Preparation method of endothelium corneum gigeriae galli peptide with function of regulating spleen

Abstract

The invention relates to the field of food processing, and discloses a preparation method of endothelium corneum gigeriae galli peptide with a spleen and stomach regulating function, which comprises the following steps: step 1, raw material pretreatment: drying and crushing endothelium corneum gigeriae galli until the particle size is less than or equal to 100m, and soaking the endothelium corneum gigeriae galli in a 60% ethanol solution according to a liquid-solid ratio of 10: 1 for 2 hours for degreasing; step two, enzymatic hydrolysis reaction: a) preparing a substrate solution with the protein mass concentration of the endothelium corneum gigeriae galli being 15-25 mg / mL, adjusting the pH value to 3.0-3.5, and adding pepsase with the enzyme concentration being 1.0%-2.0% of the mass of the substrate; b) the pH value is adjusted to 6.5-7.0, trypsin and chymotrypsin are added, and the concentration of the trypsin is 1.0%-1.5% of the mass of the substrate; and c) heating to 85 DEG C, keeping the temperature for 10 minutes, and terminating the enzymolysis reaction. Through a multi-enzyme synergetic enzymolysis technology, enzymolysis conditions and an enzyme ratio are dynamically adjusted, and the generation efficiency of the small molecule peptide is remarkably improved. The obtained endothelium corneum gigeriae galli peptide is easy to absorb, has the effects of regulating spleen and stomach, promoting digestion and improving gastrointestinal functions, can be used as a functional food raw material, and is used for prevention and adjuvant treatment of spleen and stomach related diseases.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, in particular to a method for preparing chicken gizzard peptide with spleen and stomach regulating function. Background Art

[0002] With the rapid development of the functional food and health care product market, small molecule peptides have attracted widespread attention because they are easily absorbed by the human body and have multiple biological activities. As a traditional food and medicine material, chicken gizzard lining is rich in protein, peptides and multiple biologically active ingredients, and is an important raw material for the preparation of functional small molecule peptides. Through appropriate processing technology, chicken gizzard lining protein can be used to prepare small molecule peptides with a specific molecular weight range, which can significantly improve its effect of regulating spleen and stomach function, while expanding its application in the field of functional foods and health care products.

[0003] In the prior art, the preparation of chicken gizzard peptides mostly adopts a single enzymatic hydrolysis method or an enzymatic hydrolysis process under fixed conditions. In these processes, the use of a single protease often fails to fully cut different peptide chains in the protein, resulting in insufficient enzymatic hydrolysis and low production of target small molecule peptides. In addition, separation and purification usually uses a single ultrafiltration membrane to separate large molecular proteins from small molecular peptides, but this method lacks precise control over the target molecular weight range, resulting in high impurity content and low purity of the target peptide, further affecting the quality and application value of the final product.

[0004] However, the problems with existing technologies are mainly reflected in the limitations of the enzymatic hydrolysis process. Due to the lack of dynamic regulation and multi-enzyme synergy, the degradation efficiency of proteins under single enzymatic hydrolysis conditions is low, and the enrichment rate of target small molecule peptides cannot meet the requirements of high-quality products. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for preparing chicken gizzard peptide with spleen and stomach regulating function, which solves the problem that the existing preparation method lacks dynamic regulation and multi-enzyme synergistic enzymatic hydrolysis technical solutions, and the protein degradation efficiency is low under single enzymatic hydrolysis conditions.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for preparing chicken gizzard peptide with spleen and stomach regulating function, comprising the following steps: Step 1, raw material pretreatment: dry and crush the chicken gizzard lining to a particle size of ≤100 μm, soak in 60% ethanol solution at a liquid-to-solid ratio of 10:1 for 2 hours for degreasing, filter and dry for standby use; Step 2: Enzymatic reaction: a) Prepare substrate solution with a protein concentration of 15-25 mg / mL, adjust the pH to 3.0-3.5, add pepsin with an enzyme concentration of 1.0%-2.0% of the substrate mass, and react at 40-45°C for 30-40 minutes; b) adjusting the pH value to 6.5-7.0, adding trypsin and chymotrypsin, the concentration of trypsin is 1.0%-1.5% of the substrate mass, and the concentration of chymotrypsin is 0.5%-1.0% of the substrate mass, and continuing the reaction at 45-50°C for 60-90 minutes; c) Heat to 85°C and keep warm for 10 minutes to terminate the enzymatic reaction; Step 3, separation and purification: Use 1kDa ultrafiltration membrane to separate macromolecular proteins with a molecular weight greater than 1kDa, and retain small molecular peptides with a molecular weight in the range of 100-500Da; Step 4: Stabilization treatment: add 1.0%-3.0% glycine and 0.5%-1.5% tranexamic acid to the enzymatic hydrolysate, mix, and freeze-dry to obtain chicken gizzard peptide powder.

[0007] Preferably, the moisture content of the chicken gizzard lining powder after defatting is controlled at 6%-8%, the protein content is ≥85%, and the particle size range after crushing is 80-100 .

[0008] Preferably, the enzymatic hydrolysis conditions of pepsin in step 2 are: Enzyme activity is 500-800U / mg; The pH value of the substrate solution was adjusted to 3.2-3.4; The enzymatic hydrolysis temperature is 42-44°C; The enzymatic hydrolysis time is 35-40 minutes.

[0009] Preferably, the enzymatic hydrolysis conditions of trypsin and chymotrypsin in step 2 are: Trypsin activity is 2000-3000U / mg; Chymotrypsin activity is 800-1500U / mg; The enzymatic hydrolysis temperature is 45-48°C; The enzymatic hydrolysis time is 70-90 minutes; The pH value adjustment range is 6.7-6.9.

[0010] Preferably, when heating to terminate the enzymatic hydrolysis reaction in step 2, the heating temperature is 80-90° C. and the insulation time is 8-12 minutes.

[0011] Preferably, the pore size of the 1 kDa ultrafiltration membrane used in step 3 is 1.0±0.1 kDa, the pressure range during filtration is 0.1-0.3 MPa, and the filtration temperature is controlled at 25-35°C.

[0012] Preferably, the ratios of glycine and tranexamic acid added in step 4 are 2.0%-2.5% and 0.8%-1.2% respectively, and the mixture is treated under freeze-drying conditions at -50°C to -60°C, with a vacuum degree of 0.1-0.3 mbar, and the moisture content of the final product is controlled at ≤4%.

[0013] Preferably, the molecular weight range of the small molecule peptide separated and purified in step three is 150-400 Da, the purity of the target peptide after purification is ≥85%, and the recovery rate of the target peptide is ≥75%.

[0014] Preferably, in the enzymatic hydrolysis reaction of step 2, the stirring speed is controlled at 150-200 rpm, and a magnetic stirring device is used for uniform mixing.

[0015] Preferably, after mixing glycine and tranexamic acid in step 4, the uniform mixing time is 20-30 minutes, and after freeze drying is completed, the mixture is packaged and stored under light-proof and sealed conditions.

[0016] The present invention provides a method for preparing chicken gizzard peptide with spleen and stomach regulating function. It has the following beneficial effects: 1. The present invention adopts multi-enzyme synergistic enzymolysis technology, dynamically adjusts the enzymolysis conditions and reasonably controls the ratio and activity of pepsin, trypsin and chymotrypsin to achieve the technical effect of efficiently generating target small molecule peptides. Compared with the technical solutions of single enzymolysis or fixed enzymolysis in the prior art, it solves the problems of insufficient enzymolysis and low enrichment efficiency of target peptides.

[0017] 2. The present invention introduces multi-stage ultrafiltration membrane screening in the separation and purification stage, and achieves efficient enrichment of the target peptide molecular weight range by accurately controlling the series operation of 1kDa, 100Da and 500Da membranes. Compared with the technical solution of single ultrafiltration membrane separation in the prior art, it solves the shortcomings of low product purity and difficulty in removing non-target peptide impurities, and significantly improves the functionality and applicability of the final product.

[0018] 3. The molecular weight of the small molecule peptide prepared by the present invention is precisely controlled within the range of 100-500Da, and the bioavailability of the target peptide is significantly improved through optimized enzymatic hydrolysis and separation technology. Combined with in vitro gastrointestinal simulation experiments and functional verification tests, it is proved that the small molecule peptide has a strong effect in regulating gastrointestinal function. Compared with the non-specific molecular weight peptides prepared by the prior art, the peptide product of the present invention shows better gastrointestinal mucosal repair ability and digestive system regulation effect, solving the problem of unstable effect of existing products in functional applications.

[0019] 4. The present invention optimizes freeze-drying parameters, precisely controls drying temperature, vacuum degree and time, and retains the biological activity and structural integrity of the target peptide to the greatest extent. Compared with the technical solutions of spray drying or conventional drying methods in the prior art, the technical deficiencies of high moisture content, easy hygroscopicity and unstable physical properties of the product are solved, providing a more reliable foundation for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The present invention is a flow chart of the method. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Please refer to the attached Figure 1 The embodiment of the present invention provides a method for preparing chicken gizzard peptide having the function of regulating spleen and stomach, comprising the following steps: Step 1, raw material pretreatment: dry and crush the chicken gizzard lining to a particle size of ≤100 μm, soak in 60% ethanol solution at a liquid-to-solid ratio of 10:1 for 2 hours for degreasing, filter and dry for standby use; Step 2: Enzymatic reaction: a) Prepare substrate solution with a protein concentration of 15-25 mg / mL, adjust the pH to 3.0-3.5, add pepsin with an enzyme concentration of 1.0%-2.0% of the substrate mass, and react at 40-45°C for 30-40 minutes; b) adjusting the pH value to 6.5-7.0, adding trypsin and chymotrypsin, the concentration of trypsin is 1.0%-1.5% of the substrate mass, and the concentration of chymotrypsin is 0.5%-1.0% of the substrate mass, and continuing the reaction at 45-50°C for 60-90 minutes; c) Heat to 85°C and keep warm for 10 minutes to terminate the enzymatic reaction; Step 3, separation and purification: Use 1kDa ultrafiltration membrane to separate macromolecular proteins with a molecular weight greater than 1kDa, and retain small molecular peptides with a molecular weight in the range of 100-500Da; Step 4: Stabilization treatment: add 1.0%-3.0% glycine and 0.5%-1.5% tranexamic acid to the enzymatic hydrolysate, mix, and freeze-dry to obtain chicken gizzard peptide powder.

[0023] The moisture content of chicken gizzard powder is controlled at 6%-8% after defatting, the protein content is ≥85%, and the particle size range after crushing is 80-100 .

[0024] The enzymatic hydrolysis conditions of pepsin in step 2 are: Enzyme activity is 500-800U / mg; The pH value of the substrate solution was adjusted to 3.2-3.4; The enzymatic hydrolysis temperature is 42-44°C; The enzymatic hydrolysis time is 35-40 minutes.

[0025] The enzymatic hydrolysis conditions of trypsin and chymotrypsin in step 2 are: Trypsin activity is 2000-3000U / mg; Chymotrypsin activity is 800-1500U / mg; The enzymatic hydrolysis temperature is 45-48°C; The enzymatic hydrolysis time is 70-90 minutes; The pH value adjustment range is 6.7-6.9.

[0026] When heating to terminate the enzymatic hydrolysis reaction in step 2, the heating temperature is 80-90° C. and the insulation time is 8-12 minutes.

[0027] The pore size of the 1 kDa ultrafiltration membrane used in step 3 is 1.0 ± 0.1 kDa, the pressure range during filtration is 0.1-0.3 MPa, and the filtration temperature is controlled at 25-35°C.

[0028] In step 4, glycine and tranexamic acid are added in proportions of 2.0%-2.5% and 0.8%-1.2% respectively, and the mixture is freeze-dried at -50°C to -60°C with a vacuum degree of 0.1-0.3 mbar, and the moisture content of the final product is controlled at ≤4%.

[0029] The molecular weight range of the small molecule peptide separated and purified in step 3 is 150-400Da, the purity of the target peptide after purification is ≥85%, and the recovery rate of the target peptide is ≥75%.

[0030] In the enzymatic hydrolysis reaction of step 2, the stirring speed is controlled at 150-200 rpm, and a magnetic stirring device is used for uniform mixing.

[0031] After the glycine and tranexamic acid are mixed in step 4, the uniform mixing time is 20-30 minutes. After freeze drying, the mixture is packaged and stored in a light-proof and sealed condition.

[0032] In the present embodiment, the pretreatment of the gizzard lining of chicken's gizzard includes drying, crushing, defatting and drying. The raw material is first dried to remove excess moisture to ensure that it does not stick during the crushing process. Generally, the drying temperature is controlled at 40-60°C, and the time is adjusted to a final moisture content of 5%-10% according to the initial moisture content of the gizzard lining of chicken's gizzard.

[0033] During the crushing operation, a high-speed crusher is used to process the dried chicken gizzard into a particle size of ≤100 As an option, the particle size can be adjusted to 80-100 according to the specific requirements of the subsequent enzymatic hydrolysis process. Specifically, a particle size that is too large may reduce the contact efficiency between the enzyme and the substrate, while a particle size that is too small may lead to an increased risk of membrane fouling in the subsequent filtration step.

[0034] After crushing, in order to improve the protein purity of the raw material and remove lipid impurities that affect enzyme activity, defatting treatment is performed. Generally, a 60% ethanol solution is used for soaking at a liquid-to-solid ratio of 10:1, and the temperature is controlled at 20-25°C. The defatting time is generally 1.5-2.5 hours to ensure that the lipid substances are fully dissolved. In some embodiments, the soaking time can be appropriately extended to 3 hours according to the fat content of the raw material, but it should not be too long to avoid protein dissolution loss.

[0035] After defatting, the ethanol solution is separated from the solid by vacuum filtration. As an option, positive pressure filtration equipment can be used to further improve the filtration efficiency. The solid raw material obtained after filtration needs to be dried again, and the drying temperature is controlled at 40-60°C to avoid protein denaturation. Specifically, the drying time is adjusted according to the moisture content of the raw material, and the final moisture content is controlled at 6%-8%.

[0036] In a possible implementation, in order to further improve the enzymatic hydrolysis efficiency of the raw materials, a low concentration of acidic solution (such as 0.1%-0.3% citric acid) can also be added during the degreasing process to dissolve metal ions or other impurities attached to the surface of the protein. The use of acidic solution needs to ensure that the structural integrity of the protein is not destroyed, while avoiding excessive acidification that affects the pH adjustment of subsequent enzymatic hydrolysis.

[0037] Generally, after the above treatment, the protein content of the defatted chicken gizzard lining powder is increased to more than 85%, and the moisture content is controlled at 6%-8%. In some embodiments, in order to ensure the long-term storage performance of the raw materials, the defatted raw materials can also be stored in a nitrogen environment to prevent oxidation.

[0038] In the present embodiment, the pretreatment of the gizzard lining of chicken's gizzard includes drying, crushing, defatting and drying. The raw material is first dried to remove excess moisture to ensure that it does not stick during the crushing process. Generally, the drying temperature is controlled at 40-60°C, and the time is adjusted to a final moisture content of 5%-10% according to the initial moisture content of the gizzard lining of chicken's gizzard.

[0039] During the crushing operation, a high-speed crusher is used to process the dried chicken gizzard into a particle size of ≤100 As an option, the particle size can be adjusted to 80-100 according to the specific requirements of the subsequent enzymatic hydrolysis process. Specifically, a particle size that is too large may reduce the contact efficiency between the enzyme and the substrate, while a particle size that is too small may lead to an increased risk of membrane fouling in the subsequent filtration step.

[0040] After crushing, in order to improve the protein purity of the raw material and remove lipid impurities that affect enzyme activity, defatting treatment is performed. Generally, a 60% ethanol solution is used for soaking at a liquid-to-solid ratio of 10:1, and the temperature is controlled at 20-25°C. The defatting time is generally 1.5-2.5 hours to ensure that the lipid substances are fully dissolved. In some embodiments, the soaking time can be appropriately extended to 3 hours according to the fat content of the raw material, but it should not be too long to avoid protein dissolution loss.

[0041] After defatting, the ethanol solution is separated from the solid by vacuum filtration. As an option, positive pressure filtration equipment can be used to further improve the filtration efficiency. The solid raw material obtained after filtration needs to be dried again, and the drying temperature is controlled at 40-60°C to avoid protein denaturation. Specifically, the drying time is adjusted according to the moisture content of the raw material, and the final moisture content is controlled at 6%-8%.

[0042] In a possible implementation, in order to further improve the enzymatic hydrolysis efficiency of the raw materials, a low concentration of acidic solution (such as 0.1%-0.3% citric acid) can also be added during the degreasing process to dissolve metal ions or other impurities attached to the surface of the protein. The use of acidic solution needs to ensure that the structural integrity of the protein is not destroyed, while avoiding excessive acidification that affects the pH adjustment of subsequent enzymatic hydrolysis.

[0043] Generally, after the above treatment, the protein content of the defatted chicken gizzard lining powder is increased to more than 85%, and the moisture content is controlled at 6%-8%. In some embodiments, in order to ensure the long-term storage performance of the raw materials, the defatted raw materials can also be stored in a nitrogen environment to prevent oxidation.

[0044] In this embodiment, the solution after the enzymatic hydrolysis reaction is first subjected to coarse filtration to remove insoluble particles and impurities. Generally, a filter cloth or a metal screen can be used for preliminary filtration, and the filter pore size is controlled to be 50-100 To prevent solid particles from clogging the subsequent ultrafiltration membrane. In one possible implementation, a pre-filtration unit, such as 0.45 The filter membrane can further remove suspended impurities and ensure the permeability of the solution.

[0045] The solution after coarse filtration is adjusted and enters the ultrafiltration system. Specifically, the pore size of the ultrafiltration membrane is 1kDa, which is used to separate macromolecular proteins and peptides larger than 1kDa and retain components with a molecular weight less than 1kDa. As an option, the operating pressure of the ultrafiltration system can be set between 0.1-0.3MPa to avoid excessive pressure causing membrane damage or reduced filtration efficiency. The filtration temperature is controlled at 25-35℃ to take into account the tolerance of the membrane material and the stability of the enzymatic hydrolysis product.

[0046] In some embodiments, to further improve the filtration efficiency, a dynamic ultrafiltration mode can be used to prevent the accumulation of membrane surface contamination by periodically adjusting the pressure or flow rate. Specifically, reverse cleaning or pulse operation can be performed regularly during the filtration process to extend the service life of the membrane and maintain a high separation performance.

[0047] The permeate after ultrafiltration contains the target small molecule peptide and a small amount of impurities. In order to further ensure the purity of the target molecular weight range (100-500Da), molecular weight screening is required. In general, graded filtration can be used to accurately separate by connecting ultrafiltration membranes of different pore sizes in series. As a possible implementation method, a 500Da ultrafiltration membrane is first used to retain components with a molecular weight lower than 500Da, and then a 100Da ultrafiltration membrane is used to remove small molecule impurities lower than 100Da, thereby obtaining a target peptide with a more concentrated molecular weight distribution.

[0048] Specifically, in order to avoid degradation of the product during the separation process, the solution needs to be operated under low temperature conditions. In a possible improvement, a cooling circulation system can be combined to control the solution temperature at 10-20°C to further protect the functional structure of the target peptide.

[0049] After ultrafiltration separation, the permeate needs to be further concentrated. Generally, a vacuum concentration device can be used to reduce the volume of the solution to 1 / 3-1 / 5 of the original volume to increase the concentration of the target product. As an option, the operating pressure of the vacuum concentration is controlled at 0.08-0.1MPa and the temperature does not exceed 50°C to prevent the product from being inactivated due to high temperature or excessive operation time.

[0050] In some embodiments, in order to reduce the loss of target peptides, the concentrated solution can be selectively purified by adsorption resin. The selection of resin needs to be screened in combination with the physicochemical properties of the peptides. For example, weak cation exchange resins are suitable for separating small molecule peptides with positive charges. In one possible implementation, non-target components are further removed by gradient elution, retaining target peptides with molecular weight ranges that meet the requirements.

[0051] In the present embodiment, the stabilization treatment first includes adding a stabilizer to the purified small molecule peptide solution and mixing it evenly. Generally, the selection of the stabilizer needs to comprehensively consider the physicochemical properties and stability requirements of the target peptide. In the present invention, the stabilizer is mainly glycine and tranexamic acid, and the addition ratios of the two are 1.0%-3.0% and 0.5%-1.5%, respectively. As a choice, the addition ratio of glycine can be controlled at about 2.0%, and the ratio of tranexamic acid can be controlled at about 1.0%, so as to take into account both antioxidant and anti-degradation capabilities.

[0052] Specifically, glycine can effectively reduce the risk of peptide oxidation by forming hydrogen bonds with peptide molecules, and at the same time plays a certain buffering role in the solution; while tranexamic acid, as a protease inhibitor, can reduce the further decomposition of the target peptide by residual proteases. In a possible implementation, the stabilizer can be added twice, first adding glycine and stirring evenly before adding tranexamic acid to ensure that the two components are more evenly distributed in the solution.

[0053] The solution stirring operation can be carried out using a magnetic stirring device, and the stirring speed is generally controlled at 150-200 rpm. The stirring time is usually 20-30 minutes, and the specific time can be adjusted appropriately according to the viscosity of the solution. In some embodiments, in order to avoid the solution temperature rising due to long-term stirring, the solution temperature can be controlled by a circulating cooling device during the stirring process, and the temperature range can be set at 15-20°C.

[0054] The small molecule peptide solution after uniform mixing needs to be subjected to low-temperature drying treatment. In this embodiment, freeze-drying technology is used to protect the activity and structural integrity of the target peptide to the greatest extent. Specifically, the solution is first frozen at -50°C to -60°C until completely solidified, and then heated to 10-20°C under vacuum conditions for sublimation drying. As a possible option, the vacuum degree of the freeze-drying system can be controlled within the range of 0.1-0.3mbar to ensure that the water is fully removed while avoiding heat damage to the product.

[0055] Generally, the total time of the freeze drying process is 24-48 hours, and the specific time depends on the initial volume and solid content of the solution. In some embodiments, in order to speed up the drying process, a multi-stage temperature control mode can be used, for example, first freezing at -50°C for 12 hours, then vacuum drying at -40°C for 12 hours, and finally completing sublimation drying at -20°C.

[0056] In a possible implementation, in order to improve the uniformity and antioxidant properties of the product, a small amount of maltose or lactose can be added before freeze drying as a protective excipient, and the amount added can be controlled at 0.5%-1.0%. This excipient not only helps to improve the fluidity of the powder particles, but also further enhances the stability of the product during storage.

[0057] After freeze drying, the powder is finely sieved, and the mesh size is generally set to 80-100 mesh to remove incompletely dried agglomerates and improve the particle uniformity of the powder. As an option, the sieved powder can be placed in a vacuum sealed environment for short-term buffer storage to avoid direct exposure to air and moisture absorption.

[0058] The moisture content of the final product is usually controlled at ≤4% to ensure that the powder does not clump or deteriorate during storage. In some embodiments, to further increase the shelf life of the product, the powder can be packaged in a nitrogen-filled sealed bag and stored in a refrigerated environment at 4-10°C.

[0059] Example 1 In this embodiment, a small molecule peptide powder of Gallus gallus domesticus was prepared, and the specific steps and process parameters were as follows.

[0060] First, take the raw material of chicken gizzard lining and place it in a drying oven at 45°C for 8 hours to reduce the moisture content to 7%. Then, place the dried raw material in a pulverizer and grind it to control the powder particle size to ≤100 The powder was soaked in 60% ethanol solution at a liquid-solid ratio of 10:1 for 2 hours at a soaking temperature of 25°C. After filtration, defatted protein powder was obtained and dried for later use.

[0061] Next, the defatted protein powder was dissolved in deionized water at a concentration of 20 mg / mL, the pH was adjusted to 3.2, and pepsin was added at an enzyme concentration of 1.5% of the substrate mass. The solution was reacted at 42°C for 35 minutes, and the reaction conditions were controlled by constant temperature stirring in a water bath. Subsequently, the pH of the solution was adjusted to 6.8, and trypsin and chymotrypsin were added at 1.2% and 0.8% of the substrate mass, respectively. The temperature was raised to 47°C and the reaction was continued for 80 minutes.

[0062] After the enzymatic hydrolysis was completed, the reaction solution was heated to 85°C and kept warm for 10 minutes to terminate the enzymatic hydrolysis reaction. After cooling to room temperature, the reaction solution was first passed through 0.45 The membrane was pre-filtered and then separated using a 1 kDa ultrafiltration membrane. The filtration pressure was controlled at 0.2 MPa and the temperature was 30°C, and the permeate was collected. Subsequently, the 100 Da and 500 Da ultrafiltration membranes were connected in series for graded filtration to obtain a small molecule peptide solution with a molecular weight range of 100-500 Da.

[0063] Add glycine (2.0% of the mass of the solution) and tranexamic acid (1.0%) to the above permeation solution, stir evenly, and stir for 25 minutes. Place the mixed solution in a freeze dryer, set the temperature to -55°C, control the vacuum degree at 0.2 mbar, and dry for 36 hours. After freeze drying, a chicken gizzard lining small molecule peptide powder with a moisture content of 3.8% is obtained.

[0064] Example 2 This example adopts slightly adjusted process conditions to prepare Gallus gallus domesticus small molecule peptide powder, and the specific operation is as follows.

[0065] After drying at 50℃ for 6 hours, the water content of the raw material of chicken gizzard lining was reduced to 6%. The dried chicken gizzard lining was crushed to a particle size of 80 , then soaked in 60% ethanol solution at a liquid-to-solid ratio of 9:1 for 1.5 hours at a soaking temperature of 22°C, defatted and filtered to obtain defatted powder with a protein content of 86%.

[0066] The defatted powder was dissolved in deionized water to prepare a solution with a substrate concentration of 18 mg / mL, and the pH was adjusted to 3.0. Pepsin (enzyme concentration was 1.2% of the substrate mass) was added, the reaction temperature was 40°C, and the reaction time was 30 minutes. Subsequently, the pH was adjusted to 7.0, and trypsin and chymotrypsin were added in amounts of 1.0% and 0.5%, respectively. The reaction temperature was raised to 45°C, and the reaction time was 70 minutes.

[0067] When the enzymatic hydrolysis operation is terminated, the reaction solution is heated to 80°C for 8 minutes. After preliminary filtration with the filter membrane, purification was performed with a 1 kDa ultrafiltration membrane. The filtration pressure was controlled at 0.1 MPa and the filtration temperature was 25°C. The permeate was then concentrated to 1 / 5 of the original volume by a vacuum concentrator.

[0068] Glycine (addition amount is 1.5%) and tranexamic acid (addition amount is 0.8%) were added to the concentrate, and the stirring time was 20 minutes. The solution was placed in a freeze dryer, the freezing temperature was -50°C, the drying temperature was -20°C, and the drying time was 48 hours. After drying, a chicken gizzard lining small molecule peptide powder with a moisture content of 3.6% was obtained.

[0069] Example 3 In this example, the process was optimized by adjusting the temperature and enzymatic hydrolysis time to prepare the small molecule peptide from Gallus gallus domesticus, and the operation was as follows.

[0070] The raw material of chicken gizzard lining was dried at 45℃ for 10 hours until the moisture content was 7%. After crushing, the particle size was 90 The protein content of the defatted powder obtained by soaking in 60% ethanol solution at a liquid-to-solid ratio of 11:1 at 20°C for 2.5 hours and filtering was 87%.

[0071] The defatted powder was dissolved in deionized water at a concentration of 25 mg / mL, the pH of the solution was adjusted to 3.5, and pepsin was added at an enzyme concentration of 2.0%. The reaction temperature was 43°C and the reaction time was 40 minutes. The pH was then adjusted to 6.5, and trypsin and chymosin were added at 1.5% and 1.0%, respectively. The reaction temperature was 48°C and the reaction time was 90 minutes.

[0072] The reaction solution was heated to 90°C and kept warm for 12 minutes to complete the enzyme inactivation operation. After cooling, the reaction solution was separated by a 1kDa ultrafiltration membrane at a pressure of 0.3MPa and a filtration temperature of 32°C. The permeate was separated in series by 100Da and 500Da ultrafiltration membranes to obtain a small molecule peptide solution in the target molecular weight range.

[0073] Glycine and tranexamic acid were added in proportions of 2.5% and 1.2%, respectively, and the stirring time was 30 minutes. The freeze-drying conditions were -60°C, the vacuum degree was 0.1 mbar, and the total drying time was 30 hours. Finally, a chicken gizzard lining small molecule peptide powder with a water content of 3.2% was obtained.

[0074] Example 4 In this example, the purity of small molecule peptides was improved by further adjusting the enzymatic hydrolysis and separation parameters.

[0075] The particle size of chicken gizzard lining after crushing is 85 During degreasing, the ethanol concentration is 55%, the liquid-to-solid ratio is 10:1, the soaking time is 2 hours, and the degreasing temperature is 23°C. The protein content after filtration is 85%, and then it is dried for use.

[0076] Dissolve the defatted powder to prepare a substrate solution at a concentration of 22 mg / mL. Adjust the pH to 3.3, add pepsin (1.8%), and react at 44°C for 35 minutes. Then, adjust the solution pH to 6.7, add trypsin (1.2%) and chymotrypsin (0.7%), and react at 46°C for 75 minutes.

[0077] After the enzymatic hydrolysis was completed, the solution was heated to 85°C, kept warm for 10 minutes, then cooled to room temperature and purified using a 1kDa ultrafiltration membrane. The filtration temperature was controlled at 30°C and the pressure was 0.2MPa. The permeate was then separated by a 500Da ultrafiltration membrane, and the purity of the target peptide was increased to 87%.

[0078] Add 2.0% glycine and 1.0% tranexamic acid, stir evenly for 25 minutes. Freeze-drying conditions are -55°C, vacuum degree is 0.2 mbar, and drying time is 36 hours. The final moisture content is 3.5%.

[0079] Example 5 In this example, freeze-drying parameters were adjusted to improve the fluidity of the powder.

[0080] The defatted powder was prepared into a solution at a concentration of 20 mg / mL, the pH was adjusted to 3.2, 1.5% pepsin was added, the reaction temperature was 42°C, and the reaction time was 30 minutes. The pH was then adjusted to 7.0, and trypsin and chymotrypsin were added at a ratio of 1.2% and 0.8%, respectively. The reaction temperature was 47°C, and the reaction time was 80 minutes.

[0081] The reaction solution was heated to 85°C, kept warm for 10 minutes, filtered with a 1 kDa ultrafiltration membrane, and then glycine (1.8%) and tranexamic acid (1.0%) were added. The freeze-drying condition was -60°C, and the drying time was 40 hours to obtain a powder with a water content of 3.4%.

[0082] Comparative Example 1 This comparative example is based on Example 1, except that the amount of pepsin added and the reaction conditions during the enzymatic hydrolysis were adjusted, and the remaining steps were the same as in Example 1.

[0083] The specific implementation process is as follows: First, grind the chicken gizzard into particles ≤100 After that, it was degreased with 60% ethanol solution at 25°C for 2 hours, filtered and dried for later use. The defatted powder was dissolved in deionized water, the substrate concentration was 20 mg / mL, the pH was adjusted to 3.5, and pepsin was added at an enzyme amount of 0.5% of the substrate mass. The solution was reacted at 40°C for 20 minutes, and the other parameters remained unchanged.

[0084] Subsequently, the pH of the solution was adjusted to 6.8, and trypsin and chymotrypsin were added in amounts of 1.2% and 0.8%, respectively. The reaction temperature was 47°C and the reaction time was 80 minutes. When the enzymolysis was terminated, the solution was heated to 85°C and kept warm for 10 minutes. The subsequent separation and purification steps were the same as in Example 1.

[0085] Comparative Example 2 This comparative example is based on Example 2, except that the filtration conditions in the ultrafiltration separation step are adjusted, and the remaining steps are the same as in Example 2.

[0086] The specific implementation process is as follows: the enzymatic hydrolysate is prepared according to Example 2. During the separation and purification process, a 5kDa ultrafiltration membrane is used instead of a 1kDa ultrafiltration membrane for separation. The filtration pressure is controlled at 0.1MPa and the filtration temperature is 25°C. No fractional purification of the molecular weight range is performed, and the permeate is directly collected. The addition ratios of glycine and tranexamic acid to the permeate are 1.5% and 0.8%, respectively, and the mixture is stirred evenly and then freeze-dried. The freeze-drying parameters are the same as those in Example 2.

[0087] Comparative Example 3 This comparative example is based on Example 3, except that the freeze-drying step is changed to spray drying, and the remaining steps are the same as Example 3.

[0088] The specific implementation process is as follows: After completing the enzymatic hydrolysis and separation and purification steps according to Example 3, glycine (2.0%) and tranexamic acid (1.0%) are directly added after obtaining the target peptide solution, and stirred evenly for 20 minutes. The stirred solution is placed in a spray drying device, and the inlet air temperature of the spray drying is 120°C, the outlet air temperature is 80°C, and the atomization pressure is 0.2MPa to obtain a dry powder.

[0089] Comparative Example 4 This comparative example is based on Example 4, except that the molecular weight range selectivity step is omitted and the remaining steps are the same as those of Example 4.

[0090] The specific implementation process is as follows: The defatting and enzymolysis steps were completed according to Example 4. After the enzymolysis reaction was completed, the reaction solution was directly separated using a 1kDa ultrafiltration membrane, and 100Da and 500Da ultrafiltration membranes were not used for graded filtration. The permeate was directly concentrated to 1 / 5 of the original volume, and glycine and tranexamic acid were added in proportions of 2.0% and 1.0%, respectively. The freeze-drying conditions were the same as in Example 4.

[0091] Comparative Example 5 This comparative example is based on Example 5, except that glycine and tranexamic acid are removed during the stabilization treatment, and the remaining steps are the same as those of Example 5.

[0092] The specific implementation process is as follows: the defatting, enzymatic hydrolysis and separation and purification steps are completed according to Example 5, and the obtained small molecule peptide solution is directly freeze-dried. The freeze-drying condition is -60°C, the drying time is 40 hours, and a powder product is finally obtained. No stabilizer is added, and other parameters are the same as in Example 5.

[0093] Comparative Example 6 This comparative example is based on Example 1, except that the pH adjustment range of the enzymatic hydrolysis is changed to a fixed value, and the remaining steps are the same as those of Example 1.

[0094] The specific implementation process is as follows: the defatting step is completed according to Example 1, and the defatted powder is dissolved in deionized water, and the substrate concentration is 20 mg / mL. The pH of the solution is adjusted to 3.0, and pepsin (addition amount 1.5%) is added. The reaction temperature is 42°C and the reaction time is 35 minutes. The pH value is not dynamically adjusted, and trypsin (1.2%) and chymosin (0.8%) are directly added, and the reaction is continued for 80 minutes at pH 3.0. The remaining steps are the same as in Example 1.

[0095] Comparative Example 7 This comparative example is based on Example 2, except that the filtration temperature in the separation and purification is adjusted, and the remaining steps are the same as in Example 2.

[0096] The specific implementation process is as follows: the enzymatic hydrolysate is prepared according to Example 2. During the ultrafiltration separation process, the filtration temperature is increased from 25°C to 45°C, and the filtration pressure is maintained at 0.1 MPa. No cooling protection is performed, and the permeate is directly collected. The subsequent stabilization treatment and freeze-drying steps are the same as those in Example 2.

[0097] Experiment 1: Effect of enzyme concentration on the purity and recovery of small molecule peptide products Experimental steps: Sample preparation: Take chicken gizzard lining powder, soak it in 60% ethanol solution at a liquid-to-solid ratio of 10:1 at 25°C for 2 hours, defatted and dried for later use.

[0098] A substrate solution was prepared with a substrate concentration of 20 mg / mL. The pH of the solution was adjusted to 3.2 (Example 1) or 3.5 (Comparative Example 1) according to the process conditions of Example 1 and Comparative Example 1, respectively.

[0099] Pepsin was added, the amount of pepsin added was 1.5% in Example 1 and 0.5% in Comparative Example 1. The solutions were reacted at 42° C. (Example 1) and 40° C. (Comparative Example 1) for 35 minutes.

[0100] Post-enzymatic treatment: adjust the solution pH to 6.8, add trypsin and chymotrypsin, the addition ratio and conditions are consistent: trypsin 1.2%, chymotrypsin 0.8%, react at 47°C for 80 minutes.

[0101] Heat to 85°C and keep warm for 10 minutes to terminate the enzymatic reaction.

[0102] Separation and purification: The enzymatic hydrolysate was separated by a 1 kDa ultrafiltration membrane, the filtration pressure was set to 0.2 MPa, and the temperature was controlled at 30 °C.

[0103] The permeate was collected and graded and screened through 100Da and 500Da ultrafiltration membranes to obtain the target small molecule peptide solution.

[0104] Detection and analysis: HPLC was used to determine the proportion of 100-500Da target peptides in the sample as a purity indicator.

[0105] The total recovery of the peptide product was determined by the total mass weighing method, and the recovery was calculated as the percentage of the output mass to the substrate protein mass.

[0106] Table 1: Effects of different pepsin concentrations on the purity and recovery of small molecule peptides High concentration of pepsin obviously plays a key role in the initial enzymatic hydrolysis process. By cutting specific peptide chains in macromolecular proteins, the 1.5% enzyme addition significantly improved the initial fragmentation efficiency of the polypeptide chain. This optimized initial decomposition condition can provide a good foundation for subsequent multi-enzyme synergy, especially the production of small molecule peptides (100-500Da) has been greatly enhanced. Compared with the control example, the 0.5% enzyme addition failed to fully break the macromolecular structure, resulting in a significant decrease in the enrichment of the target peptide.

[0107] Further analysis of the results revealed that the enzyme concentration not only affects the purity of the peptide, but also has an effect on the overall recovery rate. High concentrations of pepsin ensure that more large molecular proteins are converted into small molecular peptides that can be screened, while under low concentration conditions, some undegraded protein residues are retained by the ultrafiltration membrane during the separation process, resulting in a decrease in the recovery rate. In fact, this loss caused by incomplete protein decomposition directly affects the industrial preparation value of the product.

[0108] Combining experimental data and mechanism analysis, an obvious conclusion can be drawn: Properly increasing the concentration of pepsin is a scientific strategy to achieve efficient protein degradation and target peptide production. This strategy not only increases the proportion of products that meet the standard molecular weight range, but also reduces the waste of unreacted substrates, providing a sufficient basic guarantee for subsequent separation and drying processes.

[0109] Experiment 2: Effect of molecular weight screening on the distribution range of target peptides Experimental steps: Sample preparation: Take chicken gizzard powder, and perform defatting and enzymolysis treatment according to the methods of Example 4 and Comparative Example 4. The enzymolysis conditions of the two groups of experiments are the same: the concentration of the substrate solution is 22 mg / mL, the amount of pepsin added is 1.8%, and the reaction is carried out at 43°C for 35 minutes, followed by the addition ratios of trypsin and chymotrypsin of 1.2% and 0.7% respectively, the reaction temperature is 46°C, and the time is 75 minutes.

[0110] After the enzymatic hydrolysis was completed, the reaction solution was heated to 85°C, kept warm for 10 minutes, and cooled to room temperature.

[0111] Separation and screening: Example 4: After preliminary filtration using a 1 kDa ultrafiltration membrane, the permeate was passed through 500 Da and 100 Da ultrafiltration membranes in series for graded purification, and the target small molecule peptide solution was collected.

[0112] Comparative Example 4: Only 1 kDa ultrafiltration membrane was used for preliminary filtration without graded screening, and the permeate was directly collected.

[0113] Detection method: Gel permeation chromatography (GPC) was used to analyze the molecular weight distribution of the samples and to calculate the proportion of peptides in each molecular weight range.

[0114] The molecular weight ranges were divided into: less than 100 Da, 100-500 Da (target range) and greater than 500 Da. The effect of graded screening was evaluated by calculating the percentage of peptides in each range.

[0115] Table 2: Effect of molecular weight screening on the distribution range of small molecule peptides The effect of graded screening was very obvious in the experiment. By combining the series operation of 100Da and 500Da ultrafiltration membranes, Example 4 greatly increased the proportion of peptides in the target molecular weight range. Compared with the control example, the enrichment degree of the target range was increased by more than 20%. This refined screening strategy obviously makes the distribution of small molecule peptides more concentrated, and effectively reduces the interference of low molecular weight (<100Da) fragments and incompletely decomposed polypeptides (500Da). It can be seen that ultrafiltration classification has an important contribution to optimizing the functional ingredients of the product.

[0116] From a mechanistic analysis, the single 1kDa ultrafiltration membrane only completed the coarse screening function in the comparative example, and was unable to accurately separate substances in the non-target molecular weight range. The high residual ratio of large molecular peptides (500Da) indicates that the purity of the target peptide is more easily affected by impurities in the system that has not undergone graded screening. The increase in the proportion of small molecule fragments (<100Da) may also be due to the fact that some excessive degradation products were not separated in the ungraded screening.

[0117] The multi-stage separation strategy in the present invention is further verified by experiments to be scientific and necessary. This design not only significantly increases the proportion of target peptides in the 100-500Da range, but also reduces the interference of non-target components on downstream applications. The introduction of graded screening upgrades the process from simple separation to precise separation, allowing the entire system to be simultaneously optimized in terms of functionality and industrial feasibility.

[0118] Experiment 3: Effect of drying method on product stability Experimental steps: Sample preparation: Enzyme hydrolysates were prepared according to the processes of Example 3 and Comparative Example 3. The enzymatic hydrolysis conditions were substrate concentration 25 mg / mL, pepsin 2.0%, trypsin 1.5%, chymotrypsin 1.0%, reaction temperature 43°C (preliminary enzymatic hydrolysis) and 48°C (deep enzymatic hydrolysis), and enzymatic hydrolysis time was 40 minutes and 90 minutes, respectively.

[0119] After the enzymatic hydrolysis was completed, the solution was heated to 90°C and kept warm for 12 minutes to terminate the enzymatic hydrolysis reaction. The solution was then filtered using a 1 kDa ultrafiltration membrane and the permeate was collected to obtain the target small molecule peptide solution.

[0120] Drying treatment: Example 3: The target small molecule peptide solution was placed in a freeze dryer. The freezing temperature was set to -60°C, the sublimation temperature was controlled at 10-20°C, the vacuum degree was 0.1 mbar, and the drying time was 40 hours, and finally a dry powder was obtained.

[0121] Comparative Example 3: The target small molecule peptide solution was spray dried. The inlet air temperature was set to 120°C, the outlet air temperature was 80°C, and the atomization pressure was 0.2 MPa to obtain a spray dried powder.

[0122] Detection and analysis: Determine the initial moisture content and oxidation degree of the powder (with malondialdehyde content as an indicator).

[0123] The two groups of samples were stored in a refrigerated environment at 4°C for 30 days, and the changes in hygroscopicity and oxidation products were measured. Hygroscopicity was expressed as the percentage of powder weight gain, and oxidation products were expressed as the change in malondialdehyde concentration.

[0124] Table 3: Effect of drying method on initial properties and storage stability of powders Freeze drying obviously shows a significant advantage in maintaining a low moisture content of the powder. The final moisture content in Example 3 is significantly lower than that of the spray-dried product in Comparative Example 3, which is mainly attributed to the characteristics of low-temperature sublimation during the freeze drying process. Due to the high temperature environment of spray drying, it may be difficult to completely remove some residual moisture. At the same time, high temperature conditions can easily lead to slight degradation of peptide molecules, thereby increasing the initial concentration of oxidation products. Such differences have been fully reflected in the initial data.

[0125] Further observation of the changes during storage revealed that the drying method not only affects the initial properties of the powder, but also significantly affects its storage stability. After 30 days of refrigerated storage, the changes in hygroscopicity and oxidation products of the freeze-dried powder were significantly smaller, indicating that freeze-drying can more effectively protect the peptide molecular structure and reduce the impact of environmental humidity on the properties of the powder. However, due to the high residual moisture content of the spray-dried powder, the hygroscopicity increased significantly during storage, causing the powder to easily agglomerate, and the accumulation rate of oxidation products was also faster. This suggests that high-temperature drying conditions may induce more oxidation-sensitive sites in peptide molecules.

[0126] From a mechanistic point of view, freeze drying retains the original structure of the target peptide to the greatest extent by achieving water sublimation under low temperature and vacuum conditions, avoiding slight oxidation or cross-linking reactions between proteins and amino acids during high-temperature drying. At the same time, the powder generated by freeze drying has a looser particle structure, which reduces the contact area between its surface and moisture in the air. This physical property is the core reason why freeze-dried powder performs better in hygroscopicity and oxidative stability. For functional peptide products that require high purity and high stability, freeze drying is obviously more suitable as the final drying method.

[0127] Experiment 4: Effect of stabilizer addition on target peptide degradation Experimental steps: Sample preparation: The target small molecule peptide solutions were prepared according to the processes of Example 5 and Comparative Example 5. The enzymatic hydrolysis conditions were as follows: substrate concentration 20 mg / mL, pepsin addition amount 1.5%, trypsin and chymotrypsin addition amounts 1.2% and 0.8%, respectively, reaction temperature 42°C (preliminary enzymatic hydrolysis) and 47°C (deep enzymatic hydrolysis), time 30 minutes and 80 minutes, respectively.

[0128] After the enzymatic hydrolysis was completed, the mixture was heated to 85°C and kept warm for 10 minutes to terminate the enzymatic hydrolysis reaction. The permeate was separated and collected using a 1 kDa ultrafiltration membrane, and the permeate was directly used for subsequent treatment.

[0129] Example 5: Glycine (addition amount 2.0%) and tranexamic acid (addition amount 1.0%) were added to the permeation solution, stirred for 25 minutes, and freeze-dried after being homogenized.

[0130] Comparative Example 5: No stabilizer was added and freeze drying was performed directly. The drying conditions were the same as those in Example 5.

[0131] Simulated gastrointestinal digestion: prepare simulated gastric fluid (pH 2.0, 37°C) and simulated intestinal fluid (pH 7.4, 37°C).

[0132] The two groups of samples were dissolved in simulated gastric fluid at a concentration of 10 mg / mL, placed in a 37°C water bath and stirred, and samples were taken at regular intervals (0, 30, 60, and 120 minutes). After the samples were neutralized, the concentration of the residual target peptide was determined by HPLC.

[0133] Under the same operation, the samples were placed in simulated intestinal fluid to measure the changes in the concentration of residual target peptides over time.

[0134] Data analysis: Record the concentration of target peptide at different time points and analyze the residual ratio. Compare the degradation rate and residual stability of target peptide in the digestive environment with and without stabilizer.

[0135] Table 4: Residual ratio of target peptides in simulated gastric fluid and intestinal fluid (%) The addition of stabilizers obviously plays a significant role in alleviating the degradation of the target peptide in gastrointestinal digestion. The synergistic effect of glycine and tranexamic acid in Example 5 effectively inhibits the activity of the protease, thereby slowing down the degradation rate of the target peptide. In the simulated gastric fluid environment, the residual ratio of the target peptide in Example 5 is much higher than that in Comparative Example 5, which indicates that glycine may shield the sensitive sites on the peptide chain by forming protective hydrogen bonds with the active peptide, thereby reducing the hydrolysis efficiency of pepsin.

[0136] Experiments in the intestinal fluid environment also show that the protease inhibition of tranexamic acid is of great significance to the stability of the target peptide. Compared with Comparative Example 5 without adding a stabilizer, the residual ratio of the target peptide in Example 5 after 120 minutes is still close to 70%. This shows that tranexamic acid can further reduce the degradation of the target peptide by interfering with the binding of the enzyme to the substrate, thereby protecting its functionality. This protective effect is particularly significant in intestinal fluid.

[0137] From a mechanistic point of view, the synergistic effect of glycine and tranexamic acid not only enhances the chemical stability of the peptide molecule, but also inhibits the effect of the external environment on its degradation to a certain extent. The embodiment of this dual protection effect provides support for the long-term functional maintenance of the target peptide. In contrast, the lack of stabilizer protection in Comparative Example 5 leads to a significant increase in the degradation rate of the target peptide in the gastrointestinal environment, and its biological activity may be difficult to continue to the actual application stage. This difference not only verifies the necessity of the stabilizer, but also highlights the technical advantages of the present invention.

[0138] Experiment 5: Effect of filtration conditions on target peptide recovery Experimental steps: Sample preparation: Take chicken gizzard powder, and carry out raw material treatment and enzymolysis according to the process of Example 2 and Comparative Example 2. The enzymolysis conditions are substrate concentration 18 mg / mL, pepsin addition amount 1.2%, trypsin and chymotrypsin addition amounts 1.0% and 0.5%, respectively, reaction temperature 40°C (preliminary enzymolysis) and 45°C (deep enzymolysis), time 30 minutes and 70 minutes, respectively.

[0139] After the enzymatic hydrolysis was completed, the enzymatic hydrolysis reaction was terminated by heating at 85° C. for 10 minutes to obtain an enzymatic hydrolysis product solution.

[0140] Filtration operation: Example 2: Use 1 kDa ultrafiltration membrane for separation, control the filtration pressure at 0.2 MPa, maintain the temperature at 25° C., and collect the permeate. The permeate contains the target peptide with a molecular weight of less than 1 kDa.

[0141] Comparative Example 2: 5 kDa ultrafiltration membrane was used for separation, the filtration pressure was controlled at 0.1 MPa, the temperature was kept at 25°C, and the permeate was collected.

[0142] Detection and analysis: Determine the total peptide mass in the permeate (weighed in the form of dry powder) and calculate the total recovery rate (output mass / substrate protein mass × 100%).

[0143] High performance liquid chromatography (HPLC) was used to determine the proportion of target peptides (100-500 Da) in the permeate, and the purity of the target peptides and the actual recovery rate of the target peptides in the permeate (purity × total recovery rate) were calculated.

[0144] Table 5: Effect of different ultrafiltration conditions on the recovery of target peptides Different filtration conditions directly affect the recovery efficiency and purity of the target peptide. This difference reflects the key role of ultrafiltration membrane pore size selection in the separation process. In Example 2, the selection of 1kDa ultrafiltration membrane achieves effective retention of macromolecular impurities, while preferentially permeating 100-500Da target peptides, ultimately obtaining a higher purity and actual recovery rate. In Comparative Example 2, although the 5kDa ultrafiltration membrane improves the overall recovery rate, it also allows more non-target peptide components (>500Da) to enter the permeate, resulting in a significant decrease in the purity of the target peptide.

[0145] From a mechanistic point of view, the pore size of the 1kDa ultrafiltration membrane is set just near the upper molecular weight limit of the target peptide. This screening characteristic can accurately filter out large molecular impurities exceeding 1kDa while effectively retaining small molecular peptides. In contrast, the pore size of the 5kDa ultrafiltration membrane is too large to fully intercept high molecular peptides, resulting in impurities entering the permeate, which directly lowers the purity of the target peptide. Although its total recovery rate is high, the proportion of target peptides effectively recovered is lower than that in Example 2.

[0146] Different filtration conditions not only affect the purity of the product, but also have a potential impact on the downstream process links. The lower purity of the target peptide will increase the difficulty of subsequent grading and purification steps, thereby increasing production costs. The presence of high-molecular impurities may further precipitate or aggregate during storage, reducing the stability of the final product. The optimization of the 1kDa filtration conditions not only improves the enrichment efficiency of the target peptide, but also provides a better raw material basis for subsequent processing. Therefore, in the preparation process of functional small molecule peptides, the precise selection of the pore size of the ultrafiltration membrane is one of the core to achieve efficient separation, and its reasonable design can significantly improve the overall performance of the process.

[0147] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing chicken gizzard peptide having spleen and stomach regulating function, characterized in that: The following steps are involved: Step 1: Raw material pretreatment: Dry and crush the chicken gizzard lining to a particle size of ≤100 m, soak in 60% ethanol solution at a liquid-to-solid ratio of 10:1 for 2 hours for degreasing, filter and dry for later use; Step 2: Enzymatic reaction: a) preparing a substrate solution, wherein the protein mass concentration of chicken gizzard lining is 15-25 mg / mL, adjusting the pH value to 3.0-3.5, adding pepsin, the enzyme concentration is 1.0%-2.0% of the substrate mass, and reacting at 40-45° C. for 30-40 minutes; b) adjusting the pH value to 6.5-7.0, adding trypsin and chymotrypsin, the concentration of trypsin is 1.0%-1.5% of the substrate mass, and the concentration of chymotrypsin is 0.5%-1.0% of the substrate mass, and continuing the reaction at 45-50° C. for 60-90 minutes; c) Heat to 85°C and keep warm for 10 minutes to terminate the enzymatic reaction; Step 3, separation and purification: Use 1kDa ultrafiltration membrane to separate macromolecular proteins with a molecular weight greater than 1kDa, and retain small molecular peptides with a molecular weight in the range of 100-500Da; Step 4: Stabilization treatment: add 1.0%-3.0% glycine and 0.5%-1.5% tranexamic acid to the enzymatic hydrolysate, mix, and freeze-dry to obtain chicken gizzard peptide powder.

2. The method for preparing chicken gizzard peptide having spleen and stomach regulating function according to claim 1, characterized in that: The chicken gizzard lining powder has a moisture content of 6%-8% after defatting, a protein content of ≥85%, and a particle size range of 80-100 after crushing. .

3. The method for preparing chicken gizzard peptide having spleen and stomach regulating function according to claim 1, characterized in that: The enzymatic hydrolysis conditions of pepsin in step 2 are: Enzyme activity is 500-800U / mg; The pH value of the substrate solution was adjusted to 3.2-3.4; The enzymatic hydrolysis temperature is 42-44°C; The enzymatic hydrolysis time is 35-40 minutes.

4. The method for preparing chicken gizzard peptide having spleen and stomach regulating function according to claim 1, characterized in that: The enzymatic hydrolysis conditions of trypsin and chymotrypsin in step 2 are: Trypsin activity is 2000-3000U / mg; Chymotrypsin activity is 800-1500U / mg; The enzymatic hydrolysis temperature is 45-48°C; The enzymatic hydrolysis time is 70-90 minutes; The pH value adjustment range is 6.7-6.

9.

5. The method for preparing chicken gizzard peptide having spleen and stomach regulating function according to claim 1, characterized in that: When heating to terminate the enzymatic hydrolysis reaction in step 2, the heating temperature is 80-90° C. and the insulation time is 8-12 minutes.

6. The method for preparing chicken gizzard peptide having spleen and stomach regulating function according to claim 1, characterized in that: The pore size of the 1 kDa ultrafiltration membrane used in step 3 is 1.0±0.1 kDa, the pressure range during filtration is 0.1-0.3 MPa, and the filtration temperature is controlled at 25-35°C.

7. The method for preparing chicken gizzard peptide having spleen and stomach regulating function according to claim 1, characterized in that: In the step 4, the ratios of glycine and tranexamic acid added are 2.0%-2.5% and 0.8%-1.2% respectively, and the product is freeze-dried at -50°C to -60°C with a vacuum degree of 0.1-0.3 mbar, and the moisture content of the final product is controlled at ≤4%.

8. The method for preparing chicken gizzard peptide having spleen and stomach regulating function according to claim 1, characterized in that: The molecular weight range of the small molecule peptide separated and purified in step three is 150-400Da, the purity of the target peptide after purification is ≥85%, and the recovery rate of the target peptide is ≥75%.

9. The method for preparing chicken gizzard peptide having spleen and stomach regulating function according to claim 1, characterized in that: In the enzymatic hydrolysis reaction of step 2, the stirring speed is controlled at 150-200 rpm, and a magnetic stirring device is used for uniform mixing.

10. The method for preparing chicken gizzard peptide having spleen and stomach regulating function according to claim 1, characterized in that: After the glycine and tranexamic acid are mixed in the step 4, the uniform mixing time is 20-30 minutes, and after freeze drying, the mixture is packaged and stored in a light-proof and sealed condition.

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