point, allowing volatile fishy-smelling substances to vaporize and escape at a lower temperature, thus removing a large amount during the denaturation stage. This prevents fishy-smelling substances from being encapsulated or reacting during subsequent enzymatic hydrolysis, reducing the fishy smell and improving the taste from the source.

[0016] 3. The low-pressure denaturation is mild and reversible, and does not flocculate or precipitate after enzymatic hydrolysis. It is easy to filter, has a high yield, and excellent solubility. Compared with high-pressure steam explosion, this invention has lower equipment costs and higher safety.

[0017] 4. This invention uses a single-enzyme hydrolysis method, but strictly controls the hydrolysis conditions. The hydrolysis is carried out at 60-65℃ and pH 8.0-8.5 for 3 hours to avoid over-hydrolysis into amino acids. Finally, the enzyme is inactivated at 85-90℃ for 15 minutes to terminate the reaction and retain the active peptide structure. 5. In the subsequent filtration, a two-stage filtration process is adopted. Ceramic membrane microfiltration removes large molecular proteins and residues, ensuring that the proportion of molecules smaller than 10,000 Da is greater than or equal to 95%. Then, the product is concentrated by nanofiltration membrane, which simultaneously desalts, dehydrates, and removes free amino acids, enriching high-purity active peptides. Since the product prepared by the method of this invention is mainly oligopeptides smaller than 1,000 Da, without complex spatial structures, and the peptide bond heat resistance temperature reaches above 120℃, it can be sterilized at ultra-high temperature of 121-125℃ for a short time without significant decrease in peptide activity after sterilization.

[0018] 6. The peptides with a relative molecular mass <1000Da account for ≥95% of the product prepared by this invention. In addition to improving the heat resistance temperature of peptide bonds as mentioned above, it also makes the product a small molecule, which can quickly penetrate the gastrointestinal mucosa and reach tissues such as the prostate, resulting in higher bioavailability.

[0019] 7. In the first enzymatic hydrolysis stage, the pH value is adjusted to the lower limit of the optimal range for alkaline proteases. This avoids localized protein denaturation caused by a strongly alkaline environment, which leads to protein clumping and the masking of enzyme binding sites. This preserves sufficient active sites for subsequent secondary hydrolysis and prevents excessive cleavage of hydrophobic amino acid peptide bonds by alkaline proteases under a strongly alkaline environment, thus avoiding the premature production of large amounts of bitter peptides. Then, in the second enzymatic hydrolysis stage, the pH value is adjusted to the upper limit of the optimal range for alkaline proteases. At this point, the denatured protein's network structure has been successfully opened, and large protein molecules have been initially broken down. The protein structure is now loose and will not clump due to high pH. Furthermore, sufficient enzyme binding sites are exposed, reducing the likelihood of uneven hydrolysis. In addition, the enzyme is added in batches during the second enzymatic hydrolysis, avoiding localized over-hydrolysis caused by a single high enzyme dosage, further mitigating the defects of increased bitterness and peptide chain oxidation.

[0020] 8. The present invention performs a centrifugation and impurity removal step before S5 to remove fat, bone residue and colloids in advance, which significantly reduces membrane fouling and extends membrane life.

[0021] 9. Vacuum concentration will continuously increase the concentration and viscosity of the liquid. If the solution is unclear or has poor solubility, precipitation, flocculation, and viscosity will occur during concentration, which can easily lead to wall adhesion, uneven heating, local overheating, browning, and scorching. The isoelectric point of sturgeon cartilage collagen peptides is mostly around 6.0. This invention adjusts the pH to 5.5-6.5, which can keep the peptide molecules in an electrostatically stable state with moderate intermolecular repulsion, making them less prone to aggregation and sedimentation. In the subsequent vacuum concentration process, the heating is uniform, gentle, and stable, maximizing the protection of the active peptides.

[0022] 10. After sterilization, the liquid material is discharged under high pressure and instantaneously, resulting in large flow fluctuations. Spray drying requires the liquid material to enter the drying tower with stable pressure and uniform flow; otherwise, it will lead to uneven atomization and incomplete drying. This invention adds a closed aseptic temporary storage step between sterilization and spray drying to stabilize pressure and flow fluctuations. After sterilization, the liquid material temperature is high (121-125℃). If it enters the spray drying tower directly, the high temperature will cause the droplets to evaporate too quickly after atomization, which can easily lead to uneven powder particles. Moreover, temperature fluctuations will affect the balance of the inlet and outlet air temperatures in spray drying. This invention places the liquid material briefly in a low temperature environment of 4℃-10℃, which can quickly stabilize it to a suitable drying temperature range, so that the temperature of the liquid material entering the spray drying tower is uniform and consistent, ensuring atomization effect and drying quality.

[0023] 11. The product prepared by this invention contains 12 characteristic active peptide sequences, which have good effects in the preparation of products that enhance immunity and improve male sexual function. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the preparation method of the present invention; Figure 2 HE staining of prostate tissue; Figure 3 Immunohistochemical staining of prostate tissue. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Example

[0026] Preparation of Sample 1: S1. Take 100kg of sturgeon cartilage from artificially hatched Chinese sturgeon, thaw it, remove impurities, blood, and floating oil, mince it into meat paste no larger than 2mm, add 350kg of pure water at a material-to-liquid ratio of 1:3.5, and pump it into an enzymatic hydrolysis tank. S2, sealed low pressure 0.07MPa, heated to 88℃ for 18min for denaturation; S3. Cool to 61℃, adjust pH to 8.1 with NaOH, add 1.5‰ alkaline protease (protease type Alcalase 2.4L (Novozymes), enzyme activity 2.4AU / g, appearance is light yellow powder, moisture content ≤8%, storage conditions 2-8℃ sealed storage), and hydrolyze at constant temperature for 85 min; raise the temperature of the solution to 64℃ in situ, adjust pH to 8.4, add 1.1‰ alkaline protease, and hydrolyze at constant temperature for 95 min.

[0027] S4. Add 0.5% activated carbon (particle size 100-200 mesh, specific surface area ≥1000m² / g, methylene blue adsorption value ≥150mg / g, conforming to GB2760-2014 "National Food Safety Standard for the Use of Food Additives"), decolorize at 60℃ for 35min, filter the activated carbon; centrifuge at 9000r / min for 12min. S5, ceramic membrane microfiltration, membrane inlet pressure 0.3 MPa, membrane outlet pressure 0.1 MPa, 3 washes; pH adjusted to 6.0 with citric acid; nanofiltration membrane concentration, membrane inlet pressure 1.4 MPa, membrane outlet pressure 1.2 MPa, temperature 58℃; S6, vacuum concentrated at 80℃ to 30% solids; S7. Sterilization and drying: Sterilize at 123℃ for 10 seconds; temporarily store aseptically at 6℃ for 15 minutes; spray dry to obtain the finished product.

[0028] Sample 2: The difference between Sample 2 and Sample 1 is that Sample 2 is denatured at 88℃ under normal pressure for 18 minutes.

[0029] Sample 3: The difference between Sample 3 and Sample 1 is that in S3, the temperature was lowered to 65℃, the pH was adjusted to 8.2, and 2.5 kg of alkaline protease was added at once for 3 hours of enzymatic hydrolysis; after the enzymatic hydrolysis was completed, the enzyme was inactivated at 88℃ for 15 minutes.

[0030] Sample 4: The difference between Sample 4 and Sample 1 is that the enzymatic hydrolysis time is 150 min, and the enzymatic hydrolysis time for each stage is 50 min.

[0031] Sample 5: The difference between Sample 5 and Sample 1 is that the enzymatic hydrolysis time is 210 min, and the enzymatic hydrolysis time for each stage is 70 min.

[0032] Sample 6: The difference between Sample 6 and Sample 1 is that S5 does not perform the pH adjustment step before nanofiltration.

[0033] Sample 7: The difference between Sample 7 and Sample 1 is that Sample 7 is directly spray-dried after sterilization, without aseptic temporary storage.

[0034] Comparative Example 1 S1. Raw material pretreatment: Take 100kg of sturgeon cartilage, thaw it, mince it, add 350kg of purified water at a material-to-liquid ratio of 1:3.5, and pump it into an enzymatic hydrolysis tank.

[0035] S2. Protein denaturation: Directly heat to 88°C under normal pressure and open conditions, and hold for 18 minutes to denature the protein.

[0036] S3. Cool to a constant temperature of 62℃, adjust pH to 8.2, add 2.5kg of alkaline protease, and hydrolyze at a constant temperature for 3 hours; after hydrolysis, raise the temperature to 90℃ and keep warm for 15 minutes to inactivate the enzyme.

[0037] S4. Decolorization: Add 0.5% activated carbon by weight of the raw material, stir at 60℃ for 35 min to decolorize, and remove the activated carbon by plate and frame filtration (filter cloth pore size 10μm, material polypropylene) to obtain coarse filtrate.

[0038] S5. Concentrate at 80°C under normal pressure until the solid content is about 30%.

[0039] S6. After sterilization at 123℃ for 10 seconds, spray drying is performed to obtain the sturgeon peptide product.

[0040] Result Validation The physicochemical properties and molecular weight distribution of samples 1-7 and the comparative example were tested and obtained as shown in Table 1 below:

[0041] Sample 1 employed a closed-loop low-pressure denaturation, gradient enzymatic hydrolysis, and multi-stage membrane separation process, effectively removing large molecular weight proteins and achieving a 99.2% proportion of components <10000Da, with a high proportion of <1000Da oligopeptides reaching 96.2%, demonstrating optimal molecular weight controllability. Sample 2 underwent atmospheric pressure denaturation, and Sample 3, due to insufficient enzymatic hydrolysis and uneven digestion, resulted in a significant decrease in the proportion of small molecular weight oligopeptides. Sample 4 had insufficient enzymatic hydrolysis time, leaving a large amount of large molecular weight residue; Sample 5 had excessive enzymatic hydrolysis time, leading to excessive degradation of active peptides into free amino acids, resulting in a decrease in the proportion of oligopeptides instead of an increase. Comparative Example 1, using a traditional process, exhibited a wide molecular weight distribution and a low proportion of small molecules, significantly different from the present invention.

[0042] Samples 1-7 were analyzed by high-performance liquid chromatography (HPLC). Detection conditions: C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile-0.1% trifluoroacetic acid aqueous solution; gradient elution; detection wavelength: 220 nm; flow rate: 1.0 mL / min; column temperature: 30 °C. Twelve short peptide sequences were determined. Based on the total peptide content of sturgeon (total peptide content of sample 1: 75.2 g / 100 g), the following values ​​were calculated: VLPEDGRP: 1.8%–2.2%; ASYLDCIK: 2.1%–2.5%; DGLGDVAFVK: 1.5%–1.9%. APNHAVVSRK: 2.3%~2.7%; DDTKCLVDLR: 1.6%~2.0%; NSEPWAKDLK: 1.9%~2.3%; GSNFQLNELQGK: 2.0%~2.4%; AVKWCALSHHER: 2.2%~ 2.6%; YLGADYIKAVSNLR: 1.7%~2.1%; CSTSRLLEACTFHK: 1.4%~1.8%; TVRWCAVNDHEASK: 1.6%~2.0%; QETIFENLPSKDER: 1.9%~2.3%.

[0043] The enzymatic hydrolysis efficiency and sensory quality of samples 1-7 and the comparative example were evaluated, and the results are shown in Table 2 below:

[0044] Table 2 shows that the low-pressure environment of Sample 1 promotes complete collagen unwinding, significantly improving enzymatic hydrolysis efficiency by 21.3% compared to the traditional process. Simultaneously, low pressure removes volatile odor substances and inhibits oxidative browning, resulting in a product with uniform color and a very mild odor. Sample 2, with its insufficient expansion of denatured collagen under normal pressure, showed limited improvement in enzymatic hydrolysis efficiency and a more pronounced odor. Sample 3 exhibited a odor due to uneven enzymatic hydrolysis, leading to localized over-hydrolysis. Insufficient enzymatic hydrolysis in Sample 4 and over-hydrolysis in Sample 5 both resulted in decreased enzymatic hydrolysis efficiency and sensory quality. Comparative Example 1, with its crude process, exhibited low enzymatic hydrolysis efficiency, dark color, strong odor, and poor quality.

[0045] The drying effects of samples 1-7 and the comparative example were evaluated, and the results are shown in Table 3 below:

[0046] Table 3 shows that Sample 1, after pH fine-tuning to near the isoelectric point region, exhibited high system stability, with no precipitation or flocculation during concentration and optimal solubility. Combined with aseptic temporary storage to stabilize the material temperature and flow rate, it resulted in no char powder and uniform particle size after drying. Sample 3, due to strong alkali causing protein agglomeration, resulted in precipitation, slight char powder during drying, and reduced solubility. Sample 6, without pH adjustment, showed easy peptide aggregation, slight flocculation, and decreased solubility. Sample 7, lacking aseptic temporary storage, experienced fluctuations in material temperature and flow rate, leading to a small amount of char powder. Comparative Example 1, without pH control or buffering steps, resulted in easy precipitation during concentration, significant char powder during drying, and poor solubility.

[0047] The immune function of samples 1-7 and Comparative Example 1 was verified, and the results are shown in Table 4 below:

[0048] Table 4 shows that, compared with the immunodeficiency and kidney yang deficiency model groups, each sample group had varying degrees of improvement on the thymus index and spleen index of mice. Among them, sample 1 had the most significant improvement effect, which was close to the level of the blank normal group.

[0049] Sample 2 underwent denaturation under normal pressure, resulting in insufficient collagen unwinding, a large amount of residual macromolecular proteins, and insufficient release of active peptides. Therefore, the increase in the immune organ index was significantly lower than that of Sample 1.

[0050] Due to protein clumping, the enzyme binding sites of sample 3 were obscured, resulting in insufficient enzymatic digestion and uneven molecular weight distribution. Consequently, the immune enhancement effect of sample 3 was weaker than that of sample 1, which underwent gradient enzymatic digestion.

[0051] Sample 4 was enzymatically hydrolyzed for only 2 hours, resulting in incomplete protein degradation, low yield of active peptides, and limited immune-improving effects.

[0052] When the enzymatic hydrolysis time of sample 5 was extended to 4 hours, excessive degradation occurred, and some active peptides were broken down into free amino acids, resulting in a decrease in immune efficacy.

[0053] Sample 6 was not pH adjusted, resulting in poor stability of the solution. Some peptides aggregated, and bioavailability decreased slightly, but overall it remained at a high level.

[0054] Sample 7 was not kept in a sterile environment. Fluctuations in the temperature and flow rate of the liquid caused a small amount of peptides to be lost due to heat. The immunization effect was slightly lower than that of sample 1, but the difference was not significant.

[0055] Comparative Example 1 uses a traditional, extensive process, resulting in a wide molecular weight distribution, numerous macromolecular impurities, and low content of active ingredients, thus having a weak effect on improving immune organ indices.

[0056] Sexual function indicators of samples 1-7 and comparative example 1 were verified, and the results are shown in Table 5 below:

[0057] As shown in Table 4, Sample 1 was significantly better than other groups in improving the testicular index and epididymal index and shortening the erection latency in mice, indicating that it had the most outstanding effect in improving male sexual function and warming and tonifying kidney yang.

[0058] Sample 2, denatured under normal pressure, resulted in excessive residue of odorous substances, insufficient protein structure expansion, and incomplete release of active ingredients, significantly reducing its effect on improving sexual function. Sample 3 reduced enzymatic hydrolysis efficiency and peptide uniformity, decreased the proportion of effective active peptides, and showed a weaker improvement in reproductive organ indices compared to Sample 1. Sample 4, with insufficient enzymatic hydrolysis and limited release of active peptides, showed a weaker improvement in sexual function-related indicators. Sample 5, with excessive enzymatic hydrolysis, destroyed characteristic functional oligopeptides, resulting in reduced efficacy. Although Samples 6 and 7 lacked pH adjustment and aseptic storage steps, respectively, they had minimal impact on the core active ingredients and therefore maintained a strong effect. Comparative Sample 1, with its crude process, low content of small molecule oligopeptides, and poor retention of effective ingredients, showed extremely weak effects on improving sexual function.

[0059] In summary, this invention, through precise restriction enzymatic hydrolysis, efficient deodorization, and precise molecular weight control, can retain the characteristic active peptides related to sexual function to the greatest extent, enabling them to effectively improve male sexual function.

[0060] The anti-inflammatory indicators of samples 1-7 and comparative example 1 were verified, and the results are shown in Table 6 below:

[0061] Table 6 shows that sample 1 can significantly reduce the levels of TNF-α and IL-6 in the prostate tissue of model mice, and has the best anti-inflammatory and prostate-protective effects.

[0062] During the normal pressure denaturation process, the oxidation and Maillard reaction of Sample 2 were aggravated, resulting in the loss of some active ingredients and a significantly weaker anti-inflammatory effect compared to Sample 1. Sample 3 resulted in uneven enzymatic digestion, leading to a decrease in the proportion of small-molecule oligopeptides that could penetrate prostate tissue, thus reducing its anti-inflammatory effect. Sample 4 exhibited insufficient enzymatic digestion, resulting in inadequate release of anti-inflammatory peptides and limited inhibitory ability against inflammatory factors. Excessive degradation in Sample 5 disrupted the structure of the anti-inflammatory oligopeptides, reducing the decrease in inflammatory factors. Samples 6 and 7 showed slightly lower inhibitory effects on inflammatory factors than Sample 1, but overall remained at a high level, indicating that pH adjustment and aseptic storage primarily affected stability and quality, with less impact on the core anti-inflammatory activity. Comparative Sample 1, due to its larger molecular weight, higher impurity content, and lower effective peptide content, resulted in a smaller amount entering prostate tissue to exert its anti-inflammatory effect, exhibiting weak inhibitory effects on TNF-α and IL-6.

[0063] Using sample 1, the above experiment was repeated at peptide doses of 0.4 g / kg·d and 1.6 g / kg·d, respectively, and the results are shown in Table 7.

[0064] Based on the results of sample 1 in Table 6 and Table 7, it can be seen that the sturgeon peptide of this application has a significant therapeutic effect on nonbacterial prostatitis and exhibits a dose-dependent effect. The therapeutic effect of medium dose (0.8g / kg) and above intervention doses is not inferior to that of the positive control.

[0065] Sampling and HE staining were performed according to the methods described in Tables 6 and 7, such as... Figure 2 As shown, the blank control group had intact prostate tissue structure, with glandular epithelial cells arranged in a single layer, a small amount of normal secretions in the lumen, and no obvious inflammatory infiltration in the stroma surrounding the acini. The model control group had abnormal cell structure, with papillary thickening of glandular epithelial cells, partial shedding of some areas, increased secretions in the stroma surrounding the acini, fibrosis, and significant lymphocyte infiltration. The positive control group, and the low, medium, and high-dose drug intervention groups all showed varying degrees of improvement in tissue lesions and inflammatory infiltration, with the positive control group and the high-dose drug intervention group showing the most significant improvement.

[0066] Immunohistochemical results were observed based on the sampling methods described in Tables 6 and 7. Figure 3As shown in Figure 8 and Table 8, compared with the blank control group, the expression level of NF-κB (p65) in prostate tissue of the model control group was significantly increased (P < 0.01); compared with the model control group, the expression level of NF-κB (p65) in prostate tissue of both the positive drug and the test drug intervention groups was significantly decreased (P < 0.01); compared with the positive drug group, the expression level of NF-κB (p65) in the low-dose drug intervention group was significantly increased (P < 0.01), there was no significant difference in the medium-dose drug intervention group (P > 0.05), and the expression level in the high-dose drug intervention group was significantly decreased (P < 0.01). Among the drug intervention groups, the expression level of NF-κB (p65) in prostate tissue gradually decreased with increasing drug dosage, and the difference was statistically significant (P < 0.05).

[0067]

[0068] In summary, the sturgeon peptide prepared by this invention is based on a high proportion of small molecule oligopeptides <1000Da and retains a high level of active peptide components. It can effectively penetrate into prostate tissue, significantly reduce the level of inflammatory factors, and achieve the functions of protecting the prostate and improving the inflammatory state.

[0069] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing sturgeon peptide, characterized in that, Includes the following steps: S1. Raw material pretreatment: The raw material is one or more of sturgeon cartilage, fish bone, and fish skin. After thawing, it is minced and added to purified water at a material-to-liquid ratio of 1:3 to 1:

4. The mixture is then pumped into an enzymatic hydrolysis tank. S2. Protein denaturation: The enzymatic hydrolysis vessel is heated to 85-90°C under a closed, low-pressure condition of 0.06-0.08 MPa and maintained for 15-20 minutes to fully denature the protein. S3. Enzymatic hydrolysis: Cool to 60-65℃, adjust pH to 8.0-8.5, add 2‰-3‰ alkaline protease to the raw material, and hydrolyze at a constant temperature for 170-190 min; after the enzymatic hydrolysis is completed, raise the temperature to 85-90℃ and maintain it for 14-16 min to inactivate the enzyme. S4. Decolorization: Add an appropriate amount of activated carbon to the enzymatic hydrolysate, and filter to remove the activated carbon after decolorization. S5. Filtration and Concentration: Remove large protein molecules and impurities, desalt, dehydrate, and remove free amino acids to obtain a concentrated solution; S6. Vacuum concentration: Concentrate the concentrate at 75-85℃ to 25%-35% under vacuum; S7. Sterilization and drying: After sterilization at 121-125℃ for 5-15 seconds, the sturgeon peptide product is obtained by spray drying.

2. The preparation method according to claim 1, characterized in that: After S5 microfiltration, the proportion of peptides with a relative molecular mass <10000Da in the filtrate is ≥95%.

3. The preparation method according to claim 1, characterized in that: In S3, the temperature is lowered to 60-62℃, the pH is adjusted to 8.0-8.2, and 1.2‰-1.8‰ of alkaline protease is added. The mixture is then hydrolyzed at a constant temperature for 80-90 minutes. The temperature of the solution is then raised to 63-65℃ in situ, the pH is adjusted to 8.3-8.5, and 1.0‰-1.2‰ of alkaline protease is added. The mixture is then hydrolyzed at a constant temperature for 90-100 minutes.

4. The preparation method according to claim 1, characterized in that: In step S4, the enzymatic hydrolysate is transferred to a decolorizing tank, and 0.5% of the raw material weight of powdered activated carbon is added to the hydrolysate. The decolorization reaction is carried out at 55-65℃ for 30-40 minutes.

5. The preparation method according to claim 1, characterized in that: In S5, ceramic membrane microfiltration is first used, with the membrane inlet and outlet pressure difference controlled at 0.1–0.3 MPa. The filter is circulated and washed with water 2–4 times to remove large molecular proteins and impurities, obtaining filtrate. Then, nanofiltration membrane concentration is used, with the membrane inlet pressure controlled at 1.3–1.5 MPa, the membrane outlet pressure at 1.1–1.3 MPa, and the feed temperature at 55–60℃, to desalinate, dehydrate, and remove free amino acids, obtaining concentrated solution.

6. The preparation method according to claim 5, characterized in that: Before using nanofiltration membrane concentration, a pH fine-tuning step is also included: the pH of the concentrate is adjusted to 5.5-6.5 using citric acid.

7. The preparation method according to claim 1, characterized in that, Before S5, there is also a centrifugation step to remove impurities: centrifuge at 8000-10000 r / min for 10-15 min to remove fat, bone residue, and colloidal impurities.

8. The preparation method according to claim 1, characterized in that, A closed aseptic temporary storage step is added between sterilization and spray drying, where the liquid is temporarily stored in an aseptic environment at 4℃~10℃ for 5min~30min.

9. The sturgeon peptide prepared by any one of claims 1-8, characterized in that: Protein ≥78.5g / 100g, peptide content ≥75.2g / 100g, arginine ≥5.80g / 100g, chondroitin sulfate ≥11.1%; relative molecular mass less than 10000Da greater than 98%, oligopeptides less than 1000Da greater than 95%; its active short peptides include VLPEDGRP, ASYLDCIK, DGLGDVAFVK, APNHAVVSRK, DDTKCLVDLR, NSEPWAKDLK, GSNFQLNELQGK, AVKWCALSHHER, YLGADYIKAVSNLR, CSTSRLLEACTFHK, TVRWCAVNDHEASK, QETIFENLPSKDER.

10. The application of the sturgeon peptide according to claim 8 in the preparation of products that enhance immunity, improve male sexual function, or improve prostatitis, wherein the product is a health food, functional food, or dietary supplement.

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