A preparation method of leech peptide

Abstract

The present invention discloses a method for preparing leech peptides, which mainly comprises the following steps: leech beating → slurry centrifugation → supernatant fermentation → sterilization → ultrafiltration separation → nanofiltration concentration → freeze-drying. The leech peptides prepared by the method of the present invention have the characteristics of high purity and concentrated molecular weight distribution, which can greatly reduce the difficulty of separation and purification. It is very beneficial for extracting leech peptides with a molecular weight of 1000-1500Da for medical and pharmaceutical use. The method of the present invention has mild process conditions, simple operation, and is environmentally friendly, making it very suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biological preparations, and in particular to a method for preparing leech peptide. Background Art

[0002] In the 2020 edition of the Pharmacopoeia, leeches are defined as the dried whole body of Whitmania pigra Whitman, Hirudonipponica Whitman, or Whitmania acranulata Whitman, all members of the Hirudinidae family. They are caught in summer and autumn, scalded to death in boiling water, and then sun-dried or low-temperature dried. Functions and indications: They promote blood circulation and menstruation, dissipate blood stasis, and resolve symptoms. They are used to treat amenorrhea due to blood stasis, lumps and masses, hemiplegia caused by stroke, and injuries from falls.

[0003] Hirudin, an active peptide extracted from leeches, possesses high medicinal value. Numerous studies have demonstrated that leech peptides with a molecular weight of 1000-1500 Da exhibit potent thrombin inhibition and can be used in anticoagulant drugs. Hirudin peptides have also demonstrated promising results in the treatment of unstable angina, coronary angina (blood stasis syndrome), and the inhibition of atherosclerosis. Hirudin peptides also hold broad application prospects in other areas, such as their potential as a repairing and anti-aging ingredient in skincare products.

[0004] Currently, there are two main methods for obtaining leech peptides. One involves stimulating live leeches in various ways to induce them to secrete saliva, collecting the peptides, then continuing to cultivate the saliva, and then reproducing it in a cyclical process. The other involves solvent extraction, followed by separation and purification. The former is cumbersome and yields low, making it unsuitable for large-scale production. The latter, in modern biotechnology, mostly uses enzymatic extraction to produce leech peptides. The drawback of this process is not only that the highly active enzymes are relatively expensive, hindering cost reduction and efficiency, but more importantly, the molecular weight of the leech peptides obtained by enzymatic hydrolysis is not concentrated, but rather dispersed within a range of 500-2500 Da or even wider. However, the molecular weight of leech peptides for medical and pharmaceutical use is required to be between 1000-1500 Da. Such leech peptides have good thrombin inhibition and can be used in anticoagulant drugs. Therefore, the dispersed molecular weight distribution of leech peptides increases the difficulty of separation and purification, resulting in waste of raw materials and low efficiency. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the above-mentioned defects and provide a low-cost method for preparing leech peptides. The prepared leech peptides have concentrated molecular weights, are conducive to separation and purification, reduce resource waste, lower costs, and improve resource utilization.

[0006] In order to solve the above technical problems, the preparation method of the leech peptide of the present invention comprises the following steps:

[0007] (1) beating leeches, taking leeches, adding 5-10 times the weight of the leeches in a deep eutectic solvent, and beating into a leech slurry; the deep eutectic solvent is prepared by adding urea and glucose in a molar ratio of 1:1 to water, and the mass proportion of the urea and glucose in the deep eutectic solvent is 0.1-0.5%;

[0008] (2) Slurry centrifugation step (1): centrifuging the leech slurry to remove residue and retain the clear liquid;

[0009] (3) The clear liquid obtained in step (2) is transferred to a fermentation tank, and potassium dihydrogen phosphate, ferrous sulfide, manganese sulfate, sodium chloride, and magnesium chloride are added at 0.01-0.05% by weight of the clear liquid, respectively. The mixture is sterilized at 115-121°C for 15 minutes, cooled to below 45°C, and then added with Monascus culture solution at 5-10% by weight of the total liquid in the fermentation tank for microbial fermentation. The fermentation conditions are as follows: pH value 4.0-8.0, temperature 35°C-45°C, and time 30-40 hours to obtain a fermentation liquid.

[0010] (4) sterilizing the fermentation liquid by centrifuging to remove the bacterial cells and retaining the fermentation clear liquid;

[0011] (5) Ultrafiltration separation: The fermentation supernatant is separated using a 3KD ultrafiltration membrane, and the permeate is retained;

[0012] (6) Nanofiltration concentration step (5): The permeate obtained is concentrated and impurities removed using a 500Da nanofiltration membrane, and the retentate is retained. The solid content of the retentate is greater than 5%;

[0013] (7) Freeze-drying step (6): The retentate obtained is transferred to a freeze dryer and dried until the moisture content is less than 0.5%, thereby obtaining the finished product of leech peptide freeze-dried powder.

[0014] Furthermore, in the step (1) of beating the leeches of the present invention, the leeches are frozen fresh leeches, dehydrated leeches or leech powder; and the urea and glucose are of industrial grade.

[0015] Furthermore, in step (3) of the present invention, in the fermentation of the clear liquid, the potassium dihydrogen phosphate, ferrous sulfide, manganese sulfate, sodium chloride and magnesium chloride added to the fermentation tank are of analytical grade.

[0016] The beneficial effects of the present invention are mainly reflected in the following aspects:

[0017] 1. The method of the present invention uses a deep eutectic solvent (DES) to extract protein from leeches. The DES is composed of urea and glucose. This DES system can fully dissolve leech protein without dissolving fat, eliminating the traditional degreasing process using flammable and explosive organic solvents. The urea in the DES system serves as a partial nitrogen source for the initial growth of the fermentation bacteria during the subsequent fermentation process, metabolizing into ammonia, carbon dioxide, and water. The glucose serves as a carbon source during the fermentation process. The urea and glucose in the DES system have dual functions in the entire process: dissolving and extracting protein in the first step and providing nutrients required by the bacteria during the fermentation process.

[0018] 2. The method described herein converts leech protein into leech peptides (converting large-molecule proteins into small-molecule protein peptides) through fermentation and metabolism by specific bacterial species. Monascus is a saprophytic fungus that secretes a variety of enzymes, including proteases. During the fermentation process, a portion of the leech protein serves as a nitrogen source for the growth of the fungus and is metabolized into small-molecule leech peptides. The remaining portion of the leech protein is enzymatically hydrolyzed into small-molecule leech peptides by proteases produced by the fungus. This dual-pathway production of small-molecule leech peptides facilitates extraction and utilization.

[0019] 3. The method of the present invention first uses a 3KD ultrafiltration membrane to remove most of the non-small molecule peptide water-soluble impurities, and then uses a 500Da nanofiltration membrane to remove residual impurities such as urea, glucose and ash, thereby effectively improving the purity of the leech peptide.

[0020] The method of the present invention is economical and can save 30% to 40% of the cost compared with conventional enzymatic hydrolysis and extraction methods. The leech peptides prepared by the method of the present invention have concentrated molecular weights, which is conducive to the extraction of leech peptides with a molecular weight of 1000-1500 Da for medical and pharmaceutical use. It can greatly reduce the difficulty of separation and purification, improve the utilization rate of raw materials, reduce resource waste, and achieve the purpose of reducing costs and increasing efficiency. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below with reference to the examples.

[0022] The method for preparing leech peptide of the present invention mainly comprises the following steps:

[0023] Leech beating → slurry centrifugation → supernatant fermentation → sterilization → ultrafiltration separation → nanofiltration concentration → freeze drying.

[0024] The raw materials were pretreated: 7 kg of freeze-dried Philippine leech (Golden Edge Leech) was crushed to 80-100 mesh using a grinder (brand Azera) to obtain leech powder, which was set aside. The following examples and comparative examples all used this leech powder as the raw material to ensure the uniformity of the raw materials used in each experiment.

[0025] In the method of the present invention, the raw material selection is not limited to leech powder, and frozen fresh leeches or dehydrated leeches can be used. As long as the raw materials are crushed and pulped, similar effects can be achieved.

[0026] The deep eutectic solvent is prepared by adding industrial-grade urea and glucose in a molar ratio of 1:1 into water and mixing them, wherein the mass proportion of the urea and glucose in the deep eutectic solvent is 0.1-0.5%.

[0027] Example:

[0028] (1) Leech beating: Take 1.20 kg of pre-made leech powder, add 6 kg of prepared deep eutectic solvent, and beat it into a slurry using a beater (brand Azera) (taking about 30 seconds).

[0029] (2) Centrifugation of slurry: The leech slurry was passed through a high-speed centrifuge (model TGL-20M) at a speed of 12,000 r / m for 10 min. The insoluble matter was discarded to obtain 6.24 kg of clear liquid. A sample was taken and marked as 1a for later use.

[0030] The method of the present invention can be carried out in a butterfly centrifuge for large-scale production at a rotation speed of 7000 r / m to centrifuge the slurry, and the centrifugation can be repeated twice.

[0031] (3) Fermentation of the supernatant: The supernatant obtained in step (2) was transferred to a fermentation tank (model BLBI0-10SJ), and 3.1 g of analytical grade potassium dihydrogen phosphate, 3.1 g of ferrous sulfide, 3.1 g of manganese sulfate, 3.1 g of sodium chloride, and 3.1 g of magnesium chloride were added. The mixture was sterilized at 115-121°C for 15 min, cooled to below 45°C, and then inoculated with 500 ml of a culture medium of Monascus purpureus for fermentation. The fermentation conditions were: pH 6.0 ± 0.5, temperature 40 ± 0.5°C, and fermentation time 40 hours. At the end of fermentation, 6.76 kg of fermentation liquid was collected (weight gain due to steam sterilization).

[0032] (4) Sterilization: The fermentation liquid was centrifuged to remove the bacteria (centrifugation parameters were the same as in step 2), and 6.56 kg of fermentation liquid was obtained (the loss was the bacteria and the residue of the equipment during the operation).

[0033] (5) Ultrafiltration separation: The fermentation supernatant was separated by 3KD ultrafiltration membrane (membrane model 1812, material PES, the whole machine was produced by Sichuan Hecheng Filtration Equipment Co., Ltd.), and 5.94 kg of permeate was retained (the loss part was the interception material and equipment pipeline residue), and the sample was marked as 1b.

[0034] (6) Nanofiltration concentration: The permeate obtained was concentrated to about 1 kg through a 500 Da nanofiltration membrane (membrane model 1812 material composite membrane), removing impurities such as ash and amino acids. The concentrate was then diluted with 10 kg of pure water and continued to be concentrated to 2 kg. The solid content of the obtained concentrate was 8.5% (solid content determination: 105 ° C, 2 h).

[0035] (7) Freeze drying: The concentrated solution was transferred to a freeze dryer and freeze-dried until the moisture content was less than 0.5%, obtaining 168 g of finished leech peptide. The sample was marked as 1c.

[0036] Control 1: Pure water extraction, the purpose is to compare with sample 1a, and compare the extraction rate of leech protein by DES extraction with the extraction rate of leech protein by pure water.

[0037] (1) Leech beating: Take 1.20 kg of pre-made leech powder, add 6 kg of pure water, and beat it into a slurry using a beater (brand Azera) (taking 30 seconds).

[0038] (2) Slurry centrifugation: The centrifugation parameters were the same as in the example. The insoluble matter was discarded to obtain 6.17 kg of clear liquid. A sample was taken and marked as control 1.

[0039] Control 2: The leech pulping extraction and slurry centrifugation steps were the same as those in the embodiment, without the fermentation process. Finally, the leech protein and peptide 3KD ultrafiltration membrane pass rates were compared with those in the embodiment, thereby demonstrating the protein conversion effect of fermentation in the process.

[0040] (1) Leech beating: same as in the embodiment.

[0041] (2) Centrifugation of slurry: Same as in Example 1, obtaining 6.19 kg of clear liquid.

[0042] (3) Ultrafiltration separation: The clear liquid obtained in step (2) was filtered and separated through a 3KD ultrafiltration membrane, and 5.54 kg of permeate was retained (about 600 g of residual large molecules were retained in the pipeline). A sample was taken and marked as control 2.

[0043] Control 3: This comparative example does not include the DES extraction and centrifugation step. After extraction with pure water, the product is directly fermented together with the residue. The purity of leech peptide (total leech peptide content) is compared with that of sample 1c to verify the effect of the DES extraction step on improving the purity (content) of leech peptide.

[0044] (1) Leech beating and fermentation: 1.20 kg of pre-made leech powder was taken, 6 kg of pure water was added, and the mixture was beaten into a slurry using a beating machine (brand: Azera) (taking 30 seconds), and then put into a fermentation tank. 3.1 g of analytically pure potassium dihydrogen phosphate, 3.1 g of ferrous sulfide, 3.1 g of manganese sulfate, 3.1 g of sodium chloride, and 3.1 g of magnesium chloride were added, and the mixture was sterilized (sterilization parameters were the same as those in the example). After the mixture was cooled to below 45°C, 500 ml of a Monascus culture solution was added and fermented for 40 hours (fermentation was the same as in the example). The total weight of the fermentation liquid discharged after the fermentation was 7.76 kg (the residue and steam entering the tank for sterilization resulted in weight gain).

[0045] (2) Sterilization: The obtained fermentation liquid was centrifuged to remove the bacterial cells (centrifugation parameters were the same as in the example), and 7.61 kg of fermentation liquid was obtained (the loss was the residue, bacterial cells and the residue of the equipment during the operation).

[0046] (3) Ultrafiltration separation: 3KD ultrafiltration was carried out in the same manner as in Example 1, and 7.01 kg of permeate was retained (the loss was due to the retentate and the residue in the equipment pipeline).

[0047] (4) Nanofiltration concentration: 500Da nanofiltration concentration was carried out in the same manner as in the previous embodiment, and the concentrate was concentrated to about 1 kg. The concentrate was then diluted with 10 kg of pure water and concentrated to 2 kg. The solid content of the obtained concentrate was 9.1% (solid content determination: 105°C, 2 h).

[0048] (5) Freeze drying: The concentrated solution was transferred to a freeze dryer for freeze drying to obtain 181 g of leech peptide. A sample was taken and marked as control 3.

[0049] Control 4: The extraction steps of this comparative example are the same as those of the example, except that the fermentation step of the example is replaced by a conventional enzymatic hydrolysis process. The molecular weight distribution of the leech peptide prepared is compared with that of the leech peptide prepared in the example to verify the difference between the leech peptide prepared by the method of the present invention and the leech peptide prepared by the enzymatic hydrolysis method.

[0050] (1) Leech beating: same as in the embodiment.

[0051] (2) Centrifugation of slurry: Same as in Example 1; 6.21 kg of clear liquid was obtained.

[0052] (3) Enzymatic hydrolysis of the clear liquid: The clear liquid was transferred to an enzymatic hydrolysis tank, the pH value was controlled at 7.0-10.0, 2.4 g of trypsin (alkaline protease, activity ≥ 50,000 u / g) was added, and the enzymatic hydrolysis was carried out at 50°C for 5 h.

[0053] (4) Ultrafiltration separation: The fermentation broth was filtered through a 3KD ultrafiltration membrane (parameters were the same as in the example), and 5.94 kg of permeate was retained (the loss was the retentate and the residue in the equipment pipeline).

[0054] (5) Nanofiltration concentration: The permeate obtained was concentrated by 500Da nanofiltration in the same manner as in the previous embodiment to about 1kg. The concentrate was then diluted with 10kg of pure water and concentrated to 2kg. The solid content of the concentrate obtained was 8.1% (solid content determination: 105℃, 2h).

[0055] (6) Freeze drying: The concentrated solution was transferred to a freeze dryer for freeze drying to obtain 159 g of leech peptide, and a sample was taken and marked as control 4.

[0056] The leech protein and leech peptide (small molecule protein peptide) content and leech peptide molecular weight distribution of the relevant samples were determined, and the products prepared in the Examples and Comparative Examples were analyzed. Protein and peptide content were determined using the third method, the biuret method, as specified in the 2020 edition of the Chinese Pharmacopoeia for protein determination; molecular weight distribution was determined using gel permeation chromatography (GPC).

[0057] The results of the determination of the content (i.e., purity) of leech proteins and leech peptides (small molecule protein peptides) in the relevant samples are shown in Table 1. The results of the determination of the molecular weight distribution of leech peptides are shown in Table 2.

[0058] Table 1: Total content of leech proteins and peptides in relevant samples and leech protein extraction rate

[0059]

[0060] In Table 1, leech powder (leech powder obtained by pretreatment of freeze-dried leeches), Sample 1c, and Control 3 are solids, and the protein and peptide (small molecule protein peptide) contents are calculated in mass percentage (%). Sample 1a, Sample 1b, Control 1, and Control 2 are liquids, and the protein and peptide (small molecule protein peptide) contents are calculated in mass-to-volume ratio (mg / ml). The conversion relationship in the formula is: 1 mg / ml = 0.1%.

[0061] The formula for calculating the leech protein extraction rate is: sample weight × total protein and peptide content of the sample) ÷ (leech powder weight × total protein and peptide content of leech powder)

[0062] According to the formula, the leech protein extraction rate of sample 1a =

[0063] (6.24 kg x 29.6 kg x 0.1%) ÷ (1.20 kg x 16.5%) = 93.3%

[0064] The leech protein extraction rates of the remaining samples were calculated using this method, see Table 1.

[0065] Result analysis:

[0066] 1. The extraction rate of leech protein and peptide in sample 1a was about 93.3%, and the extraction rate of leech protein and peptide in control 1 was about 65.1%. The comparison of the results showed that DES solvent extraction of leech protein and peptide had a significant effect compared with water extraction.

[0067] 2. The extraction rate of leech protein in sample 1b was 85.8%, that is, the amount of protein and peptides passing through the 3KD ultrafiltration membrane was approximately 85.8% of the total amount. The extraction rate of leech protein in control 2 was 31.3%, that is, the amount of protein and peptides passing through the 3KD ultrafiltration membrane was approximately 31.3% of the total amount. This shows that the fermentation step in the method of the present invention has a significant effect on the conversion of leech protein into small molecule leech peptides (the smaller the molecular weight, the higher the permeability of the 3KD ultrafiltration membrane).

[0068] 3. Compared with control 3, sample 1c (the product of a complete embodiment of the method of the present invention) has a total protein and peptide content of 98.5% in sample 1c, while the total protein and peptide content of control 3 is 85.7%. The protein and peptide purities of the two are significantly different, that is, the purity difference is large, indicating that DES solvent extraction has a specific and significant effect on improving the purity of leech peptides.

[0069] Table 2: Molecular weight distribution of leech peptides in relevant samples (Da: Daltons)

[0070] 0.5K-1KDa (%) 1K-1.5KDa (%) 1.5K-2KDa (%) 2K-2.5KDa (%) Sample 1c 15.8 64.5 9.7 5.9 Control 4 27.8 36.5 20.9 11.5

[0071] As can be seen from Table 2, the molecular weight of the leech peptides prepared by the method of the present invention is relatively concentrated in the range of 1K-1.5KDa, which is conducive to subsequent separation and purification; the molecular weight distribution of the leech peptides prepared by conventional enzymatic hydrolysis is relatively dispersed, making subsequent separation difficult, and the yield of the leech peptides in the target range is limited.

[0072] The results in Table 1 and Table 2 show that the leech peptide prepared by the DES solvent extraction plus specific bacterial fermentation process according to the present invention has the characteristics of high purity and concentrated molecular weight distribution. In addition, the method according to the present invention has mild process conditions, simple operation, and is environmentally friendly, making it very suitable for industrial production.

Claims

1. A method for preparing leech peptide, characterized in that: The method comprises the following steps: (1) beating leeches, taking leeches, adding 5-10 times the weight of the leeches in a deep eutectic solvent, and beating into a leech slurry; the deep eutectic solvent is prepared by adding urea and glucose in a molar ratio of 1:1 to water, and the mass proportion of the urea and glucose in the deep eutectic solvent is 0.1-0.5%; (2) Slurry centrifugation step (1): centrifuging the leech slurry to remove residue and retain the clear liquid; (3) The clear liquid obtained in step (2) is transferred to a fermentation tank, and potassium dihydrogen phosphate, ferrous sulfide, manganese sulfate, sodium chloride, and magnesium chloride are added at 0.01-0.05% by weight of the clear liquid, respectively. The mixture is sterilized at 115-121°C for 15 minutes, cooled to below 45°C, and then added with Monascus culture solution at 5-10% by weight of the total liquid in the fermentation tank for microbial fermentation. The fermentation conditions are as follows: pH value 4.0-8.0, temperature 35°C-45°C, and time 30-40 hours to obtain a fermentation liquid. (4) sterilizing the fermentation liquid by centrifuging to remove the bacterial cells and retaining the fermentation clear liquid; (5) Ultrafiltration separation: The fermentation supernatant is separated using a 3KD ultrafiltration membrane, and the permeate is retained; (6) Nanofiltration concentration step (5): The permeate obtained is concentrated and impurities removed using a 500Da nanofiltration membrane, and the retentate is retained. The solid content of the retentate is greater than 5%; (7) Freeze-drying step (6): The retentate obtained is transferred to a freeze dryer and dried until the moisture content is less than 0.5%, thereby obtaining the finished product of leech peptide freeze-dried powder.

2. The method for preparing leech peptide according to claim 1, characterized in that: In the step (1) of beating the leeches, the leeches are frozen fresh leeches, dehydrated leeches or leech powder; and the urea and glucose are of industrial grade.

3. The method for preparing leech peptide according to claim 1, characterized in that: In the step (3) of fermenting the clear liquid, the potassium dihydrogen phosphate, ferrous sulfide, manganese sulfate, sodium chloride and magnesium chloride added to the fermentation tank are of analytical grade.

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