Preparation method of earthworm protein peptide with low molecular weight, low arsenic and high fibrinolytic activity

Abstract

The invention discloses a preparation method of a low-molecular-weight low-arsenic high-fibrinolytic-activity earthworm protein peptide, which is characterized in that a high-fibrinolytic-activity earthworm protein peptide product is obtained through compound step-by-step enzymolysis of bromelain and trypsin, the average molecular weight of the high-fibrinolytic-activity earthworm protein peptide product is 700-900 Da, and the fibrinolytic activity is 1100-1200 U / mg; the arsenic can be specifically adsorbed through macroporous chelating resin, 100-800 g / L of pretreated macroporous chelating resin is added into earthworm protein peptide extraction supernate, the total arsenic content of the earthworm protein peptide can be reduced to 0.3 mg / kg or below from greater than 7 mg / kg, the protein recovery rate is greater than or equal to 90%, the quality standard of current regulations is met, the resin after chelating adsorption can be reused after regeneration, and the method is simple and convenient to operate. And the device can be repeatedly used for more than 10 times. The process is efficient, safe, low in cost and suitable for industrial purification of the earthworm protein.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological product processing, and particularly relates to a large-scale production process of earthworm protein peptide with low molecular weight, low arsenic content and high fibrinolytic activity. BACKGROUND

[0002] Earthworm, also known as worm, is rich in amino acids, organic acids, enzymes, fatty acids and lipids, proteins and other nutrients. It can not only be used as animal feed and for the production of medicines and cosmetics, but also be used in the field of food and health food as a "new resource food" approved in 2009. Earthworm is often used as a traditional Chinese medicinal material, and has the effects of clearing heat and calming wind, relieving asthma, dredging collaterals and diuresis. In modern times, earthworm is still widely used in the production of medicines due to its unique effects.

[0003] Modern research shows that earthworm protein has a wide impact on the coagulation system and fibrinolytic system in the body. It can not only significantly reduce the platelet adhesion rate of rats, prolong the formation of thrombus in the body and dissolve thrombus in the body, but also increase cerebral blood flow, reduce cerebral vascular resistance, improve microcirculation, reduce the harm of three highs to the human body, and also confirm its good anticoagulant activity by fibrin plate method. Compared with macromolecular earthworm protein, small molecular earthworm protein peptide is more easily absorbed by the human body. At present, the method for preparing earthworm protein peptide mainly includes the processes of inducing vomiting from fresh earthworms, water extraction, enzymolysis, ultrafiltration and / or column separation, etc. The disadvantage is that there is no simple, efficient and large-scale production process.

[0004] As prior art CN114262727B discloses that earthworm protein with a molecular weight greater than 3000 Da is first obtained by ultrasonic assistance, then alkaline protease is added for ultrasonic-assisted enzymolysis, the enzymolysis product is ultrafiltered using a 10 kDa filter membrane to collect components smaller than 10 kDa, then a HiPrep16 / 10 DEAE FF pre-packed column is used for elution, and each fraction is collected. The product obtained has a peak area of more than E+07, and 39 peptides with a Denovo Score of more than 80 points are identified (molecular weight in the range of 510-947 Da, wide molecular weight distribution range, and complex peptide segments, such as containing 5-10 amino acids), of which the proportion of high-activity peptides is low. This method needs to use ultrafiltration process and column separation, and has many process steps and complex post-treatment, which greatly increases the cost, prolongs the process cycle, and has low product yield.

[0005] In addition, due to the special living environment of earthworms, they are easily polluted by heavy metals, among which the arsenic exceeds the standard most seriously. Therefore, when earthworm protein peptides are applied in clinical, food or health products, arsenic removal process is essential. At present, the technologies for removing arsenic in industry mainly include activated carbon adsorption, macroporous resin adsorption and other methods. However, the activated carbon adsorption has the problems of low adsorption capacity, poor selectivity for arsenic and complicated post-processing. The macroporous resin adsorption has the problems of poor selectivity for arsenic and the risk of secondary pollution caused by easy desorption after adsorption. Therefore, an efficient, safe and scalable arsenic removal process is urgently needed, which can reduce the total arsenic content to below 0.3 mg / kg while retaining the content of earthworm protein peptides.

[0006] Therefore, there is a need to screen a preparation method of earthworm protein peptides with fewer process steps, simple post-processing, low cost, short process cycle, high product yield, low arsenic residue, more concentrated molecular weight and higher fibrinolytic activity. SUMMARY

[0007] The purpose of the present application is to provide a preparation method of earthworm protein peptides with fewer process steps, simple post-processing, low cost, short process cycle, high product yield, low arsenic residue, more concentrated molecular weight and higher fibrinolytic activity. The obtained earthworm protein peptides are more easily absorbed by the human body, have lower total arsenic residue, higher safety and better fibrinolytic activity, and solve the problems of existing technology, such as multiple process steps in the preparation process of earthworm protein peptides, complicated post-processing, high production cost, long process cycle, low product yield, wide distribution of earthworm protein peptide molecular weight, difficulty in removing arsenic from earthworm protein peptides and low fibrinolytic activity of earthworm protein peptides.

[0008] In order to achieve the above purpose, the present application provides a preparation method of earthworm protein peptides with low molecular weight, low arsenic and high fibrinolytic activity, which is obtained by the following steps: stepwise protease digestion of earthworm homogenate supernatant to obtain earthworm protein peptide extraction supernatant, then adding pretreated macroporous chelating resin for adsorption, collecting the filtrate, and finally drying to obtain earthworm protein peptides with an average molecular weight of 700-900 Da, a fibrinolytic activity of 1100-1200 U / mg and an arsenic content of 0.3 mg / kg or less.

[0009] Further improvement is that the earthworm homogenate supernatant is prepared by the following method: thawing and washing earthworms, homogenizing and breaking the cell wall, centrifugal separation, and collecting the earthworm homogenate supernatant.

[0010] Further improvement is that the earthworm protein peptide extraction supernatant is prepared by twice enzyme digestion, and the earthworm protein peptides with an average molecular weight of 700-900 Da and a fibrinolytic activity of 1100-1200 U / mg can be obtained without using filter membrane.

[0011] Further improvement lies in that the twice enzymolysis is one enzymolysis using bromelain and secondary enzymolysis using trypsin.

[0012] Further improvement lies in that the one enzymolysis controls pH at 7.0-9.0, enzymolysis temperature at 20-55 DEG C, adds bromelain at 1-5 g / L (mass-volume ratio of bromelain and homogenate supernatant of dilm), stirs and enzymolyzes for 4-12 h, inactivates by heating, and centrifugates to obtain one enzymolysis supernatant.

[0013] Further improvement lies in that the secondary enzymolysis controls pH at 7.5-8.5, enzymolysis temperature at 20-37 DEG C, adds trypsin at 0.1-0.5 g / L (mass-volume ratio of trypsin and homogenate supernatant of dilm), stirs and enzymolyzes for 4-12 h, inactivates by heating, and centrifugates to obtain dilm protein peptide extraction supernatant.

[0014] Further improvement lies in that the dilm is added with purified water in a mass-volume ratio of m:v = 1:1-3 after washing to crush and homogenate and break the wall.

[0015] Further improvement lies in that the chelating resin pretreatment is that the chelating resin is first rinsed with purified water, then pretreated with sodium hydroxide solution and sodium chloride solution, and rinsed with purified water until pH value is below 9.

[0016] Further improvement lies in that the pretreated macroporous chelating resin is added to the dilm protein peptide extraction supernatant in a chelating resin adding amount of 100-800 g / L to adsorb, the adsorption pH is controlled at 5-9, the adsorption temperature is controlled at 25-40 DEG C, and the adsorption time is controlled at 4-12 h.

[0017] Further improvement lies in that after the chelating resin adsorption ends, the supernatant after adsorption is treated with 0.22 mu m hollow fiber membrane to collect the clear filtrate, the clear filtrate of dilm protein peptide after adsorption is filtered and sterilized with 0.22 mu m filter core, and finally dried to obtain low-molecular-weight low-arsenic and high-fibrinolytic-activity dilm protein peptide.

[0018] Further improvement lies in that the drying is spray drying, the inlet air temperature is 180-200 DEG C, and the outlet air temperature is 80-95 DEG C to obtain low-molecular-weight low-arsenic and high-fibrinolytic-activity dilm protein peptide.

[0019] Further improvement includes a regeneration treatment step of the chelating resin, the chelating resin is first rinsed with sodium hydroxide solution and sodium chloride solution, and then rinsed with purified water until pH value is below 9.

[0020] Compared with the prior art, the application has the following beneficial effects: The application discloses a large-scale production process of low-molecular-weight low-arsenic earthworm protein peptide with good fibrinolytic activity, and has the following advantages: 1) the problem that macromolecular earthworm protein is not easy to be absorbed by the human body is solved, the step of ultrafiltration screening after enzymolysis is avoided, and the average molecular weight of the earthworm protein peptide is less than 1000 Da through two-step enzymolysis, the average molecular weight of the small-molecular-weight earthworm protein peptide prepared by the compound enzyme is 700-900 Da (the molecular weight is more concentrated), the small-molecular-weight earthworm protein peptide is more easily absorbed by the human body, has better fibrinolytic activity, and the fibrinolytic activity is 1100-1200 U / mg. 2) By changing the adding amount, pH, temperature and adsorption time of the resin, the total arsenic residue in the earthworm protein peptide is effectively controlled, the total arsenic residue is reduced from >7 mg / kg to below 0.3 mg / kg, and the protein recovery rate is greater than or equal to 90%. The chelating resin has the functions of decolorization and chelation of other heavy metals, can effectively improve the color of the earthworm protein peptide and reduce the content of other heavy metals, improve the properties and quality of the earthworm protein peptide product, and the adsorbed resin can be regenerated by sodium hydroxide and sodium chloride and can be reused more than 10 times, so that the production cost of the earthworm protein peptide is greatly reduced. In summary, the application selects a preparation method of earthworm protein peptide, which has the advantages of few process steps, simple post-treatment, low cost, short process cycle, high product yield, low arsenic residue, more concentrated molecular weight and higher fibrinolytic activity, and has the value of popularization and application in industry. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is a standard curve for fibrinolytic activity detection of the scheme; Figure 2 is a typical fibrin plate method dissolution circle photo of the scheme; Figure 3 is a standard curve for GPC molecular weight detection of the scheme; Figure 4 is a typical GPC molecular detection spectrum of the scheme; Figure 5 is a process flowchart of the scheme. DETAILED DESCRIPTION

[0022] The following examples are included to demonstrate preferred embodiments of the application. It should be appreciated by those skilled in the art that the techniques disclosed in the examples represent techniques discovered by the inventors to function well in the practice of the application. Based on the teachings provided herein, those skilled in the art should appreciate that numerous modifications can be made to the specific embodiments described herein without departing from the spirit and scope of the application. Any equivalent techniques that replace the specific implementation described herein with other technical solutions that have substantially the same effect are intended to be within the scope of the patent.

[0023] The materials, reagents and the like used in the following examples, unless otherwise specified, can be obtained commercially.

[0024] The macroporous chelating resin was pretreated by first rinsing with 2 BV of purified water, then pretreating with 2% mass concentration sodium hydroxide solution and 8% mass concentration sodium chloride solution at room temperature, and finally rinsing with purified water until the pH value was less than 9.

[0025] The macroporous chelating resin was regenerated by first rinsing with 2% mass concentration sodium hydroxide solution and 8% mass concentration sodium chloride solution at room temperature, and then rinsing with purified water until the pH value was less than 9.

[0026] The fibrinolytic activity of the earthworm protein peptide was detected by the fibrin plate method according to the method for determining the specific activity of earthworm lumbrokinase, and a standard curve was drawn using an earthworm lumbrokinase standard. Figure 1 Figure 2 The fibrin plate method was used to detect the fibrinolytic activity of the earthworm protein peptide.

[0027] Preparation of the buffer solution: 0.01 mol / L phosphate buffer solution: take 3.58 g of disodium hydrogen phosphate, dissolve and dilute to 100 ml with water to obtain solution A; take 0.78 g of sodium dihydrogen phosphate dihydrate, dissolve and dilute to 500 ml with water to obtain solution B, and mix solutions A and B to a pH of 7.8.

[0028] Working solution: mix 0.01 mol / L phosphate buffer solution (pH 7.8) and 0.9% sodium chloride solution (1:17).

[0029] 1.5% agarose solution: take 1.5 g of agarose, dissolve in 100 ml of working solution by heating.

[0030] Fibrinogen solution: take an appropriate amount of fibrinogen, and prepare a solution containing 1.5 mg of coagulable protein per 1 ml with working solution.

[0031] Thrombin solution: take thrombin, and prepare a solution containing 1 BP unit per 1 ml with 0.9% sodium chloride solution.

[0032] Preparation of the standard solution: take earthworm lumbrokinase standard, and prepare solutions containing 8000, 6000, 4000 and 2000 units of earthworm lumbrokinase per 1 ml with 0.9% sodium chloride solution.

[0033] ​Preparation of test sample solution: take the earthworm protein peptide sample prepared in the examples of the present application, add 0.9% sodium chloride solution to dissolve and dilute to a concentration within the standard curve range.

[0034] Fibrin plate method detection process: Take 39 ml of fibrinogen solution and place it in a beaker. While stirring, add 39 ml of 55℃ agarose solution and 3 ml of thrombin solution. Mix immediately and quickly pour into a 14 cm diameter plastic culture dish. Place at room temperature horizontally for 1 hour, then punch a hole. Precisely measure 10 μl of earthworm kinase standard solution and test sample solution and place them on the same dish. Cover and place in a 37℃ constant temperature incubator for 18 hours. After removal, measure the vertical diameters of the dissolution circle with a vernier caliper. Take the logarithm of the number of units of the earthworm kinase standard as the abscissa and the logarithm of the product of the vertical diameters as the ordinate. Calculate the standard curve regression equation and substitute the logarithm of the product of the vertical diameters of the test sample into the standard curve regression equation to calculate the number of units of the test sample.

[0035] Protein concentration detection method: refer to nitrogen determination method (Chinese Pharmacopoeia 2000 edition part II second method) for determination. Multiply the result by 6.25 to get the protein concentration of the test sample solution. Example 1: screening of different single proteases

[0036] Freeze the fresh earthworm and thaw it at room temperature. Wash the thawed fresh earthworm with clean water to remove mud and sticky substances. After washing, drain the excess water. Take 7 kg of washed earthworm and add purified water at a ratio of m:v=1:2 to crush and homogenize the wall for 30 minutes. Centrifuge the homogenate to collect the supernatant. Divide the supernatant into 7 groups and investigate the effects of pepsin, trypsin, neutral protease, papain, acid protease, flavor protease and bromelain on earthworm protein peptide. Add 7 kinds of proteases to the homogenate supernatant at an addition amount of 1 g / L. Adjust the pH of the enzyme solution to 2.0, 8.5, 7.0, 7.0, 3.0, 7.0 and 7.0 respectively. Adjust the enzyme solution temperature to 37℃, 37℃, 50℃, 55℃, 50℃, 50℃ and 50℃ respectively. Stir the enzyme solution for 6 hours. After enzyme hydrolysis, heat inactivate and centrifuge to obtain earthworm protein peptide extract supernatant. Treat the earthworm protein peptide extract supernatant with a 0.22 μm hollow fiber membrane. Collect the clear filtrate. Filter the clear filtrate with a 0.22 μm filter to remove bacteria. Finally, spray dry at an inlet temperature of 185℃ and an outlet temperature of 85℃ to obtain earthworm protein peptide.

[0037] Molecular exclusion method (GPC) and fibrin plate method were used to detect the average molecular weight and fibrinolytic activity of different earthworm protein peptides.

[0038] Protease species Enzymolysis pH Enzymolysis temperature (℃) Average molecular weight (Da) Fibrinolytic activity (U / mg) Pepsin 2.0 37 1657 622.30 Trypsin 8.5 37 4550 592.28 Neutral protease 7.0 50 965 572.92 Papain 7.0 55 1080 439.52 Acid protease 3.0 50 936 650.32 Flavourzyme 7.0 50 883 605.12 Bromelain 7.0 50 980 819.64 From the data of Example 1, it can be concluded that pepsin, neutral protease, papain, acid protease, flavor protease and bromelain are easy to hydrolyze earthworm protein to a lower molecular weight, mainly because pepsin, neutral protease, papain, acid protease and bromelain belong to non-specific endoprotease, flavor protease belongs to complex enzyme, which contains non-specific endoprotease and non-specific exopeptidase, and only trypsin belongs to specific endoprotease, which strictly recognizes specific amino acid sequences, has the strongest specificity, and the average molecular weight of earthworm protein peptide prepared by enzymatic hydrolysis is also the largest. In addition, non-specific protease cleaves the peptide bond between amino acids, has certain preference for amino acid hydrolysis sites, resulting in certain differences in the composition and size of amino acid hydrolysis fragments, resulting in different amino acid compositions of peptide segments and different fibrinolytic activities. At the same time, the pH value of pepsin and acid protease is too low, and more acid and alkali processing aids need to be added, which introduces inorganic salts that are not easy to remove, reducing the content of earthworm protein peptide product. Therefore, the present application selects neutral protease, papain, flavor protease and bromelain for further research. Example 2: Screening of different protease combinations

[0039] In view of the enzymatic specificity of different enzymes, the effects of one-step enzymolysis using neutral protease, papain, flavor protease and bromelain and two-step enzymolysis using specific trypsin for secondary enzymolysis were compared to prepare earthworm protein peptide, and the changes in molecular weight and fibrinolytic activity of earthworm protein peptide after stepwise enzymolysis by different enzyme combinations were investigated.

[0040] The frozen fresh leeches are thawed at room temperature, and the thawed fresh leeches are washed with clean water to remove sludge and stickiness. After washing, the leeches are drained of excess water. 6 kg of washed leeches are taken, and purified water is added to the washed leeches at a ratio of m:v = 1:2 for 30 minutes of homogenate wall breaking. The homogenate supernatant is collected by centrifugation, and the homogenate supernatant is evenly divided into 6 groups. The proteinase combinations (step-by-step enzymolysis: first neutral protease and then trypsin), (step-by-step enzymolysis: first papain and then trypsin), (step-by-step enzymolysis: first flavor protease and then trypsin), and (step-by-step enzymolysis: first bromelain and then trypsin), (one-pot enzymolysis: bromelain + trypsin), (step-by-step enzymolysis: first trypsin and then bromelain) are used to investigate the enzymolysis of leech protein. In the step-by-step enzymolysis, neutral protease, papain, flavor protease, bromelain, and trypsin are added to the homogenate supernatant at an addition amount of 1 g / L, 1 g / L, 1 g / L, 1 g / L, and 0.1 g / L, respectively, the pH of the first enzymolysis is adjusted to 7.0, 7.0, 7.0, 7.0, and 8.5, respectively, the temperature of the first enzymolysis is adjusted to 50℃, 55℃, 50℃, 50℃, and 37℃, respectively, and the stirring enzymolysis is performed for 6 hours. After the enzymolysis is completed, the first enzymolysis supernatant is obtained by heating and inactivating centrifugation. In the second enzymolysis, trypsin and bromelain are added to the different first enzymolysis supernatants in group 5 at an addition amount of 0.1 g / L and 1 g / L, respectively, the pH of the second enzymolysis is adjusted to 8.5, 8.5, 8.5, 8.5, and 7.0, respectively, the temperature of the second enzymolysis is adjusted to 37℃, 37℃, 37℃, 37℃, and 50℃, respectively, and the stirring enzymolysis is performed for 6 hours. After the enzymolysis is completed, the leech protein peptide extraction supernatant is obtained by heating and inactivating centrifugation. In addition, in the one-pot enzymolysis, bromelain and trypsin are added to the homogenate supernatant at an addition amount of 1 g / L and 0.1 g / L, respectively, the enzymolysis pH is adjusted to 7.5, the enzymolysis temperature is adjusted to 37℃, and the stirring enzymolysis is performed for 6 hours. After the enzymolysis is completed, the leech protein peptide extraction supernatant is obtained by heating and inactivating centrifugation. The 6 groups of leech protein peptide extraction supernatants are treated using a 0.22 μm hollow fiber membrane, and the leech protein peptide clear filtrate is collected. The leech protein peptide clear filtrate is filtered using a 0.22 μm filter cartridge to remove bacteria, and finally spray dried. The inlet air temperature of the spray drying is 185℃, and the outlet air temperature is 85℃. The leech protein peptide product is obtained.

[0041] The average molecular weight and fibrinolytic activity of different leech protein peptides are detected by molecular exclusion method (GPC) and fibrin plate method, respectively. Figure 3 is the standard curve of GPC molecular weight detection of the present scheme; Figure 4 is a typical spectrum of GPC molecular detection of the present scheme.

[0042] Experimental group Protease species Average molecular weight (Da) Fibrinolytic activity (U / mg) Experimental group 1 Neutral protease first, then trypsin 824 650.42 Experimental group 2 Papain first, then trypsin 852 535.12 Experimental group 3 Flavourzyme first, then trypsin 792 635.24 Experimental group 4 Bromelain first, then trypsin 832 1100.15 Experimental group 5 Trypsin first, then bromelain 865 850.46 Experimental group 6 Bromelain + trypsin one-pot method (simultaneous enzymolysis) 883 823.43

[0043] As can be seen from the data of Example 2, by stepwise enzymolysis, the average molecular weight of the leech protein peptide can be further reduced, which helps to improve the bioavailability of the leech protein peptide. As can be seen from the comparison of the fibrinolytic activity data, the leech protein peptide prepared by stepwise enzymolysis of bromelain and trypsin in experimental group 4 has the highest fibrinolytic activity. The trypsin recognizes and enzymolyzes specific amino acid sequences, and the small molecule peptides produced show stronger fibrinolytic activity. The fibrinolytic activity of the rest of experimental groups 1-3 has increased, but the increase is limited. The fibrinolytic activity of experimental group 5 and experimental group 6 is also lower than that of experimental group 4, which may be because the non-selective hydrolysis of bromelain after specific cutting changes the composition of the peptide segment, and the non-selective hydrolysis of bromelain also reduces the activity of trypsin.

[0044] Examples 3-6 further investigate the effects of different enzymolysis conditions on the average molecular weight and fibrinolytic activity of the leech protein peptide. Example 3: Fibrinolytic activity and GPC molecular weight detection 1

[0045] The frozen fresh leeches were thawed at room temperature, and the thawed fresh leeches were washed with clean water to remove mud and stickiness. After washing, the excess water was drained. 1 kg of washed leeches were taken, and purified water was added at a ratio of 1:2 to crush and homogenize the wall for 30 min. The homogenate supernatant was collected by centrifugation. The homogenate supernatant was subjected to stepwise enzymolysis. Bromelain was used for primary enzymolysis. Primary enzymolysis: 2 g / L of complex bromelain was added, the pH of enzymolysis was controlled at 7.0, the temperature of enzymolysis was controlled at 50°C, and the stirring enzymolysis was performed for 8 h. After heating and inactivation, the primary enzymolysis supernatant was obtained by centrifugation. Trypsin was used for secondary enzymolysis of the primary enzymolysis supernatant. Secondary enzymolysis: 0.2 g / L of trypsin was added, the pH of enzymolysis was controlled at 7.5, the temperature of enzymolysis was controlled at 30°C, and the stirring enzymolysis was performed for 8 h. After heating and inactivation, the leech protein peptide extract supernatant was obtained by centrifugation. The leech protein peptide extract supernatant was treated with a 0.22 μm hollow fiber membrane, and the leech protein peptide clear filtrate was collected. The leech protein peptide clear filtrate was filtered and sterilized with a 0.22 μm filter cartridge. Finally, spray drying was performed at an inlet temperature of 185°C and an outlet temperature of 85°C to obtain the leech protein peptide product.

[0046] The average molecular weight and fibrinolytic activity of the leech protein peptide were detected by molecular exclusion method (GPC) and fibrin plate method, respectively. The average molecular weight of the leech protein peptide obtained in this example was 765 Da, and the fibrinolytic activity was 1200.00 U / mg. Example 4: Fibrinolytic activity and GPC molecular weight detection 2

[0047] The frozen fresh geoducks are placed at room temperature for natural thawing, and the thawed fresh geoducks are washed with clean water to remove sludge and stickiness. After washing, the geoducks are drained of excess water. 1 kg of the washed geoducks are added with purified water in a ratio of 1:2 for 30 min of homogenate wall breaking. The homogenate supernatant is collected by centrifugation. The homogenate supernatant is subjected to step-by-step enzymatic hydrolysis. Bromelain is used for one-step enzymatic hydrolysis. One-step enzymatic hydrolysis: 5 g / L of compounded bromelain is added, the enzymatic hydrolysis pH is controlled at 7.0, the enzymatic hydrolysis temperature is controlled at 20℃, and the enzymatic hydrolysis is stirred for 12 h. The enzymatic hydrolysis is inactivated by heating, and the one-step enzymatic hydrolysis supernatant is obtained by centrifugation. Trypsin is used for two-step enzymatic hydrolysis of the one-step enzymatic hydrolysis supernatant. Two-step enzymatic hydrolysis: 0.5 g / L of trypsin is added, the enzymatic hydrolysis pH is controlled at 7.5, the enzymatic hydrolysis temperature is controlled at 20℃, and the enzymatic hydrolysis is stirred for 12 h. The enzymatic hydrolysis is inactivated by heating, and the geoduck protein peptide extraction supernatant is obtained by centrifugation. The geoduck protein peptide extraction supernatant is treated with a 0.22 μm hollow fiber membrane, and the geoduck protein peptide clear filtrate is collected. The geoduck protein peptide clear filtrate is filtered and sterilized with a 0.22 μm filter cartridge. Finally, spray drying is performed at an inlet temperature of 185℃ and an outlet temperature of 85℃ to obtain geoduck protein peptide products.

[0048] The average molecular weight and fibrinolytic activity of the geoduck protein peptide are detected by molecular exclusion method (GPC) and fibrin plate method, respectively. The average molecular weight of the geoduck protein peptide obtained in this embodiment is 784 Da, and the fibrinolytic activity is 1188.45 U / mg. Example 5: Fibrinolytic activity and GPC molecular weight detection 3

[0049] The frozen fresh geoducks are placed at room temperature for natural thawing, and the thawed fresh geoducks are washed with clean water to remove sludge and stickiness. After washing, the geoducks are drained of excess water. 1 kg of the washed geoducks is added with purified water in a ratio of 1:3 for 30 minutes of homogenate wall breaking. The homogenate supernatant is collected by centrifugation. The homogenate supernatant is subjected to step-by-step enzymatic hydrolysis. Bromelain is used for one-step enzymatic hydrolysis. One-step enzymatic hydrolysis: 5 g / L of compounded bromelain is added, the enzymatic hydrolysis pH is controlled at 9.0, the enzymatic hydrolysis temperature is controlled at 55°C, and the enzymatic hydrolysis is stirred for 4 hours. The enzyme is inactivated by heating, and the one-step enzymatic hydrolysis supernatant is obtained by centrifugation. Trypsin is used for two-step enzymatic hydrolysis of the one-step enzymatic hydrolysis supernatant. Two-step enzymatic hydrolysis: 0.5 g / L of trypsin is added, the enzymatic hydrolysis pH is controlled at 8.5, the enzymatic hydrolysis temperature is controlled at 37°C, and the enzymatic hydrolysis is stirred for 4 hours. The enzyme is inactivated by heating, and the geoduck protein peptide extraction supernatant is obtained by centrifugation. The geoduck protein peptide extraction supernatant is treated with a 0.22 μm hollow fiber membrane, and the geoduck protein peptide clear filtrate is collected. The geoduck protein peptide clear filtrate is filtered and sterilized with a 0.22 μm filter cartridge. Finally, spray drying is performed at an inlet air temperature of 185°C and an outlet air temperature of 85°C to obtain geoduck protein peptide products.

[0050] The average molecular weight and fibrinolytic activity of the geoduck protein peptide are detected by molecular exclusion method (GPC) and fibrin plate method, respectively. The average molecular weight of the geoduck protein peptide obtained in this embodiment is 865.74 Da, and the fibrinolytic activity is 1154.56 U / mg. Example 6: Fibrinolytic activity and GPC molecular weight detection

[0051] The frozen fresh earthworms are thawed at room temperature, and the thawed fresh earthworms are washed with clean water to remove mud and stickiness. After washing, the excess water is drained. 1 kg of the washed earthworms are added with purified water in a ratio of 1:1 for 30 min of homogenate wall breaking. The homogenate supernatant is collected by centrifugation. The homogenate supernatant is subjected to step-by-step enzymolysis. Bromelain is used for one-step enzymolysis. One-step enzymolysis: 1 g / L of compounded bromelain is added, the enzymolysis pH is controlled at 8.0, the enzymolysis temperature is controlled at 55°C, and the enzymolysis is stirred for 10 h. After heating and inactivation, the one-step enzymolysis supernatant is obtained by centrifugation. Trypsin is used for two-step enzymolysis of the one-step enzymolysis supernatant. Two-step enzymolysis: 0.1 g / L of trypsin is added, the enzymolysis pH is controlled at 8.5, the enzymolysis temperature is controlled at 37°C, and the enzymolysis is stirred for 12 h. After heating and inactivation, the earthworm protein peptide extraction supernatant is obtained by centrifugation. The earthworm protein peptide extraction supernatant is treated with a 0.22 μm hollow fiber membrane, and the earthworm protein peptide clear filtrate is collected. The earthworm protein peptide clear filtrate is filtered and sterilized with a 0.22 μm filter cartridge. Finally, spray drying is performed at an inlet temperature of 185°C and an outlet temperature of 85°C to obtain the earthworm protein peptide product.

[0052] The average molecular weight and fibrinolytic activity of the earthworm protein peptide are detected by molecular exclusion method (GPC) and fibrin plate method, respectively. The average molecular weight of the earthworm protein peptide obtained in this embodiment is 832.14 Da, and the fibrinolytic activity is 1108.56 U / mg.

[0053] Examples 7-11 further investigate the adsorption effect of chelating resin on arsenic in earthworm protein peptide. Example 7: Chelating resin arsenic removal research

[0054] The frozen fresh leeches are thawed at room temperature, and the thawed fresh leeches are washed with clean water to remove mud and stickiness. After washing, the excess water is drained. 5 kg of the washed leeches are taken, and purified water is added at a ratio of 1:2 to crush and homogenize the leeches for 30 minutes. The homogenate is centrifuged to collect the supernatant. The supernatant is subjected to stepwise enzymatic hydrolysis. Bromelain is used for the first enzymatic hydrolysis. The first enzymatic hydrolysis is carried out by adding 2 g / L of the compounded bromelain, controlling the pH to 7.0 and the temperature to 50°C, and stirring for 8 hours. The mixture is heated and inactivated, and centrifuged to obtain the supernatant of the first enzymatic hydrolysis. The supernatant is subjected to the second enzymatic hydrolysis using trypsin. The second enzymatic hydrolysis is carried out by adding 0.2 g / L of trypsin, controlling the pH to 7.5 and the temperature to 30°C, and stirring for 8 hours. The mixture is heated and inactivated, and centrifuged to obtain the leech protein peptide extract supernatant. The leech protein peptide extract supernatant is collected in a volume of 14 L. The 14 L supernatant is evenly divided into 5 parts (2.8 L each). Four parts are subjected to the addition of chelating resin to adsorb the leech protein peptide extract supernatant according to the steps of Examples 8-11. One part is not subjected to the addition of chelating resin, but is directly subjected to 0.22 um hollow fiber membrane treatment to collect the leech protein peptide clear filtrate. The leech protein peptide clear filtrate is filtered and sterilized using a 0.22 um filter. Finally, spray drying is performed at an inlet temperature of 185°C and an outlet temperature of 85°C to obtain 92 g of deep yellow leech protein peptide product. Example 8: Chelating resin arsenic removal study

[0055] According to the resin addition amount of 100 g / L, 2.8 L of leech protein peptide extract supernatant prepared in Example 7 is added to the pretreated macroporous chelating resin (Hubei Ruitian Environmental Protection Technology Co., Ltd., model RTA-800B). The adsorption is carried out at pH 5.0 and 25°C for 12 hours. After the adsorption of the resin is completed, solid-liquid separation is performed using suction filtration to obtain the supernatant after adsorption. The supernatant after adsorption is treated using a 0.22 um hollow fiber membrane to collect the clear filtrate. The leech protein peptide clear filtrate is sterilized and filtered using a 0.22 um filter. Finally, spray drying is performed at an inlet temperature of 185°C and an outlet temperature of 85°C to obtain 90 g of yellow leech protein peptide product. The Kjeldahl method is used to detect the protein concentration of the leech protein peptide extract supernatant and the leech protein peptide extract supernatant after chelating resin adsorption. The results show that the recovery rate of leech protein peptide after chelating resin treatment is 98%. Figure 5 is a process flow chart for preparing leech protein peptide.

[0056] The contents of lead, total arsenic and mercury in the unchelated resin treated earthworm protein peptide product obtained in Example 7 and the chelated resin treated earthworm protein peptide product obtained in Example 8 were detected by using an inductively coupled plasma mass spectrometer (ICP-MS), and the results were as follows: Detection item Example 7 Earthworm protein peptide finished product (mg / kg) Example 8 Earthworm protein peptide finished product (mg / kg) Lead 0.305 0.00253 Arsenic 7.865 0.292 Mercury 0.085 Not detected Example 9: Chelated resin arsenic removal research

[0057] According to the resin addition amount of 400 g / L, the pretreated macroporous chelated resin RTA-800B was added to 2.8 L of the earthworm protein peptide extraction supernatant prepared in Example 7, and adsorbed for 6 h at pH 7.0 and 30°C. After the resin adsorption was completed, solid-liquid separation was performed by using suction filtration to obtain the supernatant after adsorption. The supernatant after adsorption was treated by using a 0.22 μm hollow fiber membrane, and the clear filtrate was collected. The earthworm protein peptide clear filtrate was sterilized by using a 0.22 μm filter cartridge, and finally spray dried at an inlet air temperature of 185°C and an outlet air temperature of 85°C to obtain 88 g of light yellow earthworm protein peptide product. The protein concentrations of the earthworm protein peptide extraction supernatant and the earthworm protein peptide extraction supernatant after chelated resin adsorption were detected by using the Kjeldahl method, and the results showed that the recovery rate of the earthworm protein peptide after chelated resin treatment was 96%.

[0058] The contents of lead, total arsenic and mercury in the chelated resin treated earthworm protein peptide product obtained in the example were detected by using an inductively coupled plasma mass spectrometer (ICP-MS), and the results were as follows: Detection item Example 7 Earthworm protein peptide finished product (mg / kg) Example 9 Earthworm protein finished product (mg / kg) Lead 0.305 Not detected Arsenic 7.865 0.110 Mercury 0.085 Not detected Example 10: Chelated resin arsenic removal research

[0059] According to the resin addition amount of 800 g / L, the pretreated macroporous chelated resin RTA-800B was added to 2.8 L of the earthworm protein peptide extraction supernatant prepared in Example 7, and adsorbed for 4 h at pH 9.0 and 40°C. After the resin adsorption was completed, solid-liquid separation was performed by using suction filtration to obtain the supernatant after adsorption. The supernatant after adsorption was treated by using a 0.22 μm hollow fiber membrane, and the clear filtrate was collected. The earthworm protein peptide clear filtrate was sterilized by using a 0.22 μm filter cartridge, and finally spray dried at an inlet air temperature of 185°C and an outlet air temperature of 85°C to obtain 84 g of light yellow earthworm protein peptide product. The protein concentrations of the earthworm protein peptide extraction supernatant and the earthworm protein peptide extraction supernatant after chelated resin adsorption were detected by using the Kjeldahl method, and the results showed that the recovery rate of the earthworm protein peptide after chelated resin treatment was 92%.

[0060] The contents of lead, total arsenic and mercury in the earthworm protein peptide product treated by the chelating resin obtained in the embodiment were detected by using an inductively coupled plasma mass spectrometer (ICP-MS), and the results were as follows: Detection item Example 7 Earthworm protein peptide finished product (mg / kg) Example 10 Earthworm protein peptide finished product (mg / kg) Lead 0.305 Not detected Arsenic 7.865 0.045 Mercury 0.085 Not detected Example 11: Study on arsenic removal by chelating resin

[0061] According to the resin addition amount of 400 g / L, the macroporous chelating resin RTA-800B (10 batches of adsorption were used for the macroporous chelating resin to treat the earthworm protein peptide extract supernatant) was added to 2.8 L of the earthworm protein peptide extract supernatant prepared in Example 7, and the adsorption was carried out at pH 7.0 and 30℃ for 6 h. After the adsorption of the resin was completed, the solid-liquid separation was carried out by using suction filtration to obtain the supernatant after adsorption. The supernatant after adsorption was treated by using a 0.22 μm hollow fiber membrane, and the clear filtrate was collected. The clear filtrate of the earthworm protein peptide was sterilized by using a 0.22 μm filter cartridge, and finally spray drying was carried out. The inlet air temperature of the spray drying was 185℃, and the outlet air temperature was 85℃. 87 g of light yellow earthworm protein peptide product was obtained. The protein concentration of the earthworm protein peptide extract supernatant and the earthworm protein peptide extract supernatant after chelating resin adsorption was detected by using the Kjeldahl method, and the results showed that the recovery rate of the earthworm protein peptide after chelating resin treatment was 95%.

[0062] The contents of lead, total arsenic and mercury in the earthworm protein peptide product treated by the chelating resin obtained in the embodiment were detected by using an inductively coupled plasma mass spectrometer (ICP-MS), and the results were as follows: Detection item Example 7 Earthworm protein peptide finished product (mg / kg) Example 11 Earthworm protein peptide finished product (mg / kg) Lead 0.305 Not detected Arsenic 7.865 0.112 Mercury 0.085 Not detected The results of Examples 8-11 showed that the macroporous chelating resin was added to the earthworm protein peptide extract supernatant after pretreatment according to the resin addition amount of 100-800 g / L for adsorption, and the contents of lead, arsenic and mercury in the final earthworm protein peptide product met the quality requirements, and the content of arsenic was less than 0.3 mg / kg. Moreover, with the increase of the chelating resin addition amount, the content of arsenic in the earthworm protein peptide product decreased obviously.

[0063] The chelating resin of the application has the functions of decolorization and chelation of other heavy metals, can effectively reduce the color of the earthworm protein peptide and the content of other heavy metals, and improve the properties and quality of the earthworm protein peptide product.

[0064] It was found by comparing the results of Example 9 and Example 11 that the macroporous chelating resin after regeneration had a considerable arsenic chelation effect / arsenic removal effect, and the chelating resin could be used continuously for 10 batches or more, thereby greatly reducing the material cost. Example 12: Study on efficacy

[0065] The earthworm protein peptide prepared by using Example 9 was used to study its influence on carrageenan rat thrombosis.

[0066] Fifty SD rats with equivalent weight were selected, and after adaptive feeding for one week, they were randomly divided into 5 groups, 10 rats in each group, namely normal group, model control group, earthworm protein peptide low-dose group (25 mg / kg), earthworm protein peptide medium-dose group (50 mg / kg), and earthworm protein peptide high-dose group (100 mg / kg). Except for the normal control group, the rest of the groups were subcutaneously injected with 1% carrageenan (prepared with normal saline) at 50 mg / kg.bw on the back of the rats to induce thrombosis, and the environmental temperature was controlled at 17℃, and the rats were free to eat. After 24 h and 48 h of modeling, the length of the tail vein thrombus of the rats was measured with a vernier caliper, and the proportion of the rats with tail vein thrombosis was counted. The experimental results are as follows: Table Influence of earthworm protein peptide on tail vein thrombosis (x± S , n=10) Note: Compared with the model control group, *P<0.05, **P<0.01 After injection of carrageenan, some rats in each test group and the model group had different degrees of congestion and black tails. Compared with the model group, the low-dose earthworm protein peptide could significantly shorten the length of the tail vein thrombus of the rats, but did not reduce the formation rate of the tail vein thrombus of the rats. The medium-dose earthworm protein peptide and the high-dose earthworm protein peptide could both significantly shorten the average length of the tail vein thrombus of the rats after 24 h and 48 h of modeling. The high-dose group had obvious advantages in shortening the average length of the tail vein thrombus of the rats after modeling and reducing the formation rate of the tail vein thrombus, and the thrombosis formation rate was reduced by 40% and 10%, respectively. The experimental results show that the earthworm protein peptide can reduce the formation rate of thrombosis and play a thrombolytic role, and has a certain preventive effect on thrombosis.

[0067] The above only describes the embodiments of the present application, and does not limit the patent range of the present application, and any equivalent results or equivalent process transformations made by using the content of the present application specification, or direct or indirect application in other related technical fields, should be included in the protection range of the present patent.

Claims

1. A method for preparing low molecular weight, low arsenic and high fibrinolytic activity of leech protein peptide, characterized in that, The earthworm protein peptide is obtained by the following steps: stepwise protease hydrolysis of earthworm homogenate supernatant to obtain earthworm protein peptide extraction supernatant, then adding a pretreated macroporous chelating resin for adsorption, collecting the filtrate, and drying to obtain earthworm protein peptide with an average molecular weight of 700-900 Da, a fibrinolytic activity of 1100-1200 U / mg, and an arsenic content of 0.3 mg / kg or less.

2. The method for preparing low molecular weight, low arsenic and high fibrinolytic activity of leech protein peptide according to claim 1, characterized in that, The earthworm homogenate supernatant is prepared by the following method: thawing and washing the earthworm, homogenizing the broken wall, and centrifugal separation to collect the earthworm homogenate supernatant.

3. The method for preparing a low-molecular-weight, low-arsenic, and high-fibrinolytic-activity earthworm protein peptide according to claim 1, wherein: The earthworm protein peptide extraction supernatant is prepared by two times of enzyme hydrolysis, and the earthworm protein peptide with an average molecular weight of 700-900 Da and a fibrinolytic activity of 1100-1200 U / mg can be obtained without filtering membrane screening.

4. The method for preparing a low-molecular-weight, low-arsenic, and high-fibrinolytic-activity earthworm protein peptide according to claim 3, wherein: The two times of enzyme hydrolysis are one time of enzyme hydrolysis using bromelain and two times of enzyme hydrolysis using trypsin.

5. The method for preparing a low-molecular-weight, low-arsenic, and high-fibrinolytic-activity earthworm protein peptide according to claim 4, characterized in that: The one time of enzyme hydrolysis controls the pH to be 7.0-9.0, the enzyme hydrolysis temperature to be 20-55℃, and the bromelain to be added at 1-5 g / L, and the stirring enzyme hydrolysis is carried out for 4-12 h, heating inactivation, and centrifugal separation to obtain the one time of enzyme hydrolysis supernatant.

6. The method for preparing a low-molecular-weight, low-arsenic, and high-fibrinolytic-activity earthworm protein peptide according to claim 4, characterized in that: The two times of enzyme hydrolysis controls the pH to be 7.5-8.5, the enzyme hydrolysis temperature to be 20-37℃, and the trypsin to be added at 0.1-0.5 g / L, and the stirring enzyme hydrolysis is carried out for 4-12 h, heating inactivation, and centrifugal separation to obtain the earthworm protein peptide extraction supernatant.

7. The method for preparing a low-molecular-weight, low-arsenic, and high-fibrinolytic-activity earthworm protein peptide according to claim 1, characterized in that: The macroporous chelating resin pretreatment is first rinsing the chelating resin with purified water, and then pretreating the chelating resin with sodium hydroxide solution and sodium chloride solution, and rinsing the chelating resin with purified water to a pH value of less than 9.

8. The method for preparing a low-molecular-weight, low-arsenic, and high-fibrinolytic-activity earthworm protein peptide according to claim 1, characterized in that: The pretreated macroporous chelating resin is added to the earthworm protein peptide extraction supernatant in an amount of 100-800 g / L for adsorption, and the adsorption pH is controlled to be 5-9, the adsorption temperature is controlled to be 25-40℃, and the adsorption time is controlled to be 4-12 h.

9. The method for preparing a low-molecular-weight, low-arsenic, and high-fibrinolytic-activity earthworm protein peptide according to claim 9, characterized in that: After the adsorption of the chelating resin is completed, the supernatant after adsorption is treated with a 0.22 μm hollow fiber membrane, the clear filtrate is collected, the clear filtrate of the earthworm protein peptide after adsorption is filtered and sterilized with a 0.22 μm filter, and finally dried to obtain the earthworm protein peptide with low molecular weight, low arsenic content and high fibrinolytic activity.

10. The method for preparing a low-molecular-weight, low-arsenic, and high-fibrinolytic-activity earthworm protein peptide according to claim 9, characterized in that: The drying is spray drying, and the inlet air temperature is 180-200℃, and the outlet air temperature is 80-95℃.

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