Method for preparing high-activity leech peptide by subcritical water assisted microbial fermentation method
Through sub -critical water extraction and microbial fermentation hydrolysis method, the problem of low activity and poor purity difference in traditional leeches extraction methods is solved, high biological activated leech peptides are prepared, and high -efficiency, environmental protection and high purity extraction effects are achieved.
Patent Information
- Application Number
- CN202411829412.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-12
- Publication Date
- 2025-01-21
AI Technical Summary
Traditional hirudin extraction methods have problems such as low activity, poor purity, long process time and environmental pollution.
A subcritical water extraction-assisted microbial fermentation method was used to prepare highly bioactive leech peptides through the steps of crushing leeches, subcritical water extraction, microbial fermentation and hydrolysis, decolorization, deodorization, and vacuum freeze-drying.
It improves the anticoagulant activity of leeches peptides, and its activity reaches about 10,000A/G. It is high, no odor, easy to dissolve, and has a long shelf life. It is suitable for food, health care, medicine and skin care products.
Smart Images
Figure FT_1 
Figure FT_2 
Figure FT_3
Abstract
Description
Technical Field
[0001] The invention belongs to the field of leech peptide preparation, and in particular relates to a method for preparing leech peptide with high biological activity by adopting a subcritical water-assisted microbial fermentation method. Background Art
[0002] Medicinal leeches are blood-sucking leeches that have been used for medical treatment since ancient times. According to the Shennong's Classic of Medicinal Herbs, leeches have the effects of breaking blood and removing blood stasis, relaxing the meridians and unblocking collaterals in traditional Chinese medicine, and are used to treat blood stasis, amenorrhea, traumatic injuries, etc. The main active ingredient of leeches is hirudin.
[0003] Hirudin is a protein in the salivary glands of medicinal leeches and is a highly effective and specific thrombin inhibitor. Hirudin inhibits the activity of thrombin by directly binding to thrombin to form a complex. Therefore, it can inhibit fibrinogen coagulation and thrombin-catalyzed hemostasis reactions. It can be used clinically to develop antithrombotic drugs for various thrombotic diseases. In addition, hirudin also has a variety of biological activities, including wound repair, anti-fibrosis, anti-tumor, anti-hyperuricemia, etc.
[0004] Natural hirudin is a single-chain polypeptide containing 65-66 amino acids and a molecular weight of 7000 Da. The hirudin molecule contains three disulfide bonds. Its N-terminus binds to the catalytic site of thrombin, inhibiting the catalytic effect of thrombin; the C-terminus is rich in acidic amino acids and binds to the substrate recognition site of thrombin, thereby inhibiting the activation of thrombin. There are many subtypes of hirudin, including HV1, HV2 and HV3, all of which have highly similar core sequences and are highly specific thrombin inhibitors. In recent years, more and more hirudin variants have been discovered, and these variants have high stability and activity.
[0005] The traditional method of extracting hirudin is to first extract with water or alcohol, and then combine salting out, pH precipitation, enzymatic hydrolysis, ultrafiltration or chromatography to obtain the finished hirudin. These methods have the disadvantages of complex extraction, high cost, and difficulty in industrial production, and the obtained hirudin has low activity and poor purity. For example, the water extraction method has poor water solubility, low biological activity, and low extraction efficiency; although the alcohol extraction method improves the extraction efficiency, the organic solvent needs to be removed later, resulting in a long process flow and environmental pollution. Traditional hydrolysis methods include acidic hydrolysis, alkaline hydrolysis and enzymatic hydrolysis. The acid-base hydrolysis method requires extremely low and extremely high pH conditions, and some amino acids are damaged. The enzymatic hydrolysis method can keep almost all the constituent amino acids intact, but the enzymatic reaction takes a long time and the hydrolysis is incomplete.
[0006] Subcritical water is liquid water with a temperature between 100 and 374°C, a pressure lower than the critical pressure of 22.4 MPa, and a density higher than the critical density of 0.32 g / cm3. Subcritical water has a low dielectric constant and strong catalytic ability, and is rich in hydrogen ions and hydroxide ions. It has acid-base catalytic function and can quickly decompose organic matter. In addition, due to the high temperature, the solubility of protein is significantly enhanced. The subcritical water extraction method is green and environmentally friendly, has no environmental pollution, has the advantages of low production cost and high extraction rate of active ingredients, and can be used for industrial production. The microbial hydrolysis method uses the protease produced by the microorganisms themselves to hydrolyze proteins to produce polypeptides. The conditions are mild, the hydrolysis is sufficient, and the amino acids are not damaged, so that the treated polypeptides have more application space. Summary of the invention
[0007] The invention provides a method for preparing high-bioactive leech peptide by subcritical water extraction assisted microbial fermentation, so as to solve the problems of low activity, poor purity, long process time, environmental pollution and the like in the traditional process.
[0008] In order to solve the above technical problems, the specific technical scheme of the method for preparing highly active leech peptides by subcritical water-assisted enzymatic hydrolysis of the present invention is as follows: 1) Grinding: Weigh the dried leeches, grind them roughly with a mortar, and then grind them with a grinder to obtain leech powder; 2) Extraction: Add leech powder to distilled water and extract in subcritical water. After the extraction, cool to room temperature, centrifuge, and filter the supernatant to obtain the extract; 3) Hydrolysis: The pH of the extract is adjusted, and the strain is added for fermentation to obtain the fermented hydrolyzate, and the fermentation is terminated; 4) Separation: filtering the hydrolyzate to obtain a filtrate; 5) Decolorization and deodorization: The obtained hydrolyzed filtrate is decolorized and deodorized to obtain the leech peptide stock solution; 6) Drying: The leech peptide stock solution is vacuum freeze-dried to obtain leech peptide freeze-dried powder.
[0009] Preferably, the temperature of subcritical water is 230° C.-300° C., the pressure is 15-20 MPa, and the extraction time is 10-20 min.
[0010] Preferably, in step (3), the pH is 4.5-7.0.
[0011] Preferably, in step (3), the strain is Lactobacillus plantarum.
[0012] Preferably, in step (3), the fermentation condition is 30° C. for 3-6 hours.
[0013] Preferably, in step (3), the fermentation termination condition is 95° C. for 10 min.
[0014] Preferably, in step (5), the pH of the hydrolysis filtrate is adjusted to 5.0-6.0, and 1.0%-2.0% (w / v) of the filtrate volume of activated carbon is added, and the decolorization and deodorization is carried out at 50°C-60°C for 30-60 minutes.
[0015] The method for preparing highly active leech peptides by subcritical water-assisted enzymatic hydrolysis of the present invention has the following advantages.
[0016] 1. The present invention uses subcritical water extraction for the first time to extract hirudin. Compared with the traditional water extraction method, the extraction efficiency is higher and it is more economical and environmentally friendly than using organic solvents such as ethanol and trichloroacetic acid.
[0017] 2. The leech peptide prepared by the present invention is obtained by hydrolyzing hirudin through microbial fermentation, and has higher anticoagulant activity. The measured antithrombin activity is about 10,000 ATU / g, which is significantly higher than that of the traditional process.
[0018] 3. The leech peptide prepared by the present invention is obtained by microbial fermentation and hydrolysis. After hydrolysis, the amino acid structure is not destroyed, the hydrolysis efficiency is high, the hydrolysis is complete, and it is more absorbable.
[0019] 4. The leech peptide prepared by the present invention has been decolorized, deodorized and vacuum freeze-dried. The product has high purity, no peculiar smell, is easy to dissolve, has a long shelf life, and can be widely used in the fields of food, health products, medicines, skin care products, etc. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is a schematic diagram of the process for preparing highly active leech peptides of the present invention; Figure 2 This is a comparison chart of the activities of the methods of Example and Comparative Example 1; Figure 3 It is a comparison chart of the activities of the methods of Example 2, Comparative Examples 2, 3, and 4; Figure 4 It is a comparison diagram of molecular weight distribution between Example and Comparative Examples 1, 2, 3, and 4; DETAILED DESCRIPTION
[0021] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.
[0022] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.
[0023] In the examples, unless otherwise specified, all the means used are conventional means in the art.
[0024] As used herein, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or such composition, step, method, article, or apparatus is not necessarily limited to only those elements but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.
[0025] In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.
[0026] The leech peptide activity detection in the present invention adopts the thrombin titration method: Weigh 1g of leech peptide freeze-dried powder, add 5ml of physiological saline to dissolve, centrifuge (14000rpm, 10min, 4℃), and retain the supernatant. Take 100μl of supernatant, add 200μl of 0.5% fibrinogen, mix well, incubate at 37℃ for 5min, add 40NIH / ml thrombin solution, 2μl each time, add once every 2min, shake gently while adding, record the volume consumed during coagulation, and calculate the anticoagulant activity (ATU / g) of the sample to be tested according to the following formula.
[0027] U=C1V1 / C2V2 in: C1: thrombin concentration NIH / ml C2: Sample concentration g / ml V1: volume consumed by thrombin μl V2: Volume of sample added μl Note: If the sample does not solidify after being added for more than 5 times, dilute the sample by a certain multiple and then test again.
[0028] The molecular weight of leech peptide in the present invention is detected by multi-stage ultrafiltration method: The leech peptide stock solution was separated by multi-stage ultrafiltration, and UE001 (1000Da), UE003 (3500Da), and UE005 (5000Da) ultrafiltration membranes were selected to obtain components <1000Da, 1000-3500Da, 3500-5000Da, and >5000Da, respectively. The protein concentration of each component was determined by the Bradford method.
[0029] Example 1
[0030] Weigh 100g of dried leech, crush it in a mortar, and grind it in a grinder to obtain leech powder. Add 1L of distilled water at a ratio of 1:10, extract with subcritical water at 230℃ and 15MPa for 10min, cool to room temperature, centrifuge at 14000rpm / min, 16℃, 20min parameters, and take the supernatant. After filtering the supernatant with filter paper, adjust the pH to 4.5, heat to 30℃, add plant lactobacillus, ferment for 3 hours, heat to 95℃, treat for 10min to stop fermentation, cool to 50℃, filter with 0.45μm filter membrane to obtain hydrolyzate. Adjust the pH to 5.0, add 10g of activated carbon, and decolorize for 30min. After decolorization, centrifuge at 14000rpm / min, 16℃, 20min parameters to take the supernatant. After filtering with 0.45μm filter membrane, vacuum freeze-drying is performed to obtain leech peptide freeze-dried. After testing, the activity was 9875 ATU / g.
[0031] Measurement results of molecular weight distribution of leech peptide stock solution: Leech peptide stock solution components Protein concentration (mg / ml) Relative content (%) Leech peptide stock solution 27.63 100 Ultrafiltration fraction 1 (<1000Da) 22.44 81.23 Ultrafiltration fraction 2 (1000~3500Da) 2.03 7.34 Ultrafiltration fraction 3 (3500~5000Da) 1.25 4.51 Ultrafiltration fraction 4 (>5000Da) 1.90 6.88 Example 2
[0032] Weigh 100g of dried leech, crush it in a mortar, and grind it in a grinder to obtain leech powder. Add 1L of distilled water at a ratio of 1:10, extract with subcritical water at 230℃ and 15MPa for 10min, cool to room temperature, centrifuge at 14000rpm / min, 16℃, 20min parameters, and take the supernatant. After filtering the supernatant with filter paper, adjust the pH to 5.5, heat to 30℃, add plant lactobacillus, ferment for 3 hours, heat to 95℃, treat for 10min to stop fermentation, cool to 50℃, filter with 0.45μm filter membrane to obtain hydrolyzate. Adjust the pH to 5.0, add 10g of activated carbon, and decolorize for 30min. After decolorization, centrifuge at 14000rpm / min, 16℃, 20min parameters, and take the supernatant. After filtering with 0.45μm filter membrane, vacuum freeze-drying is performed to obtain leech peptide freeze-dried. After testing, the activity was 9930 ATU / g.
[0033] Measurement results of molecular weight distribution of leech peptide stock solution: Leech peptide stock solution components Protein concentration (mg / ml) Relative content (%) Leech peptide stock solution 28.25 100 Ultrafiltration fraction 1 (<1000Da) 23.57 83.42 Ultrafiltration fraction 2 (1000~3500Da) 2.01 7.11 Ultrafiltration fraction 3 (3500~5000Da) 1.22 4.32 Ultrafiltration fraction 4 (>5000Da) 1.44 5.09 Example 3
[0034] Weigh 100g of dried leech, crush it in a mortar, and grind it in a grinder to obtain leech powder. Add 1L of distilled water at a ratio of 1:10, extract with subcritical water at 230℃ and 15MPa for 10min, cool to room temperature, centrifuge at 14000rpm / min, 16℃, 20min parameters, and take the supernatant. After filtering the supernatant with filter paper, adjust the pH to 7.0, heat to 30℃, add plant lactobacillus, ferment for 3 hours, heat to 95℃, treat for 10min to stop fermentation, cool to 50℃, filter with 0.45μm filter membrane to obtain hydrolyzate. Adjust the pH to 5.0, add 10g of activated carbon, and decolorize for 30min. After decolorization, centrifuge at 14000rpm / min, 16℃, 20min parameters, and take the supernatant. After filtering with 0.45μm filter membrane, vacuum freeze-drying is performed to obtain leech peptide freeze-dried. After testing, the activity was 8950 ATU / g.
[0035] Measurement results of molecular weight distribution of leech peptide stock solution: Leech peptide stock solution components Protein concentration (mg / ml) Relative content (%) Leech peptide stock solution 25.26 100 Ultrafiltration fraction 1 (<1000Da) 19.02 75.31 Ultrafiltration fraction 2 (1000~3500Da) 2.39 9.45 Ultrafiltration fraction 3 (3500~5000Da) 2.58 10.22 Ultrafiltration fraction 4 (>5000Da) 1.26 4.98 Example 4
[0036] Weigh 100g of dried leech, crush it in a mortar, and grind it in a grinder to obtain leech powder. Add 1L of distilled water at a ratio of 1:10, extract it with subcritical water at 300℃ and 20MPa for 10min, cool it to room temperature, centrifuge it at 14000rpm / min, 16℃, 20min parameters, and take the supernatant. After filtering the supernatant with filter paper, adjust the pH to 4.5, heat it to 30℃, add plant lactobacillus, ferment it for 3 hours, heat it to 95℃, treat it for 10min to stop the fermentation, cool it to 50℃, filter it with a 0.45μm filter membrane, and obtain the hydrolyzate. Adjust the pH to 5.0, add 10g of activated carbon, and decolorize it for 30min. After the decolorization is completed, centrifuge it at 14000rpm / min, 16℃, 20min parameters, and take the supernatant. After filtering with a 0.45μm filter membrane, vacuum freeze-drying is performed to obtain leech peptide freeze-dried. After testing, the activity was 10400 ATU / g.
[0037] Measurement results of molecular weight distribution of leech peptide stock solution: Leech peptide stock solution components Protein concentration (mg / ml) Relative content (%) Leech peptide stock solution 29.33 100 Ultrafiltration fraction 1 (<1000Da) 24.75 84.37 Ultrafiltration fraction 2 (1000~3500Da) 1.34 4.58 Ultrafiltration fraction 3 (3500~5000Da) 1.66 5.67 Ultrafiltration fraction 4 (>5000Da) 1.56 5.31 Example 5
[0038] Weigh 100g of dried leech, crush it in a mortar, and grind it in a grinder to obtain leech powder. Add 1L of distilled water at a ratio of 1:10, extract it with subcritical water at 300℃ and 20MPa for 10min, cool it to room temperature, centrifuge it at 14000rpm / min, 16℃, 20min parameters, and take the supernatant. After filtering the supernatant with filter paper, adjust the pH to 5.5, heat it to 30℃, add plant lactobacillus, ferment for 3 hours, heat it to 95℃, treat it for 10min to stop fermentation, cool it to 50℃, filter it with a 0.45μm filter membrane, and obtain a hydrolyzate. Adjust the pH to 5.0, add 10g of activated carbon, and decolorize it for 30min. After the decolorization is completed, centrifuge it at 14000rpm / min, 16℃, 20min parameters, and take the supernatant. After filtering with a 0.45μm filter membrane, vacuum freeze-drying is performed to obtain leech peptide freeze-dried. After testing, the activity was 11050 ATU / g.
[0039] Measurement results of molecular weight distribution of leech peptide stock solution: Leech peptide stock solution components Protein concentration (mg / ml) Relative content (%) Leech peptide stock solution 30.90 100 Ultrafiltration fraction 1 (<1000Da) 26.12 84.52 Ultrafiltration fraction 2 (1000~3500Da) 1.68 5.43 Ultrafiltration fraction 3 (3500~5000Da) 1.57 5.08 Ultrafiltration fraction 4 (>5000Da) 1.51 4.89 Example 6
[0040] Weigh 100g of dried leech, crush it in a mortar, and grind it in a grinder to obtain leech powder. Add 1L of distilled water at a ratio of 1:10, extract it with subcritical water at 300℃ and 20MPa for 10min, cool it to room temperature, centrifuge it at 14000rpm / min, 16℃, 20min parameters, and take the supernatant. After filtering the supernatant with filter paper, adjust the pH to 7.0, heat it to 30℃, add plant lactobacillus, ferment it for 3 hours, heat it to 95℃, treat it for 10min to stop the fermentation, cool it to 50℃, filter it with a 0.45μm filter membrane, and obtain the hydrolyzate. Adjust the pH to 5.0, add 10g of activated carbon, and decolorize it for 30min. After the decolorization is completed, centrifuge it at 14000rpm / min, 16℃, 20min parameters, and take the supernatant. After filtering with a 0.45μm filter membrane, vacuum freeze-drying is performed to obtain leech peptide freeze-dried. After testing, the activity was 9910 ATU / g.
[0041] Measurement results of molecular weight distribution of leech peptide stock solution: Leech peptide stock solution components Protein concentration (mg / ml) Relative content (%) Leech peptide stock solution 28.08 100 Ultrafiltration fraction 1 (<1000Da) 23.35 83.15 Ultrafiltration fraction 2 (1000~3500Da) 2.25 8.01 Ultrafiltration fraction 3 (3500~5000Da) 1.17 4.16 Ultrafiltration fraction 4 (>5000Da) 1.31 4.65 Comparative Example 1 Weigh 100g of dried leech, crush it in a mortar, put it in a grinder and grind it to obtain leech powder. Add 1L of distilled water at a ratio of 1:10, stir and soak for 3 hours, centrifuge at 14000rpm / min, 16℃, 20min parameters, and take the supernatant. After filtering the supernatant with filter paper, adjust the pH to 6.0, heat it to 30℃, add plant lactobacillus, ferment for 6 hours, heat it to 95℃, treat it for 10min to stop fermentation, reduce it to 50℃, filter it with a 0.45μm filter membrane, and obtain the hydrolyzate. Adjust the pH to 5.0, add 10g of activated carbon, and decolorize for 30min. After the decolorization is completed, centrifuge at 14000rpm / min, 16℃, 20min parameters, and take the supernatant. After filtering with a 0.45μm filter membrane, vacuum freeze-drying is performed to obtain leech peptide freeze-dried. After testing, the activity is 3150 ATU / g.
[0042] Measurement results of molecular weight distribution of leech peptide stock solution: Leech peptide stock solution components Protein concentration (mg / ml) Relative content (%) Leech peptide stock solution 9.33 100 Ultrafiltration fraction 1 (<1000Da) 2.87 30.75 Ultrafiltration fraction 2 (1000~3500Da) 3.29 35.27 Ultrafiltration fraction 3 (3500~5000Da) 2.16 23.16 Ultrafiltration fraction 4 (>5000Da) 1.00 10.72 Comparative Example 2 Weigh 100g of dried leech, crush it in a mortar, and grind it in a grinder to obtain leech powder. Add 1L of distilled water at a ratio of 1:10, extract it with subcritical water at 300℃ and 20MPa for 10min, cool it to room temperature, centrifuge it at 14000rpm / min, 16℃, 20min, and take the supernatant. After filtering the supernatant with filter paper, add 5ml 6 mol / L HCI to hydrolyze for 2h. Filter it with a 0.45μm filter membrane to obtain a hydrolyzate. Adjust the pH to 6.0, add 20g of activated carbon, and decolorize it for 30min. After decolorization, centrifuge it at 14000rpm / min, 16℃, 20min, and take the supernatant. After filtering with a 0.45μm filter membrane, vacuum freeze-dry it to obtain leech peptide freeze-dried. After testing, the activity is 5870 ATU / g.
[0043] Measurement results of molecular weight distribution of leech peptide stock solution: Leech peptide stock solution components Protein concentration (mg / ml) Relative content (%) Leech peptide stock solution 17.49 100 Ultrafiltration fraction 1 (<1000Da) 9.25 52.86 Ultrafiltration fraction 2 (1000~3500Da) 3.74 21.36 Ultrafiltration fraction 3 (3500~5000Da) 3.26 18.62 Ultrafiltration fraction 4 (>5000Da) 1.22 6.99 Comparative Example 3 Weigh 100g of dried leech, crush it in a mortar, and grind it in a grinder to obtain leech powder. Add 1L of distilled water at a ratio of 1:10, extract it with subcritical water at 300℃ and 20MPa for 10min, cool it to room temperature, centrifuge it at 14000rpm / min, 16℃, 20min, and take the supernatant. After filtering the supernatant with filter paper, add 5ml 8 mol / L NaOH and hydrolyze it for 2h. Filter it with a 0.45μm filter membrane to obtain a hydrolyzate. Adjust the pH to 6.0, add 20g of activated carbon, and decolorize it for 30min. After decolorization, centrifuge it at 14000rpm / min, 16℃, 20min, and take the supernatant. After filtering with a 0.45μm filter membrane, vacuum freeze-dry it to obtain leech peptide freeze-dried. After testing, the activity is 5430 ATU / g.
[0044] Measurement results of molecular weight distribution of leech peptide stock solution: Leech peptide stock solution components Protein concentration (mg / ml) Relative content (%) Leech peptide stock solution 17.42 100 Ultrafiltration fraction 1 (<1000Da) 9.82 56.38 Ultrafiltration fraction 2 (1000~3500Da) 3.38 19.43 Ultrafiltration fraction 3 (3500~5000Da) 2.82 16.19 Ultrafiltration fraction 4 (>5000Da) 1.38 7.93 Comparative Example 4 Weigh 100g of dried leech, crush it in a mortar, and grind it in a grinder to obtain leech powder. Add 1L of distilled water at a ratio of 1:10, extract it with subcritical water at 300℃ and 20MPa for 10min, cool it to room temperature, centrifuge it at 14000rpm / min, 16℃, 20min, and take the supernatant. After filtering the supernatant with filter paper, adjust the pH to 8.0, heat it to 50℃, add 5g of trypsin, and enzymolyze it for 2h. Heat it to 90℃ and kill the enzyme for 15min. After cooling to 50℃, filter it with a 0.45μm filter membrane to obtain the enzymatic solution. Adjust the pH to 5.5, add 10g of activated carbon, and decolorize it for 60min. After the decolorization is completed, centrifuge it at 14000rpm / min, 16℃, 20min, and take the supernatant. After filtering with a 0.45μm filter membrane, vacuum freeze-drying is performed to obtain leech peptide freeze-dried powder. After testing, the activity was 6350 ATU / g.
[0045] Measurement results of molecular weight distribution of leech peptide stock solution: Leech peptide stock solution components Protein concentration (mg / ml) Relative content (%) Leech peptide stock solution 19.95 100 Ultrafiltration fraction 1 (<1000Da) 11.92 59.77 Ultrafiltration fraction 2 (1000~3500Da) 3.56 17.83 Ultrafiltration fraction 3 (3500~5000Da) 2.44 12.24 Ultrafiltration fraction 4 (>5000Da) 2.01 10.07 Comparative Example 1 Based on the present invention, the traditional water extraction method was used to extract leeches to evaluate the effect of subcritical water extraction on the extraction efficiency and activity.
[0046] Comparative Examples 2, 3 and 4 are based on the present invention, and the extract is hydrolyzed by acid hydrolysis, alkaline hydrolysis and enzymatic hydrolysis respectively to evaluate the effect of the hydrolysis method on the molecular weight of leech peptide.
[0047] Experimental Example 1 (Extraction Method Comparison): The activities of the leech peptides prepared in Examples 1-6 and Comparative Example 1 were compared. Figure 2As shown, the analysis results show that the activity of leech peptides using the preparation process of the present invention (Examples 1-6) is increased by about 3 times compared with the traditional extraction method (Comparative Example 1).
[0048] Experimental Example 2 (Comparison of hydrolysis methods): The activities of the leech peptides prepared in Examples 1-6 and Comparative Examples 2, 3, and 4 were compared. Figure 3 As shown, the analysis results show that the activity of leech peptides using the preparation process of the present invention (Examples 1-6) is increased by about 1.5-2 times compared with the traditional hydrolysis method (Comparative Examples 2, 3, 4).
[0049] Experimental Example 3 (Comparison of Hydrolysis Molecular Weight) The molecular weights of the leech peptides prepared in Examples 1-6 and Comparative Examples 1, 2, 3, and 4 were compared. Figure 4 As shown, the analysis results show that the short peptides with a molecular weight of less than 1000Da of the leech peptides prepared by the preparation process of the present invention (Examples 1-6) account for 75% to 85%, while the short peptides with a molecular weight of less than 1000Da of the leech peptides prepared by the traditional hydrolysis method (Comparative Examples 1, 2, 3, and 4) account for 30% to 60%.
Claims
1. A method for preparing highly active leech peptides by subcritical water-assisted microbial fermentation, characterized in that The following steps are involved: 1) Grinding: Weigh the dried leeches, coarsely grind them in a mortar, and then grind them in a grinder to obtain leech powder; 2) Extraction: Add leech powder to distilled water and extract in subcritical water. After the extraction, cool to room temperature, centrifuge, and filter the supernatant to obtain the extract; 3) Hydrolysis: The pH of the extract is adjusted, and the strain is added for fermentation to obtain the fermented hydrolyzate, and the fermentation is terminated; 4) Separation: filtering the hydrolyzate to obtain a filtrate; 5) Decolorization and deodorization: The obtained hydrolyzed filtrate is decolorized and deodorized to obtain the leech peptide stock solution; 6) Drying: The leech peptide stock solution is vacuum freeze-dried to obtain leech peptide freeze-dried powder.
2. The method according to claim 1, characterized in that In the step (2), the temperature of the subcritical water is 230°C-300°C, the pressure is 15-20 MPa, and the extraction time is 10-20 min.
3. The method according to claim 1, characterized in that In the step (3), the pH is 4.5-7.
0.
4. The method according to claim 1, characterized in that In the step (3), the strain is Lactobacillus plantarum.
5. The method according to claim 1, characterized in that In the step (3), the fermentation condition is 30° C. for 3-6 hours.
6. The method according to claim 1, characterized in that In the step (3), the fermentation termination condition is 95° C. for 10 min.
7. The method according to claim 1, characterized in that In the step (5), the pH of the hydrolysis filtrate is adjusted to 5.0-6.0, and activated carbon is added in an amount of 1.0%-2.0% (w / v) of the filtrate volume, and the mixture is decolorized and deodorized at 50°C-60°C for 30-60 minutes.