Preparation method of leech peptide

Abstract

The invention discloses a preparation method of leech peptide. The preparation method mainly comprises the following steps: leech pulping, slurry centrifugation, clear liquid fermentation, sterilization, ultrafiltration separation, nanofiltration concentration and freeze drying. The leech peptide prepared by the method provided by the invention has the characteristics of high purity and concentrated molecular weight distribution, can greatly reduce the difficulty of separation and purification, and is very beneficial to extraction of medical and medicinal leech peptide with the molecular weight of 1000-1500Da; the method disclosed by the invention is mild in process condition, simple to operate, environment-friendly and very suitable for industrial production.

Description

Technical Field

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

[0002] In the 2020 edition of the Pharmacopoeia, leeches are the dried whole body of Whitmania pigra Whitman, Hirudonipponica Whitman or Whitmania acranulata Whitman, all of which belong to the family Hirudinidae. They are caught in summer and autumn, scalded to death with boiling water, and dried in the sun or at low temperature. Functions and indications: They can break blood and promote menstruation, remove blood stasis and eliminate symptoms; they are used for blood stasis, amenorrhea, lumps and masses, hemiplegia caused by stroke, and injuries caused by falls.

[0003] Leech peptides are active peptides extracted from leeches and have high medicinal value. A large number of studies have shown that leech peptides with a molecular weight of 1000-1500Da have good thrombin inhibition effects and can be used in anticoagulant drugs; leech peptides have also shown strong performance in the treatment of unstable angina, coronary heart disease angina (blood stasis syndrome), and inhibition of atherosclerosis. Leech peptides also have broad application prospects in other fields, such as being expected to be used as functional ingredients such as repair and anti-aging in skin care products.

[0004] At present, there are two main ways to obtain leech peptides. One is to stimulate the living leech in different ways to make it secrete saliva to collect leech peptides, continue to cultivate after collecting saliva, and then carry out cyclic operation to obtain; the other is to use solvent extraction, separation and purification and other processes to prepare. The former is cumbersome and the yield is too low to meet large-scale production. The latter, that is, most modern biotechnology uses enzymatic extraction to prepare leech peptides. The disadvantage of this process is not only that the highly active enzymes are relatively expensive and not conducive to cost reduction and efficiency improvement, but more importantly, the molecular weight of the leech peptides obtained by enzymatic hydrolysis is not concentrated, and is dispersed in the range of 500-2500Da or wider, while the molecular weight of medical and pharmaceutical leech peptides is required to be between 1000-1500Da. Such leech peptides have good thrombin inhibition effect and can be used in anticoagulant drugs; therefore, the dispersed distribution of the molecular weight 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, reduce costs, and improve resource utilization.

[0006] In order to solve the above technical problems, the preparation method of leech peptide of the present invention comprises the following steps: (1) beating leeches: taking leeches, adding 5-10 times the weight of the leeches of a deep eutectic solvent, and beating into a leech slurry; the deep eutectic solvent is prepared by adding urea and glucose into water in a molar ratio of 1:1, and the mass proportion of urea and glucose in the deep eutectic solvent is 0.1-0.5%; (2) Slurry centrifugation step (1) centrifuging the leech slurry to remove residues and retain the clear liquid; (3) The clear liquid fermentation step (2) The obtained clear liquid is transferred into 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, and sterilized at 115-121° C. for 15 min, cooled to below 45° C., and added with Monascus purpurogenous culture liquid 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 bacteria and retain the fermentation clear liquid; (5) Ultrafiltration separation: The fermentation clear liquid is separated using a 3KD ultrafiltration membrane, and the permeate is retained; (6) Nanofiltration concentration step (5): the permeate obtained is concentrated and impurities are removed using a 500Da nanofiltration membrane, and a retentate is retained, wherein the retentate has a solid content 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.

[0007] Furthermore, in the step (1) of beating 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.

[0008] Furthermore, in the step (3) of the clear liquid fermentation of the present invention, the potassium dihydrogen phosphate, ferrous sulfide, manganese sulfate, sodium chloride and magnesium chloride added to the fermentation tank are of analytical grade.

[0009] The beneficial effects of the present invention are mainly reflected in the following aspects: 1. The method of the present invention uses a deep eutectic solvent Deep Eutectic Solvent (DES) to extract protein substances in leeches. The deep eutectic solvent (DES) is composed of urea and glucose. The DES system can fully dissolve leech protein but does not dissolve fat substances, thereby eliminating the degreasing process using flammable and explosive organic solvents in traditional processes. The urea in the DES system serves as a partial nitrogen source required for the initial growth of the fermentation bacteria in the subsequent fermentation process and is metabolized into ammonia, carbon dioxide and water, while 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, namely, they play a role in dissolving and extracting protein in the first step and in providing nutrients required by the bacteria during the fermentation process.

[0010] 2. The method of the present invention realizes the conversion of leech protein into leech peptide (conversion of macromolecular protein into small molecular protein peptide) through fermentation metabolism of specific strains. Monascus is a saprophytic fungus that can secrete a variety of enzymes, including proteases; during the fermentation process, part of the leech protein becomes a nitrogen source for the growth of Monascus and is metabolized into small molecular leech peptides, while the other part of the leech protein is hydrolyzed into small molecular leech peptides by proteases produced by the metabolism of Monascus; the production of small molecular leech peptides is achieved through dual pathways, which is conducive to extraction and utilization.

[0011] 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 leech peptides.

[0012] The method of the present invention is economical and can save 30% to 40% of the cost compared with the conventional enzymatic extraction method. The leech peptides prepared by the method of the present invention have concentrated molecular weights, which are conducive to extracting leech peptides with a molecular weight of 1000-1500Da for medical and pharmaceutical use, and 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

[0013] The present invention is further described in detail below with reference to the embodiments.

[0014] The method for preparing leech peptide of the present invention mainly comprises the following steps: Leech beating → slurry centrifugation → supernatant fermentation → sterilization → ultrafiltration separation → nanofiltration concentration → freeze drying. First, the raw materials were pretreated: 7 kg of freeze-dried Philippine leeches (Golden Edge Leeches) were taken and crushed to 80-100 meshes by a grinder (brand Azera) to obtain leech powder for later use; the following examples and comparative examples all used this leech powder as a raw material to ensure that the raw materials used in each experiment were uniform.

[0015] 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.

[0016] 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%.

[0017] Example: (1) Leech beating: Take 1.20 kg of pre-prepared 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).

[0018] (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.

[0019] The method of the present invention can be carried out in large-scale production by using a butterfly centrifuge with a rotation speed of 7000 r / m to centrifuge the slurry, and the centrifugation can be repeated twice.

[0020] (3) Fermentation of clear liquid: The clear liquid 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 respectively. The mixture was sterilized at 115-121°C for 15 min, cooled to below 45°C, and 500 ml of the fermentation culture solution of Monascus purpurogenum was added for fermentation. The fermentation conditions were as follows: pH 6.0±0.5, temperature 40±0.5°C, and time 40 hours. At the end of the fermentation, 6.76 kg of fermentation liquid was collected (weight gain due to steam sterilization in the tank).

[0021] (4) Sterilization: The obtained fermentation liquid is centrifuged to remove the bacteria (centrifugation parameters are the same as step 2), and 6.56 kg of fermentation liquid is obtained (the loss is the bacteria and the residue of the equipment during the operation).

[0022] (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.

[0023] (6) Nanofiltration concentration: The permeate obtained was concentrated to about 1 kg by passing through a 500Da nanofiltration membrane (membrane model 1812 material composite membrane) to remove 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 concentrate obtained was 8.5% (solid content determination: 105°C, 2h).

[0024] (7) Freeze drying: The obtained concentrate is transferred to a freeze dryer and freeze-dried until the moisture content is less than 0.5%, obtaining 168 g of finished leech peptide. The sample is marked as 1c.

[0025] 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.

[0026] (1) Leech beating: Take 1.20 kg of pre-prepared leech powder, add 6 kg of pure water, and beat it into a slurry using a beater (brand: Azera) (taking 30 seconds).

[0027] (2) Slurry centrifugation: The centrifugation parameters were the same as in Example 1. The insoluble matter was discarded to obtain 6.17 kg of clear liquid. A sample was taken and marked as Control 1.

[0028] Control 2: The leech pulping extraction and pulp centrifugation steps are the same as those in the embodiment, without the fermentation process. Finally, the leech protein and peptide 3KD ultrafiltration membrane pass rate is compared with that in the embodiment, thereby proving the protein conversion effect of fermentation in the process.

[0029] (1) Leech beating: same as in the embodiment.

[0030] (2) Centrifugation of slurry: Same as in Example 1, obtaining 6.19 kg of clear liquid.

[0031] (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 the pipeline residue plus large molecular weight retention) and a sample was taken and marked as control 2.

[0032] Control 3: This comparative example does not have the DES extraction centrifugal residue removal 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 sample 1c to verify the effect of the DES extraction step on improving the purity (content) of leech peptide.

[0033] (1) Leech beating and fermentation: 1.20 kg of pre-made leech powder was added with 6 kg of pure water, and the powder was beaten into a slurry using a pulper (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 sterilized (sterilization parameters were the same as those in the example). After the temperature was lowered to below 45° C., 500 ml of the fermentation bacteria Monascus expansion 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 weight was increased due to the residue and steam entering the tank for sterilization).

[0034] (2) Sterilization: The obtained fermentation liquid was centrifuged to remove the bacterial cells (the centrifugation parameters were the same as in the example), and 7.61 kg of fermentation liquid was obtained (the lost part was the residue, bacterial cells and the residue of the equipment during the operation).

[0035] (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 the retentate and the residue in the equipment pipeline).

[0036] (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, 2h).

[0037] (5) Freeze drying: The obtained 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.

[0038] Control 4: The extraction steps of this comparative example are the same as those of the embodiment, and the fermentation step of the embodiment is replaced by a conventional enzymatic hydrolysis process. The molecular weight distribution of the prepared leech peptide is compared with that of the leech peptide prepared in the embodiment 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. (1) Leech beating: same as in the embodiment.

[0039] (2) Centrifugation of slurry: Same as in Example 1; 6.21 kg of clear liquid was obtained.

[0040] (3) Enzymatic hydrolysis of the clear liquid: The clear liquid is transferred to an enzymatic hydrolysis tank, the pH value is controlled at 7.0-10.0, 2.4 g of trypsin (alkaline protease, activity ≥ 50,000 u / g) is added, and enzymatic hydrolysis is carried out at 50°C for 5 h.

[0041] (4) Ultrafiltration separation: The fermentation broth was filtered through a 3KD ultrafiltration membrane (parameters were the same as in Example 1), and 5.94 kg of permeate was retained (the loss was the retentate and the residue in the equipment pipeline).

[0042] (5) Nanofiltration concentration: The permeate obtained was concentrated to about 1 kg by 500 Da nanofiltration in the same manner as in the previous embodiment. The concentrate was then diluted with 10 kg of pure water and concentrated to 2 kg. The solid content of the concentrate obtained was 8.1% (solid content determination: 105°C, 2 h).

[0043] (6) Freeze drying: The concentrated solution was transferred to a freeze dryer for freeze drying to obtain 159 g of leech peptide. A sample was taken and marked as control 4.

[0044] The leech protein and leech peptide (small molecule protein peptide) content and the molecular weight distribution of leech peptide in the relevant samples were determined, and the products prepared in the examples and comparative examples were analyzed. The protein and peptide content were determined by the third method of the biuret method in the protein content determination of the 2020 edition of the Chinese Pharmacopoeia; the molecular weight distribution was determined by gel permeation chromatography (GPC).

[0045] The results of the determination of the contents (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.

[0046] Table 1: Total content of leech protein and peptides in relevant samples and leech protein extraction rate In Table 1, leech powder (leech powder obtained by pretreatment of freeze-dried Philippine leeches), sample 1c and control 3 are solid, and the contents of protein and peptide (small molecule protein peptide) are measured in mass percentage (%); sample 1a, sample 1b, control 1 and control 2 are liquid, and the contents of protein and peptide (small molecule protein peptide) are measured in mass volume ratio (mg / ml), and the conversion relationship in the formula is: 1mg / ml=0.1%.

[0047] 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) According to the formula, the leech protein extraction rate of sample 1a = (6.24Kg×29.6×0.1%)÷(1.20Kg×16.5%)=93.3% The leech protein extraction rates of the remaining samples were calculated using this method (see Table 1).

[0048] Result analysis: 1. The extraction rate of leech protein and peptide in sample 1a is about 93.3%, and the extraction rate of leech protein and peptide in control 1 is about 65.1%. The comparison of the results shows that DES solvent extraction of leech protein and peptide has a significant effect compared with water extraction.

[0049] 2. The extraction rate of leech protein in sample 1b was 85.8%, that is, the amount of protein and peptide passing through the 3KD ultrafiltration membrane was about 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 peptide passing through the 3KD ultrafiltration membrane was about 31.3% of the total amount, indicating 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).

[0050] 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 quite different, that is, the purity is quite different, indicating that DES solvent extraction has a specific and obvious effect on improving the purity of leech peptides.

[0051] Table 2: Molecular weight distribution of leech peptides of relevant samples (Da: Dalton) 0.5K-1KDa (%) 1K-1.5KDa (%) 1.5K-2KDa (%) 2K-2.5KDa (%) Sample 1c 15.8 64.5 9.7 5.9 Comparison 4 27.8 36.5 20.9 11.5 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 the conventional enzymatic hydrolysis method is relatively dispersed, which is difficult to separate subsequently, and the yield of the leech peptides in the target range is limited.

[0052] The results of Table 1 and Table 2 show that the leech peptide prepared by the DES solvent extraction plus specific strain fermentation process of the present invention has the characteristics of high purity and concentrated molecular weight distribution. In addition, the method of the present invention has mild process conditions, simple operation, and environmental friendliness, and is 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 of a deep eutectic solvent, and beating into a leech slurry; the deep eutectic solvent is prepared by adding urea and glucose into water in a molar ratio of 1:1, and the mass proportion of urea and glucose in the deep eutectic solvent is 0.1-0.5%; (2) Slurry centrifugation step (1) centrifuging the leech slurry to remove residues and retain the clear liquid; (3) The clear liquid fermentation step (2) The obtained clear liquid is transferred into 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, and sterilized at 115-121° C. for 15 min, cooled to below 45° C., and added with Monascus purpurogenous culture liquid 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 bacteria and retain the fermentation clear liquid; (5) Ultrafiltration separation: The fermentation clear liquid is separated using a 3KD ultrafiltration membrane, and the permeate is retained; (6) Nanofiltration concentration step (5): the permeate obtained is concentrated and impurities are removed using a 500Da nanofiltration membrane, and a retentate is retained, wherein the retentate has a solid content 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 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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