Method for extracting cattle spleen peptide based on natural macromolecule-photosensitive synergistic system

Abstract

The invention belongs to the field of cosmetics and health care products, and provides a method for extracting cattle spleen peptide based on a natural macromolecule-photosensitive synergistic system, and the method comprises the following steps: step 1, grinding and sieving a cattle spleen raw material, and drying for later use; step 2, adding the cattle spleen powder obtained in the step 1 into a protease solution for enzymolysis; step 3, adding natural macromolecules and photosensitive components into the enzymolysis system to carry out illumination-assisted treatment; and 4, separating and purifying the product in the step 3 to obtain the cattle spleen peptide. According to the method disclosed by the invention, the extraction rate and the activity of the cattle spleen peptide can be remarkably improved under a mild condition, the molecular weight distribution of the product is concentrated at 1000-8000Da, and the biological activity is well kept. The obtained cattle spleen peptide is green and safe, is suitable for industrial production, and is widely applied to the fields of functional foods, health-care products and medical raw materials.

Description

Technical Field

[0001] This application belongs to the field of cosmetics and health products. Specifically, this application provides a method for extracting bovine spleen peptides based on a natural macromolecule-photosensitive synergistic system. Background Technology

[0002] Peptides, as functional factors with antioxidant, anti-inflammatory, and immunomodulatory activities, have significant application value in the food, pharmaceutical, and cosmetic fields. However, existing extraction processes generally suffer from significant technical bottlenecks: First, traditional acid-base hydrolysis methods require extreme conditions of strong acid / base, which easily leads to peptide structure destruction and activity loss; second, single enzymatic hydrolysis processes suffer from low reaction efficiency and a wide molecular weight distribution of products, requiring multi-step separation and purification operations, resulting in high energy consumption and increased costs; more seriously, the wastewater treatment problem caused by chemical reagent residues has become an environmental pain point restricting the green development of the industry. Although natural macromolecules (such as cyclodextrins and steviol glycosides) have shown application potential in the field of bioactive substance delivery due to their cavity inclusion, interfacial stability, and molecular recognition characteristics, current technologies have not yet constructed an integrated extraction system based on the synergistic effect of natural macromolecules—in particular, there is a lack of innovative solutions that combine cyclodextrin-like inclusion agents, steviol glycoside-like natural surfactants, and alkaline proteolytic hydrolysis technology for auxiliary extraction. Summary of the Invention

[0003] Against this backdrop, this invention further introduces photosensitive small molecules (such as riboflavin, chlorophyll derivatives, methylene blue, indocyanine green, etc.), utilizing their efficient absorption capabilities for specific wavelengths of light (blue light 300–500 nm, red light 600–700 nm) to achieve energy transfer of light energy to the system or reactive oxygen species (ROS). 1 O2、·O2 - The process generates ·OH groups. Under mild conditions, this promotes the relaxation of protein secondary structures and the dissociation of non-covalent forces, exposing more cleavage sites. Simultaneously, the inclusion and stabilizing effects of natural macromolecules effectively prevent secondary polymerization or inactivation of small peptides. The resulting "natural macromolecule-photosensitive synergistic system" not only significantly improves the extraction efficiency and product stability of bovine spleen peptides but also possesses advantages such as being green, low-energy, and highly selective. Therefore, this invention, through the synergistic effect of natural macromolecules (cyclodextrin, steviol glycosides, etc.) and photosensitive small molecules, combined with alkaline enzymatic hydrolysis, enhances peptide extraction rate and bioactivity under mild conditions, overcoming the shortcomings of existing technologies. This invention has significant scientific value and broad application prospects, providing new ideas and technical support for the sustainable development of peptide-related industries.

[0004] On the one hand, this application provides a method for extracting bovine spleen peptides based on a natural macromolecule-photosensitive synergistic system, characterized in that the method includes:

[0005] Step 1: grind the bovine spleen raw material into powder and sieve, and dry for standby;

[0006] Step 2: add the bovine spleen powder obtained in step 1 to the protease solution for enzymolysis;

[0007] Step 3: add natural macromolecules and photosensitive components to the enzymolysis system for light-assisted treatment;

[0008] Step 4: separate and purify the product of step 3 to obtain bovine spleen peptides.

[0009] Further, the protease is trypsin and / or papain.

[0010] Further, the protease enzyme activity of the protease solution is 3000-10000 U / mL, the enzymolysis time is 60-90 minutes, the pH of the enzymolysis system is 7-8, and the enzymolysis temperature is 45-55℃.

[0011] Further, the natural macromolecule is a polysaccharide.

[0012] Further, the polysaccharide is selected from cyclodextrin and / or chitosan.

[0013] Further, the photosensitive component is riboflavin, sodium chlorophyll salt and / or methylene blue.

[0014] Further, the amount of natural macromolecule added in step 3 is 1-2 w / v%, and the amount of photosensitive component added is 0.03-0.07 w / v%.

[0015] Further, the light-assisted treatment time in step 3 is 30-45 minutes, and the light-assisted treatment temperature is the same as the enzymolysis temperature in step 2.

[0016] Further, step 4 includes one or more steps of dialysis, ultrafiltration, concentration and drying.

[0017] The membrane in dialysis and ultrafiltration can be selected according to product needs, such as but not limited to 500Da to 10,000Da, 1000-3000Da, etc., and the drying and concentration methods can be selected from various methods known in the art, including but not limited to rotary evaporation, lyophilization, hot air drying, spray drying, etc.

[0018] In another aspect, the present application provides a bovine spleen peptide product prepared by the above method.

[0019] Compared with the prior art, the method has the following advantages and technical effects:

[0020] The application provides a kind of natural macromolecule-photosensitive synergistic system based on efficient extraction method of bovine spleen peptide, by introducing cyclodextrin and other natural macromolecules, significantly improve the yield of polypeptide under mild conditions, better than traditional acid-base hydrolysis or single enzymatic method.

[0021] The application provides a method for converting protein into bovine spleen peptide, by regulating the type and concentration of natural macromolecules, photosensitive conditions and extraction temperature, obtaining polypeptide product with molecular weight concentrated in 500-8000 Da, with good solubility and bioavailability.

[0022] The application provides a kind of green and efficient extraction method of bovine spleen peptide, under the action of mild light and natural macromolecules, reducing excessive degradation of protein, protecting the structural integrity of target polypeptide, while reducing reaction temperature and energy consumption, suitable for various animal protein raw materials.

[0023] The application provides a kind of bovine spleen peptide extraction method, natural macromolecules have good embedding, stabilizing and solubilizing effect, can effectively prevent polypeptide aggregation and precipitation, improve product recovery rate and stability, and have certain antioxidant or anti-inflammatory auxiliary function. The application provides a kind of simple and green bovine spleen peptide extraction process, without using strong acid, strong base or organic solvent, avoiding environmental pollution and equipment corrosion problem, meeting the green chemistry concept, easy to realize industrialized production. DETAILED DESCRIPTION

[0024] The following examples facilitate better understanding of the application, but are not limited thereto, and these examples are only for illustrative purposes, and do not limit the protection scope of the application.

[0025] Example 1 natural macromolecule-photosensitive synergistic system extraction of bovine spleen peptide

[0026] Accurately weigh 10 g of bovine spleen powder sieved through 40 mesh, add trypsin solution (5000 U / mL) at a solid-liquid ratio of 1:30 under the condition of pH 7.5 and 50℃, and carry out preliminary enzymatic reaction for 60 minutes. Then, 1 w / v% β-cyclodextrin (w / v) and 0.05 w / v photosensitive component riboflavin (absorption wavelength blue light 450 nm) are added; power 20 W / cm 2 , continue to stir at 50℃ for 30 minutes to promote the release of polypeptide and improve the stability.

[0027] After high-temperature enzyme inactivation of the reaction solution obtained in step (1), centrifugal separation is carried out, and the supernatant is loaded into a dialysis bag with a molecular weight cutoff of 1000 Da, and dialysis is carried out in deionized water for 24 hours to remove small molecular impurities and unreacted components, and obtain high-purity bovine spleen peptide solution.

[0028] The dialysate of step (2) was concentrated by rotary evaporation, and then freeze-dried at -20°C for 48 hours. Finally, white bovine spleen peptide powder was obtained.

[0029] The extraction rate of the obtained bovine spleen peptide was 8.7%, the molecular weight was concentrated in 1000-2500 Da, the purity was 86% (protein content / powder mass), and it had good solubility and antioxidant activity.

[0030] Example 2 (no natural macromolecule, photosensitive blank control)

[0031] 10 g of bovine spleen powder was weighed and sieved through a 40-mesh sieve. Trpsin solution (5000 U / mL) was added at a solid-liquid ratio of 1:30, and the enzyme was hydrolyzed at pH 7.5 and 50°C for 2 hours without adding natural macromolecules or photosensitive components.

[0032] The enzyme hydrolysate was centrifuged, and the supernatant was subjected to subsequent processing (same as Example 1).

[0033] The concentrated solution was freeze-dried at -20°C for 48 hours to obtain bovine spleen peptide powder.

[0034] The extraction rate of the obtained bovine spleen peptide was 5.2%, the molecular weight distribution span was large (4000-9000 Da), the purity was 62%, and the solubility was poor, indicating the importance of the synergistic effect of natural macromolecules and photosensitivity.

[0035] Example 3 (single enzyme hydrolysis control)

[0036] 10 g of bovine spleen powder was accurately weighed and added with 0.05% w / v trypsin solution (5000 U / mL) at a solid-liquid ratio of 1:30 at pH 7.5 and 50°C for 120 minutes without adding natural macromolecules or photosensitive components.

[0037] After the enzyme hydrolysis was completed, the reaction solution was centrifuged, and the supernatant was directly freeze-dried (-20°C, 48 hours).

[0038] The final extraction rate of bovine spleen peptide was 5.6%, the molecular weight distribution was very wide (3500-9000 Da), the purity was 60%, and the product was a light yellow powder, indicating that single enzyme hydrolysis method was difficult to obtain high-quality bovine spleen peptide without natural macromolecules and photosensitivity.

[0039] Example 4 (photosensitivity + polysaccharide assisted extraction)

[0040] Accurately weigh 10 g of bovine spleen powder, and add compound protease (trypsin 5000 U / mL + papain 5000 U / mL) at a solid-liquid ratio of 1:25 at 55°C and pH 7.5 for 90 minutes. Then adjust the system to alkaline conditions, and add 1.5% chitosan (w / v) and photosensitive component sodium copper chlorophyllin (absorption wavelength red light 630 nm, incandescent lamp power 15 W / cm 2 , 1000 lux), and continue stirring for 45 minutes.

[0041] After high-temperature enzyme inactivation and centrifugal separation, the supernatant is loaded into a dialysis bag (1000 Da) and dialyzed with deionized water for 36 hours.

[0042] After rotary evaporation and concentration, vacuum freeze-drying is performed at a drying temperature of -25°C for 48 hours to obtain a white powder.

[0043] The obtained bovine spleen peptide extraction rate is 9.3%, the molecular weight is mainly concentrated in 1000-2500 Da, the purity is 88%, the solubility is good, and the antioxidant activity is significant.

[0044] Example 5 (natural macromolecule excess control)

[0045] Weigh 10 g of bovine spleen powder, and add compound protease (conditions are the same as in Example 4) and stevioside 5% (w / v) at a solid-liquid ratio of 1:30 at 50°C and pH 8.0 for 60 minutes without adding a photosensitive component.

[0046] After high-temperature enzyme inactivation and centrifugal separation, the supernatant is loaded into a dialysis bag (1000 Da) and dialyzed with deionized water for 36 hours.

[0047] The final bovine spleen peptide extraction rate is 5.8%, the purity is 66%, the product is viscous and easy to precipitate, and it is shown that the high concentration of natural macromolecules leads to a decrease in enzymatic efficiency.

[0048] Example 6 (traditional alkaline hydrolysis control)

[0049] Weigh 10 g of bovine spleen powder, and add 20 mL of 0.1 mol / L NaOH solution, and hydrolyze at 90°C for 2 hours. Cool to neutral, centrifugal separation, do not add a photosensitive component or a natural macromolecule, and do not dialyze or perform membrane separation.

[0050] After rotary evaporation and concentration, freeze-drying is performed at -25°C for 48 hours to obtain a powder. The bovine spleen peptide extraction rate is 7.0%, but the purity is 59%, the molecular weight distribution is 6000-10000 Da, it is dark brown and has a burnt smell, and it is shown that traditional high-temperature alkaline hydrolysis destroys the structure of polypeptides.

[0051] Example 7 (β-cyclodextrin + ultrafiltration assistance)

[0052] Take 10 g of bovine spleen powder, add complex protease (conditions same as example 4) at a solid-liquid ratio of 1:25 at pH 7.5 and 55°C for enzymolysis for 90 minutes, then add 1.5% β-cyclodextrin and photosensitive component methylene blue (absorption wavelength blue light 450 nm, incandescent lamp power 20 W / cm 2 , 1000 lux) and stir for 45 minutes.

[0053] After high-temperature enzyme inactivation and centrifugation, the supernatant was passed through an ultrafiltration membrane with a molecular weight cutoff of 2000 Da, the operating pressure was 0.3 MPa, the temperature was 40°C, and the filtration time was 2 hours to remove small molecular impurities.

[0054] After rotary evaporation and vacuum freeze-drying, the drying temperature was -25°C, and the drying time was 48 hours to obtain white bovine spleen peptide powder.

[0055] The extraction rate was 8.4%, the molecular weight was 500-2000 Da, the purity was 83%, the solubility and antioxidant activity were good, and it was suitable for the development of functional food or health products.

[0056] Table 1: Extraction rate, purity and molecular weight of polypeptides under different extraction solvents

[0057]

Claims

1. A method for extracting bovine spleen peptides based on a natural macromolecule-photosensitizer synergistic system, characterized in that, The method comprises: Step 1: grinding and sieving the bovine spleen raw material, drying for standby; Step 2: adding the bovine spleen powder obtained in step 1 into a protease solution for enzymolysis; Step 3: adding natural macromolecules and photosensitive components to the enzymolysis system for light-assisted treatment; Step 4: separating and purifying the product of step 3 to obtain bovine spleen peptide.

2. The method of claim 1, wherein the protease is trypsin and / or papain.

3. The method of claim 2, wherein the protease enzyme activity of the protease solution is 3000-10000 U / mL, the enzymolysis time is 60-90 minutes, the pH of the enzymolysis system is 7-8, and the enzymolysis temperature is 45-55℃.

4. The method of any one of claims 1-3, wherein the natural macromolecule is a polysaccharide.

5. The method of claim 4, wherein the polysaccharide is selected from cyclodextrin and / or chitosan.

6. The method of any one of claims 1-5, wherein the photosensitive component is riboflavin, sodium chlorophyll salt, and / or methylene blue.

7. The method of any one of claims 1-6, wherein the amount of natural macromolecule added in step 3 is 1-2 w / v%, and the amount of photosensitive component added is 0.03-0.07 w / v%.

8. The method of any one of claims 1-7, wherein the light-assisted treatment time in step 3 is 30-45 minutes, and the light-assisted treatment temperature is the same as the enzymolysis temperature in step 2.

9. The method of claim 1, wherein step 4 comprises one or more steps of dialysis, ultrafiltration, concentration, and drying.

10. The bovine spleen peptide product prepared by the method of any one of claims 1-9.

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