Preparation method of pig liver peptide and application thereof

Abstract

The present application belongs to the technical field of pig liver peptide preparation, and particularly relates to a preparation method of pig liver peptide and application thereof. The present application takes fresh pig liver as raw material, and fully exposes protease cleavage site through homogenization and moderate denaturation at high temperature. Protease is hydrolyzed by trypsin and alkaline protease to realize efficient hydrolysis of protein. Then, gradient concentration is performed by using >3000D and 300-1000D ultrafiltration membranes to precisely enrich high-activity small molecule peptides. Subsequently, chitooligosaccharide and trehalose complex embedding system is introduced to modify pig liver peptide by microcapsule embedding, which effectively removes the fishy smell of the product and improves the stability of the peptide structure. That is, the present application uses the whole process of high-temperature denaturation, double-enzyme synergistic hydrolysis, two-stage gradient ultrafiltration and complex embedding modification to significantly improve the degree of hydrolysis of pig liver protein and the yield of the product. The obtained product has low fishy smell, good stability, good anti-inflammatory activity and high absorption rate.

Description

Technical Field

[0001] This invention belongs to the field of porcine liver peptide preparation technology, specifically relating to a method for preparing porcine liver peptide and its application. Background Technology

[0002] Pig liver is a valuable livestock by-product, rich in high-quality animal protein, essential amino acids, vitamins, and trace elements such as iron and zinc. It is an excellent raw material for preparing natural antioxidant and anti-inflammatory bioactive peptides. Compared to artificially synthesized active ingredients, pig liver-derived bioactive peptides are safer, more biocompatible, and easily absorbed by the human body, showing broad industrialization prospects in functional foods, dietary supplements, and health foods.

[0003] Currently, research on enzymatic hydrolysis preparation processes for porcine liver peptides has been gradually carried out in the industry. For example, Chinese patents CN109957595 A and CN 122038517 A both disclose conventional porcine liver peptide preparation and compound enzymatic hydrolysis processes. Among them, CN109957595 A adopts a single protease hydrolysis combined with a single-stage ultrafiltration process. Although the process is simple and has a low threshold, it lacks a high-temperature activation and denaturation step for proteins, resulting in a dense protein spatial structure and insufficient exposure of enzyme cleavage sites. Due to the single protease hydrolysis site, it suffers from low degree of protein hydrolysis, poor preparation efficiency, and low product yield. At the same time, relying solely on simple centrifugation and single-stage ultrafiltration for impurity removal cannot effectively enrich highly active small molecule peptides, and the product has weak antioxidant and anti-inflammatory activities. In addition, this scheme does not perform any deodorization or stability modification treatment, resulting in a product with a strong inherent fishy smell and extremely poor palatability. Although CN 122038517 A improved the process by using a complex enzyme system containing alkaline protease for enzymatic hydrolysis, it did not specify precise enzyme ratios, enzyme dosages, and temperature and pH parameters. The process controllability was poor, and the hydrolysis uniformity was insufficient. It also lacked a high-temperature denaturation pretreatment step and only used conventional single-stage membrane filtration without a gradient purification process. As a result, the molecular weight distribution of the prepared porcine liver peptides was disordered, and the activity stability was poor.

[0004] It is evident that existing porcine liver peptide preparation processes suffer from the following shortcomings: 1. The lack of a precise protein activation and efficient hydrolysis system results in insufficient hydrolysis of raw materials, low production efficiency, inadequate product yield, and significant waste of livestock and poultry by-product resources; 2. The absence of a fractional purification process leads to low enrichment of small-molecule, highly active peptides, resulting in weak and unstable antioxidant and anti-inflammatory activities in the product; 3. The absence of modification treatment to address the inherent fishy odor of porcine liver results in poor palatability and limited application scenarios; 4. The prepared free porcine liver peptides exhibit poor structural stability, are easily degraded and inactivated by gastric acid and digestive enzymes, and have low bioavailability, significantly reducing the actual efficacy and commercial value of porcine liver peptides. Therefore, providing a porcine liver peptide preparation process with high yield, high activity, low fishy odor, and high bioavailability not only addresses the shortcomings of existing technologies but also enables the large-scale, efficient production of high-quality porcine liver peptides. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing porcine liver peptides and their applications, which has the advantages of high yield, high activity, low odor, and high in vivo bioavailability.

[0006] This invention provides a method for preparing porcine liver peptides, comprising the following steps: S1. Homogenize and denature the pig liver to obtain denatured pig liver slurry; S2. The denatured pig liver slurry is mixed with a complex enzyme for enzymatic hydrolysis, ultrafiltration, and concentration to obtain a concentrated peptide solution; the complex enzyme includes trypsin and alkaline protease. S3. The concentrated peptide solution is mixed with a composite embedding agent for embedding modification to obtain a modified peptide solution; the composite embedding agent includes: chitosan oligosaccharide solution and trehalose solution; S4. The modified peptide solution is sterilized and spray-dried to obtain porcine liver peptide.

[0007] Preferably, in step S2, the mass ratio of trypsin to alkaline protease is 1:1 to 1:4; the enzymatic hydrolysis temperature is 50-55℃ and the time is 1-3 hours. Based on the dry weight of the pig liver described in S1, the amount of compound enzyme added is 3000~5000U / g dry weight of pig liver.

[0008] Preferably, in step S3, water is used as a solvent to obtain chitosan oligosaccharide solution and trehalose solution respectively; in the composite encapsulating agent, the mass ratio of chitosan oligosaccharide solution and trehalose solution is 1:2 to 1:3; The mass concentration of the chitosan oligosaccharide solution is 1%~2%; The trehalose solution has a mass concentration of 8% to 10%.

[0009] Preferably, in step S3, the mass ratio of the composite encapsulating agent to the dry weight of the concentrated peptide solution is 8~15:100, based on the dry weight of the concentrated peptide solution. During the embedding modification, the film hydration temperature is 40~45℃; the reverse evaporation vacuum degree is -0.08~-0.1MPa, and the time is 30~45min.

[0010] Preferably, in step S1, the mass ratio of pig liver to water during homogenization is 1:1; the homogenization speed is 8000~10000 r / min, and the time is 5~8 min. The denaturation temperature is 90~100℃, and the time is 1~2h.

[0011] Preferably, in step S2, the ultrafiltration process is as follows: the enzymatic hydrolysate is filtered through an ultrafiltration membrane at 25±5℃ to retain components with a molecular weight >3000D, thereby obtaining a porcine liver peptide solution with a solid content of 5%±2%.

[0012] Preferably, in step S2, the concentration process is as follows: the porcine liver peptide solution is concentrated using an ultrafiltration membrane with a molecular weight cutoff of 300-1000D to obtain a concentrated peptide solution with a solid content ≥20%.

[0013] Preferably, in step S4, when performing spray drying, the inlet air temperature is 200±10℃ and the outlet air temperature is 90±5℃.

[0014] This invention provides porcine liver peptides obtained by the preparation method described in the above technical solution.

[0015] This invention provides an application of the preparation method described in the above technical solution, wherein the application includes at least one of the following (1)-(3): (1) Enhance the activity of porcine liver peptides; (2) Improve the utilization rate of porcine liver peptides; (3) Reduce the fishy smell of pig liver peptides.

[0016] Compared with the prior art, the present invention has the following advantages: 1. High preparation efficiency and excellent product yield: This invention first uses a constant temperature denaturation treatment at 100℃ to destroy the spatial folding structure of porcine liver protein and fully expose the enzyme cleavage sites; then, trypsin and alkaline protease are combined for synergistic hydrolysis. The two proteases have complementary action sites, which greatly improves the hydrolysis efficiency compared with the single enzymatic hydrolysis process, and the protein is hydrolyzed more thoroughly, effectively improving the yield of porcine liver peptide products, significantly improving the utilization rate of raw materials, and reducing production losses and costs.

[0017] 2. The product has strong bioactivity and significant antioxidant and anti-inflammatory effects: This invention adopts a two-stage gradient ultrafiltration process. First, it removes large molecular ineffective impurities through an ultrafiltration membrane of 3000D or higher, and then it accurately enriches small molecular active peptides through a 300-1000D ultrafiltration membrane. The resulting porcine liver peptides have a uniform molecular weight distribution and a very high proportion of small molecular active peptides. They have excellent DPPH free radical scavenging ability, outstanding antioxidant activity, and good anti-inflammatory bioactivity. The product has strong functional stability.

[0018] 3. Completely solves the fishy smell defect of pork liver peptides, resulting in a better product taste: This invention innovatively introduces a chitosan oligosaccharide and trehalose composite encapsulation system, combined with a thin-film hydration-reverse evaporation composite process, which can effectively encapsulate the fishy smell active substances in pork liver peptides, masking and removing the inherent fishy smell of pork liver from the source. This solves the industry problems of traditional pork liver peptide products having a strong fishy smell, poor palatability, and limited application scenarios, and greatly improves the product's eating experience and commercial value.

[0019] 4. Enhance product stability and in vivo absorption and utilization: The complex microcapsule structure formed by chitosan oligosaccharide and trehalose can form a protective film on the surface of porcine liver peptides. On the one hand, it protects the active peptides from oxidation and inactivation during spray drying and storage, thus improving the product's storage stability. On the other hand, it can resist the degradation and destruction by human gastric acid and digestive tract proteases, enabling the sustained-release absorption of active peptides, significantly improving the bioavailability of porcine liver peptides in vivo, and maximizing their antioxidant and anti-inflammatory effects.

[0020] 5. Simple and controllable process, suitable for large-scale production: The entire process of this invention adopts physical modification + biological enzymatic hydrolysis, without chemical additives, green, safe and pollution-free; the process parameters are precise and controllable, and the ultrafiltration classification, encapsulation modification and spray drying are integrated and continuous, with high production efficiency and stable product quality, which can be widely used in the large-scale production of functional foods, health products and dietary supplements. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This invention provides a graph of various inflammatory factor expression data. Detailed Implementation

[0023] In this invention, unless otherwise specified, the raw materials, equipment and methods used are conventionally selected.

[0024] The trypsin described in this invention is rTrypsin® Granulate, with an enzyme activity ≥800 USP / mg; the alkaline protease is Alcalase 2.4 L FG, with an enzyme activity ≥2.4 AU-A / g; In preparing the chitosan oligosaccharide solution and trehalose solution according to this invention, the chitosan oligosaccharide used is a food-grade white to pale yellow powder with a molecular weight of 2000~3000 Da, a degree of deacetylation ≥85%, and a moisture content ≤6.0%. After preparing the chitosan oligosaccharide solution with water as a solvent, the mass concentration of the chitosan oligosaccharide solution is 1%~2%, the pH is 5.0~7.0, and the powder purity is ≥80%. The trehalose is a food-grade white crystalline powder with a purity ≥98%, a glass transition temperature ≥110℃, and low hygroscopicity. After preparing the trehalose solution with water as a solvent, the mass concentration of the trehalose solution is 8%~10%, and the pH is 6.0~7.5.

[0025] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1 A method for preparing porcine liver peptides, comprising the following steps: S1. Raw material pretreatment: Select 2kg of fresh, unspoiled pig liver [use a rapid moisture analyzer to determine the moisture content of the pig liver, and calculate the dry weight of the pig liver = fresh pig liver mass × (1 - moisture %), the calculated dry weight of the pig liver is 0.56kg], remove the fascia and blood clots, rinse with clean water, add 2kg of purified water (i.e., the mass ratio of fresh pig liver to water is 1:1) and homogenize at 9000r / min for 6min to obtain a uniform pig liver slurry; S2. Denaturation treatment: The above-mentioned pig liver slurry was kept at a constant temperature of 100℃ for 1 hour to complete the appropriate denaturation of protein and obtain denatured pig liver slurry. S3. Complex Enzymatic Hydrolysis: The trypsin is rTrypsin® Granulate with an enzyme activity of 800 USP / mg; the alkaline protease is Alcalase 2.4 L FG with an enzyme activity of 2.4 AU-A / g; the trypsin and alkaline protease are mixed at a mass ratio of 1:1 to obtain the complex enzyme. Add a compound enzyme to the denatured pig liver slurry (when adding the compound enzyme, the amount added is based on the dry weight of pig liver calculated in step S1, which is 560g, to ensure that 5000U of compound enzyme is added per gram of dry weight of pig liver), keep the system at a constant temperature of 50℃ and pH 7.5, and continue enzymatic hydrolysis for 3 hours to obtain the enzymatic hydrolysate. S4. Coarse filtration to remove impurities: The above enzymatic hydrolysate is subjected to coarse filtration to remove impurities (using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da to remove solute components with a molecular weight cutoff greater than 3000 Da, and filtering at a constant temperature of 25°C) to remove residual enzymes and macromolecular impurities, and obtain a peptide-containing diluent with a solid content of 5% and a mass of 3.76 kg. S5. Ultrafiltration Concentration: The above peptide-containing diluent was concentrated using a 1000D ultrafiltration membrane to obtain 2.49 kg of concentrated peptide solution with a solid content of 20%. The dry weight of the concentrated peptide solution was calculated to be 498 g. S6. Encapsulation Modification: Chitosan oligosaccharide with a molecular weight of 3000 Da, a degree of deacetylation of 90%, and a moisture content of ≤6.0% was selected; after preparing the chitosan oligosaccharide solution with water as a solvent, the mass concentration of the chitosan oligosaccharide solution was 1%, the pH was 7.0, and the powder purity was ≥80%; Trehalose with a purity of ≥98%, a glass transition temperature of ≥110℃, and low hygroscopicity was selected; after preparing the trehalose solution with water as a solvent, the mass concentration of the trehalose solution was 10%, and the pH was 7.5.

[0027] The above-mentioned chitosan oligosaccharide solution and trehalose solution were mixed at a mass ratio of 1:2.5 to obtain a composite encapsulating agent. The composite encapsulating agent was added to the above-mentioned concentrated peptide solution (the amount of composite encapsulating agent added was based on the dry weight of the concentrated peptide solution in step S5, which was 498g; the amount of composite encapsulating agent added was 10% of the dry weight of the concentrated peptide solution, i.e., 49.8g) to obtain a mixture. The mixture was stirred and mixed evenly under a water bath at 42℃. Then it was transferred to a rotary evaporator and rotated at 42℃ and normal pressure for 15 minutes to perform thin film hydration. Subsequently, the vacuum degree was controlled at -0.09MPa and 42℃ and the rotation was continued for 40 minutes for counter-evaporation, so that chitosan oligosaccharide-trehalose formed dense microcapsules on the surface of the concentrated peptide solution, achieving odor encapsulation and activity protection. After the completion, the vacuum was slowly broken to obtain the modified peptide solution. S7. Sterilization filtration: The modified peptide solution above is sterilized by pressurized filtration using a 0.22μm PES filter cartridge to obtain sterilized peptide solution; S8. Spray drying: The above-mentioned sterilizing peptide solution is spray dried at an inlet air temperature of 200℃ and an outlet air temperature of 90℃ to obtain pig liver peptide powder; the pig liver peptide powder is aseptically sealed and packaged to obtain 486g of finished product.

[0028] Comparative Example 1 A method for preparing porcine liver peptides, comprising the following steps: S1. Raw material pretreatment: Select fresh, non-deteriorated pig liver, remove fascia and blood clots, rinse with clean water, then add water to the cleaned pig liver (the mass ratio of liver to water is 1:1), homogenize at 9000r / min for 6min to obtain a uniform pig liver slurry. S2. Degreasing treatment: Add alkaline lipase (purchased from Novozymes, model Lipozyme TL 100L, enzyme activity 10000U / g, enzyme addition amount is 1% of the weight of pig liver slurry) to the above pig liver slurry and mix. Enzymatic hydrolysis is carried out at 40-45℃ and pH 8.5 for 60 min. The enzyme is inactivated 15 min after hydrolysis. After the above degreasing treatment, most of the fat impurities in the pig liver are removed, and the defatted pig liver slurry is obtained. S3. Single protease hydrolysis: The defatted pig liver slurry was hydrolyzed using a single alkaline protease (enzyme activity 200,000 U / mL). The amount of alkaline protease added was 0.3% of the weight of the pig liver slurry. The hydrolysis temperature was 45-50℃, pH 8.0, and the hydrolysis time was 4 hours. After enzyme inactivation, the hydrolysate was obtained.

[0029] S4. Centrifugation to remove impurities: After the enzymatic hydrolysis is completed, adjust the pH of the above enzymatic hydrolysis system to 7.0, and collect the supernatant by high-speed centrifugation; S5. Ultrafiltration treatment: The supernatant above was filtered using a single 3000Da ultrafiltration membrane, and the permeate was collected to obtain liver peptide solution. S6. Spray drying: The above liver peptide solution is spray dried at an inlet air temperature of 200-240℃ and an outlet temperature of 100℃ to obtain porcine liver peptide.

[0030] Comparative Example 2 The difference from Example 1 is that step S6 is omitted in Comparative Example 2, and the steps are as follows: S1. Raw material pretreatment: Same as S1 in Example 1; S2, denaturation treatment: Same as S2 in Example 1; S3, Compound Enzymatic Hydrolysis: Same as S3 in Example 1; S4. Coarse filtration to remove impurities: Same as S4 in Example 1; S5, Ultrafiltration Concentration: The method is the same as S5 in Example 1; S6. Sterilization filtration: The above concentrated peptide solution is sterilized by pressurized filtration using a 0.22μm PES filter element to obtain sterilized peptide solution; S7. Spray drying: Same as S8 in Example 1.

[0031] Comparative Example 3 The difference from Example 1 is that S6 is omitted in Comparative Example 3, and only a single alkaline enzyme is used for enzymatic hydrolysis in S3. The steps are as follows: S1. Raw material pretreatment: Same as S1 in Example 1; S2, denaturation treatment: Same as S2 in Example 1; S3, Single alkaline enzymatic hydrolysis: Alcalase 2.4 L FG, enzyme activity 2.4 AU-A / g; Add alkaline protease to the denatured pig liver slurry (the amount added is based on the dry weight of pig liver calculated in step S1, ensuring that 5000U of alkaline protease is added per gram of dry weight of pig liver), keep the system at a constant temperature of 50℃ and pH 7.5, and continue enzymatic hydrolysis for 3 hours to obtain the enzymatic hydrolysate. S4. Coarse filtration to remove impurities: Same as S4 in Example 1; S5, Ultrafiltration Concentration: The method is the same as S5 in Example 1; S6. Sterilization filtration: The above concentrated peptide solution is sterilized by pressurized filtration using a 0.22μm PES filter element to obtain sterilized peptide solution; S7. Spray drying: Same as S8 in Example 1.

[0032] Comparative Example 4 The difference from Example 1 is that S6 is omitted in Comparative Example 4, and only a single trypsin is used in S3. The steps are as follows: S1. Raw material pretreatment: Same as S1 in Example 1; S2, denaturation treatment: Same as S2 in Example 1; S3. Single trypsin: The trypsin is rTrypsin® Granulate with an enzyme activity of 800 USP / mg; add trypsin to the denatured pig liver slurry (the amount added is based on the dry weight of pig liver calculated in step S1, ensuring that 5000U of trypsin is added per gram of dry weight of pig liver), keep the system at a constant temperature of 50℃ and pH 7.5, and continue enzymatic hydrolysis for 3 hours to obtain the enzymatic hydrolysate; S4. Coarse filtration to remove impurities: Same as S4 in Example 1; S5, Ultrafiltration Concentration: The method is the same as S5 in Example 1; S6. Sterilization filtration: The above concentrated peptide solution is sterilized by pressurized filtration using a 0.22μm PES filter cartridge to obtain sterilized peptide solution; S7. Spray drying: Same as S8 in Example 1.

[0033] The product yields of each porcine liver peptide in Example 1 and Comparative Examples 1-4 were statistically analyzed, and their efficacy was verified, as follows: (1) Determination of degree of hydrolysis (DH) The amino nitrogen content of the porcine liver peptides prepared in Example 1 and Comparative Examples 1-4 was determined according to GB 5009.5-2025 "National Food Safety Standard - Determination of Protein in Food", and the degree of protein hydrolysis was calculated. The formula for calculating the degree of hydrolysis (DH) is as follows: DH=

[0034] The determination of ammonia nitrogen content was carried out in accordance with the formaldehyde potentiometric titration method in GB / T 5009.39-2003.

[0035] (2) Determination of DPPH free radical scavenging rate Referring to the methods in GB / T 39100-2020 "Determination of Antioxidant Activity of Polypeptides: DPPH and ABTS Methods", the DPPH free radical scavenging rate of the porcine liver peptides prepared in Example 1 and Comparative Examples 1-4 was determined, and the calculation formula is as follows: Sweep rate = (1- ) 100% Where: A1: Absorbance of sample with DPPH solution; A2: Absorbance of sample with 95% ethanol solution; A3: Absorbance of DPPH solution with 95% ethanol solution.

[0036] (3) Sensory evaluation The porcine liver peptides prepared in Example 1 and Comparative Examples 1-4 were subjected to blind testing. Lower scores in later stages indicated a stronger fishy odor; the relevant scoring criteria are shown in Table 1. Table 1 Scoring criteria for porcine liver peptides

[0037] The blind test requirements are as follows: 1. Evaluators: Select 20 sensory evaluators (male to female ratio 1:1, age 20-45 years old). Requirements: normal sense of smell and taste, no rhinitis or cold, no alcohol, no spicy / strong-smelling food, and no perfume / fragrance in the 24 hours before the test; unified training on scoring standards should be conducted in advance to ensure consistent understanding.

[0038] 2. Sample preparation and numbering Samples: Porcine liver peptides prepared in Examples 1 and Comparative Examples 1-4 were used to prepare a 10% (w / w) sample solution of the above porcine liver peptides with purified water at room temperature. The solution was stirred until completely dissolved and allowed to stand without layering or precipitation.

[0039] Blind sample numbering: A three-digit random number code (such as 127, 351, 806) is used. The original label of the sample is removed. The code and the correspondence between the sample are kept only by the person in charge of the experiment. The evaluators are unaware of the code throughout the process.

[0040] Dispensing: Dispense equal amounts into uniform, colorless, odorless, and transparent sample cups, cover the cups (to prevent odor from evaporating), and randomly shuffle the order of placement.

[0041] Supporting supplies: Room temperature purified water (for rinsing mouth), disposable paper cups, rating sheets, pens, and trash cans.

[0042] 3. Environmental Requirements The sensory evaluation was conducted in a standard sensory evaluation room with good ventilation, no odor, soft and uniform lighting, and a room temperature of 20-25°C, while avoiding external noise and odor interference.

[0043] The blind testing procedure is as follows (execute step by step): 1. Sample placement: Randomly place the coded sample cups on the evaluation table. Each evaluator receives a complete set of samples (5 sets) + water.

[0044] 2. Evaluation rules and reminders to evaluators: This evaluation only assesses the intensity of the fishy smell, and scores should be based on the standards in Table 1; rinsing the mouth is required between each set of samples to avoid cross-contamination of odors.

[0045] 3. Item-by-item evaluation and scoring: ① Remove the lid of the sample cup, smell the odor first, and then taste the sample solution in small sips; ② Based on the perceived degree of fishy smell, fill in the corresponding score on the scoring sheet according to the scoring criteria; ③ After evaluating one sample, immediately drink water to rinse your mouth thoroughly, wait 60 seconds, and wait until the odor in your mouth and nasal cavity has completely dissipated before evaluating the next sample; ④ After all five samples have been evaluated and the scoring is confirmed to be correct, submit the scoring sheet.

[0046] 4. Round Arrangement: After all evaluators have completed the first round of evaluation, the sample placement order can be shuffled to conduct a second round of retesting, thereby improving the reliability of the data.

[0047] The results of the above efficacy verification are shown in Table 2.

[0048] Table 2 Efficacy evaluation of different treatments

[0049] Based on the data in Table 2, under the same raw materials and testing conditions, the core performance indicators of the porcine liver peptide products prepared in Example 1 and Comparative Examples 1-4 of this invention were tested, and the comparison results are as follows: 1. Degree of hydrolysis and product yield: The highest degree of hydrolysis in Comparative Examples 1-4 was only 71.5%, and the product yield was relatively low; the degree of hydrolysis in the embodiments of this invention reached 85%. It can be seen that the technical solution of this application is conducive to improving the product yield, that is, the raw material utilization rate is significantly higher and the preparation efficiency is greatly optimized.

[0050] 2. Antioxidant activity: The DPPH free radical scavenging rate of the product in Comparative Example 1 was only 65.2%, that of Comparative Example 2 was 73.8%, that of Comparative Example 3 was 71.5%, and that of Comparative Example 4 was 73.4%; the DPPH free radical scavenging rate of the porcine liver peptide prepared in Example 1 of this invention was 79.2%, and its antioxidant activity was significantly better than that of the existing comparative process, with outstanding enrichment effect of small molecule active peptides.

[0051] 3. Sensory flavor: The pork liver peptide powders prepared in Comparative Examples 1-4 all had a distinct pork liver odor, poor palatability, and limited product application; while the product prepared in Example 1 of this invention had no obvious odor, excellent flavor, and stronger commercial applicability.

[0052] (4) Test of anti-inflammatory effect The anti-inflammatory effect was tested according to the method in T / SPMA 003-2023 "Test Method for Soothing Efficacy of Cosmetics: Determination of Nitric Oxide Content Based on Lipopolysaccharide-Induced Macrophage Cell Line Inflammatory Cell Model".

[0053] The porcine liver peptides prepared in Example 1 and Comparative Examples 1-2 were diluted with sterile water to obtain 5 mg / mL porcine liver peptide sample solutions. The anti-inflammatory effect was determined using a mouse macrophage RAW264.7, LPS (lipopolysaccharide, which induces inflammation), TNF-α, and IL-6 inflammatory factor detection kit.

[0054] Mouse macrophages RAW264.7 cells were seeded into culture plates and cultured until they adhered to the plate. The blank group, model group, Example 1, Comparative Example 1, and Comparative Example 2 were labeled. Except for the blank group, LPS was added to the other groups to induce inflammation in the cells. Then, porcine liver peptide sample solution was added to each group, and the blank group and model group were added with an equal amount of culture medium and cultured for a longer period of time. After culture, the cell supernatant was collected, and the levels of tumor necrosis factor TNF-α and interleukin IL-6 were detected using an ELISA kit. With the relative expression level of the blank group set at 1.00, the relative levels of TNF-α and IL-6 were measured, and the inhibition rate of inflammatory factors was calculated to evaluate the anti-inflammatory effect. The formula for calculating the inhibition rate of inflammatory factors (%) is as follows: Inhibition rate (%) = (1 - expression level of sample group / expression level of model group) × 100%.

[0055] The expression data of various inflammatory factors are shown in Table 3 and Figure 1 As shown (in) Figure 1 In the examples, Case 1 corresponds to Example 1, Comparative Example 1 corresponds to Comparative Example 1, and Comparative Example 2 corresponds to Comparative Example 2. The anti-inflammatory performance results are shown in Table 4.

[0056] Table 3. Expression data of various inflammatory factors

[0057] Table 4 Anti-inflammatory properties under different treatments

[0058] Based on the data in Tables 3 and 4, after preparing the corresponding products using the preparation methods in the comparative examples, each product showed weak inhibitory effects on inflammatory factors and poor functional stability. However, after using the technical solution in Example 1, the prepared products could significantly inhibit the expression of inflammatory factors, exhibiting excellent and stable anti-inflammatory properties and more comprehensive functional attributes.

[0059] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing porcine liver peptides, characterized in that, Includes the following steps: S1. Homogenize and denature the pig liver to obtain denatured pig liver slurry; S2. The denatured pig liver slurry is mixed with a complex enzyme for enzymatic hydrolysis, ultrafiltration, and concentration to obtain a concentrated peptide solution; the complex enzyme includes trypsin and alkaline protease. S3. The concentrated peptide solution is mixed with a composite embedding agent for embedding modification to obtain a modified peptide solution; the composite embedding agent includes: chitosan oligosaccharide solution and trehalose solution; S4. The modified peptide solution is sterilized and spray-dried to obtain porcine liver peptide.

2. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of trypsin to alkaline protease is 1:1 to 1:4; the enzymatic hydrolysis temperature is 50-55℃ and the time is 1-3 hours. Based on the dry weight of the pig liver described in S1, the amount of compound enzyme added is 3000~5000U / g dry weight of pig liver.

3. The preparation method according to claim 1, characterized in that, In step S3, water is used as a solvent to obtain chitosan oligosaccharide solution and trehalose solution respectively; in the composite encapsulating agent, the mass ratio of chitosan oligosaccharide solution and trehalose solution is 1:2 to 1:3; The mass concentration of the chitosan oligosaccharide solution is 1%~2%; The trehalose solution has a mass concentration of 8% to 10%.

4. The preparation method according to claim 1, characterized in that, In step S3, the mass ratio of the composite encapsulating agent to the dry weight of the concentrated peptide solution is 8~15:100, based on the dry weight of the concentrated peptide solution. When performing the embedding modification, the film hydration temperature is 40~45℃; the reverse evaporation vacuum degree is -0.08~-0.1MPa, and the time is 30~45min.

5. The preparation method according to claim 1, characterized in that, In step S1, when homogenizing, the mass ratio of pig liver to water is 1:1; the homogenization speed is 8000~10000 r / min, and the time is 5~8 min. The denaturation temperature is 90~100℃, and the time is 1~2h.

6. The preparation method according to claim 1, characterized in that, In step S2, the ultrafiltration process is as follows: the enzymatic hydrolysate is filtered through an ultrafiltration membrane at 25±5℃ to retain components with a molecular weight >3000D, resulting in a porcine liver peptide solution with a solid content of 5%±2%.

7. The preparation method according to claim 1, characterized in that, In step S2, the concentration process is as follows: the porcine liver peptide solution is concentrated using an ultrafiltration membrane with a molecular weight cutoff of 300-1000D to obtain a concentrated peptide solution with a solid content of ≥20%.

8. The preparation method according to claim 1, characterized in that, In step S4, during spray drying, the inlet air temperature is 200±10℃ and the outlet air temperature is 90±5℃.

9. Porcine liver peptide obtained by the preparation method according to any one of claims 1-8.

10. The application of the preparation method according to any one of claims 1-8, wherein the application comprises at least one of the following (1)-(3): (1) Enhance the activity of porcine liver peptides; (2) Improve the utilization rate of porcine liver peptides; (3) Reduce the fishy smell of pig liver peptides.

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