Preparation method of high-activity pancreatin
By using a stepwise extraction method involving compound enzymatic hydrolysis, gradient activation, and ELP self-precipitation, the problems of defatting, extraction, and stability in the preparation of pancreatic enzymes in existing technologies have been solved, achieving the preparation of pancreatic enzymes with high activity and high yield.
Patent Information
- Application Number
- CN202511124614.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-12
- Publication Date
- 2025-11-07
AI Technical Summary
Existing technologies for pancreatic enzyme preparation suffer from defects in the defatting process, oil hindering extraction and penetration, low yield, and insufficient stability, resulting in low pancreatic enzyme activity and yield.
Lipase, trypsin, and amylase were extracted stepwise using a combination of enzymatic hydrolysis, gradient activation, and ELP self-precipitation. Specific activators were used at different temperatures through gradient activation, and stepwise purification was performed using an ELP-fused lipase scavenger.
It significantly improves the activity and yield of trypsin, pancreatic lipase and pancreatic amylase in pancreatic enzymes, reduces fat residue, meets pharmaceutical grade standards, and is green, safe and free of chemical residues.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological medicine, and particularly relates to a preparation method of high-activity pancreatin. BACKGROUND
[0002] As a multi-enzyme complex, pancreatin mainly contains trypsin, pancreatic amylase and pancreatic lipase, etc. The preparation of pancreatin in the prior art has the following technical bottlenecks: 1. Defects in defatting process: the patent document with the publication number CN107338235B proposes to freeze the raw material first and then defat, thereby reducing the amount of acetone, but still needs 2-3 times of defatting, which cannot completely avoid residues; 2. Oil and fat hinder extraction and penetration: the patent document with the publication number CN107338235B points out that the pancreas slurry without defatting contains a large amount of oil and fat, which hinders the penetration of activators (such as CaCl2) and solvents during direct extraction, and the extraction rate is reduced by 30%; 3. Low yield: the existing technology uses ammonium sulfate fractionation precipitation to separate part of the enzyme components, but introduces high-concentration ions (NH4 + >1M), which needs an additional dialysis step, and the yield is reduced by 12%; 4. Insufficient stability: the use of spray drying process causes the enzyme activity to decrease by 15%-20% due to high temperature (inlet air temperature >120℃), and the product is prone to moisture absorption and caking.
[0003] Based on the above technical defects, it is of great significance to develop a new preparation method of pancreatin to improve the activity and yield of trypsin, pancreatic lipase and pancreatic amylase in pancreatin. SUMMARY
[0004] The present application is to solve the technical problem of low product activity and yield in the preparation of pancreatin in the prior art, and aims to provide a preparation method of high-activity pancreatin, which effectively improves the activity and yield of trypsin, pancreatic lipase and pancreatic amylase in pancreatin through complex enzymolysis, defatting, gradient activation, and finally step-by-step extraction of lipase, trypsin and amylase by ELP self-precipitation.
[0005] The present application is realized through the following technical scheme.
[0006] A preparation method of high-activity pancreatin, characterized in that it comprises the following steps: Breaking and enzymolysis of pig pancreas, defatting to obtain defatted pancreas slurry; Adding first activator CaCl2+MgCl2+glutathione to the defatted pancreas slurry, and performing first-stage activation at 3-5℃, and then increasing the temperature to 13-17℃, and adding second activator bile salt+trehalose to perform second-stage activation; adding the ELP fusion lipase capture agent to the activated pancreatic plasma to obtain a magnetic bead-lipase complex precipitate and a first supernatant; removing the magnetic beads from the magnetic bead-lipase complex precipitate to obtain lipase; centrifuging the first supernatant to obtain an ELP-trypsin complex precipitate and a second supernatant; removing the ELP from the ELP-trypsin complex precipitate to obtain trypsin; adding PEG 6000 to the second supernatant to obtain amylase.
[0007] The present application first crushes, enzymatically digests and degreases a pig pancreas to obtain degreased pancreatic plasma, then performs gradient activation, and finally uses the ELP self-precipitation method to stepwise extract lipase, trypsin and amylase, thereby significantly improving the activity and yield of trypsin, pancreatic lipase and pancreatic amylase in pancreatic enzymes.
[0008] In the first stage of the gradient activation, CaCl2+MgCl2+glutathione is used for activation treatment at 3-5℃, Ca 2+ The trypsinogen is activated, the enzyme-substrate binding force is enhanced by MgCl2, and the sulfhydryl active center is protected by glutathione as a reducing agent to protect the activity of the enzyme. In the second stage, bile salt+trehalose is used for activation treatment at 13-17℃. The bile salt activates the pro-lipase, and trehalose inhibits the thermal denaturation of amylase by glass transition. Therefore, the present application can activate the enzymes by gradient activation under different temperature conditions and using specific activators in different stages, thereby improving the activity of the enzymes.
[0009] In the stepwise extraction of pancreatic enzymes, an ELP fusion lipase capture agent is used for treatment. The ELP fusion lipase capture agent is made of anti-ELP tag nanobodies (KD=10 -9 M) coupled with magnetic beads. The magnetic beads combine with lipase to form a magnetic bead-lipase complex precipitate, thereby separating the lipase from other enzymes. Further centrifugation of the supernatant obtains an ELP-trypsin complex precipitate, thereby separating the trypsin from amylase (in the second supernatant). Finally, the magnetic bead-lipase complex precipitate, the ELP-trypsin complex precipitate and the second supernatant are purified and extracted, respectively, to obtain lipase, trypsin and amylase, respectively. Therefore, the present application uses the ELP fusion lipase capture agent for treatment, which can stepwise separate and purify lipase, trypsin and amylase, thereby effectively improving the yield of pancreatic enzymes.
[0010] Note that ELP is an artificial polypeptide derived from elastin, with the sequence formula (Val-Pro-Gly-Xaa-Gly)_n (Xaa is any amino acid except Pro, and n is the number of repetitions), which has amphiphilic properties: hydrophobic residues (such as Val) and hydrophilic residues (Gly) are arranged alternately, and the Xaa site in the pentapeptide unit can be finely regulated by selecting different amino acids (hydrophilic or hydrophobic) to balance the hydrophilic and hydrophobic levels of the entire molecule. Below the critical phase transition temperature (T t ), ELP is dissolved in water and is in a disordered coiled state, and above the critical phase transition temperature (T t ), ELP collapses and self-assembles into insoluble β-helix-like aggregates.
[0011] Further, the enzymatic hydrolysis refers to adding a complex enzymatic hydrolysis system to the broken mince slurry, and the complex enzymatic hydrolysis system includes lipase and phospholipase A2, and the addition amounts are 0.5% to 1.0% and 0.2% to 1.5% of the weight of the mince slurry, respectively.
[0012] Among them, the lipase is derived from Aspergillus oryzae, with an enzyme activity of ≥10000 U / g, which specifically catalyzes the gradual hydrolysis of intracellular triglycerides to generate free fatty acids (FFA), diglycerides and monoglycerides; the phospholipase A2 is derived from pig pancreas, with an enzyme activity of ≥5000 U / g, which can selectively hydrolyze the ester bond at the sn-2 position of the cell membrane phospholipid to generate lysophospholipids, thereby increasing the permeability of the cell membrane; the use of the two in combination can synergistically destroy the structure of the fat cell membrane and improve the enzymatic hydrolysis efficiency. The complex enzymatic hydrolysis system is prepared by dissolving the lipase and the phospholipase A2 in a Tris-HCl buffer (0.05 M to 0.2 M) with a pH of 6.8.
[0013] Further, the enzymatic hydrolysis is performed under a nitrogen protection environment for 2 to 5 hours. During the enzymatic hydrolysis process, the concentration of the nitrogen gas should be maintained at a dissolved oxygen of ≤0.5 mg / L, so as to reduce the activity loss caused by oxidation, increase the activity recovery rate of oxygen-sensitive enzymes such as trypsin and lipase, and block the vicious cycle of fat oxidation-enzyme inactivation-odor generation from the root, so that the finished product meets the odor standard of the pharmaceutical grade, in addition, the traditional antioxidant (such as BHT) can be replaced, and the risk of chemical residues can be eliminated, which is green and safe.
[0014] Further, the defatting includes three-stage treatment: First-stage centrifugation: remove the upper layer of free oil under the condition of 3-5℃; Second-stage centrifugation: adjust the pH to 4.5 to 6.0 to precipitate undegraded lipid proteins, and remove the intermediate layer of flocculent material by centrifugation; Third-stage refining: use membrane filtration to obtain a clear enzymatic hydrolysate.
[0015] The application adopts centrifuges to carry out two-stage centrifugal treatment, the first-stage centrifugal treatment removes upper free oil (about 68% of total lipid), the collected crude oil is refined by molecular distillation (120 DEG C / 0.1 Pa), and the acid value (AV) is less than or equal to 1.5 mg KOH / g, so that the crude oil can be used as a cosmetic raw material; the second-stage centrifugal treatment further precipitates undegraded lipoprotein, the intermediate layer flocculation is removed by centrifugation, and then the clarified enzymatic hydrolysate is obtained by filtering through a filter membrane, and the final degreasing rate is greater than or equal to 92%, and the residual fat content is less than or equal to 1.8%, compared with the traditional acetone method for degreasing, the application has a higher degreasing rate and a lower fat residue.
[0016] Further, the adding amount of CaCl2, MgCl2 and glutathione in the first-stage activator formula accounts for 0.3% to 1.0%, 0.1% to 0.5% and 0.05% to 0.2% of the weight of the defatted pancreatic juice respectively.
[0017] Further, the adding amount of bile acid salt and trehalose in the second-stage activator formula accounts for 0.2% to 1.0% and 1.0% to 2.0% of the weight of the defatted pancreatic juice respectively.
[0018] Further, the application further comprises adding 30% to 50% (v / w, according to the weight of pig pancreas) of phosphate buffer to the activated pancreatic juice for extraction, centrifugation, collection of the third supernatant, and then adding 0.1% to 0.2% (w / v, according to the volume of the third supernatant) of ELP fusion lipase capture agent. This step is carried out before the step-by-step extraction of pancreatic enzymes, and serves to pre-adjust the ionic strength, so as to create conditions for the subsequent precipitation of the ELP fusion lipase capture agent.
[0019] Further, the lipase obtained after removing the magnetic beads from the magnetic bead-lipase complex precipitate is as follows: glycine-HCl buffer is added to the magnetic bead-lipase complex precipitate, stirring is uniformly carried out at 22-28 DEG C, the magnetic beads in the solution are separated out by using a magnet, the supernatant is collected, and freeze-drying is carried out to obtain the finished lipase.
[0020] Further, the trypsin obtained after removing the ELP from the ELP-trypsin complex precipitate is as follows: the ELP-trypsin complex precipitate is dissolved in TEC buffer, filter membrane filtration is carried out, the filtrate is collected, and freeze-drying is carried out to obtain the finished trypsin.
[0021] Further, the amylase obtained by adding PEG 6000 to the second supernatant is as follows: the pH of the second supernatant is adjusted to 6-7, PEG 6000 is added, stirring is carried out at 22-28 DEG C, the precipitate is collected after centrifugation, the precipitate is dissolved in PBS, the filtrate is collected after centrifugation again, and freeze-drying is carried out on the filtrate to obtain the finished amylase.
[0022] Compared with the prior art, the present application has the following advantages and beneficial effects.
[0023] 1. The present application first crushes, enzymatically hydrolyzes and degreases the pig pancreas to obtain degreased pancreatic juice, then performs gradient activation, and finally uses the ELP self-precipitation method to extract lipase, trypsin and amylase in steps, which significantly improves the activity and yield of trypsin, pancreatic lipase and pancreatic amylase in pancreatic enzymes.
[0024] 2. The present application uses gradient activation, the first stage uses CaCl2+MgCl2+glutathione for activation treatment at 3-5℃, Ca 2+ activates trypsinogen, MgCl2 enhances enzyme-substrate binding, and glutathione protects the sulfhydryl active center as a reducing agent to ensure enzyme activity, and the second stage uses bile salt+trehalose for activation treatment at 13-17℃, bile salt activates pro-lipase, and trehalose inhibits amylase thermal denaturation through glass transition, therefore, the present application can activate the enzymes by gradient activation at different temperature conditions and use specific activators at different stages, so that the activation effect is achieved and the enzyme activity is improved.
[0025] 3. In the step-by-step extraction of pancreatic enzymes, the present application uses ELP fusion lipase capture agent for treatment, which is made of anti-ELP tag nanobody (KD=10 -9 M) coupled magnetic beads, the magnetic beads combine with lipase to form magnetic bead-lipase complex precipitate, so as to separate lipase from other enzymes, then the supernatant is further centrifuged to obtain ELP-trypsin complex precipitate, so as to separate trypsin from amylase (in the second supernatant), and finally the magnetic bead-lipase complex precipitate, ELP-trypsin complex precipitate and the second supernatant are purified and extracted, so as to obtain lipase, trypsin and amylase respectively. Therefore, the present application uses ELP fusion lipase capture agent for treatment, which can achieve the purpose of step-by-step separation and purification of lipase, trypsin and amylase, and effectively improves the yield of pancreatic enzymes.
[0026] 4. The present application uses a complex enzymolysis system of lipase and phospholipase A2 group layers, which can synergistically destroy the structure of fat cell membranes and improve the enzymolysis efficiency.
[0027] 5. In the enzymolysis process, the present application introduces nitrogen, which can reduce the activity loss caused by oxidation, improve the activity recovery rate of oxygen-sensitive enzymes such as trypsin and lipase, and block the vicious cycle of fat oxidation-enzyme inactivation-odor generation from the root, so that the finished product meets the odor standard of pharmaceutical grade. In addition, it can replace traditional antioxidants (such as BHT) to eliminate the risk of chemical residues, and is green and safe, which meets the requirements of FDA 21 CFR 111 regulations.
[0028] 6. The defatting step of the present application adopts centrifuge to carry out twice centrifugal treatment, and then carries out filtration through a filter membrane to obtain a clear enzymatic hydrolysate, and the final defatting rate is greater than or equal to 92%, and the residual fat content is less than or equal to 1.8%, compared with the traditional defatting method using acetone, the defatting rate is effectively improved and the residual fat amount is reduced. DETAILED DESCRIPTION
[0029] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with embodiments. Obviously, the illustrative embodiments of the present application and their descriptions are only used to explain the present application and not as a limitation of the present application.
[0030] The following detailed description of the embodiments of the present application will be made. However, there will be cases where unnecessary detailed descriptions are omitted. For example, there will be cases where detailed descriptions of matters that are well known and repeated descriptions are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate understanding by those skilled in the art.
[0031] The ranges disclosed in the present application are limited by the lower limit and the upper limit in the form of a range, and the given range is limited by selecting a lower limit and an upper limit, and the selected lower limit and upper limit limit the boundaries of the particular range. The range defined in this way can include or not include the end value, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range.
[0032] If not specifically stated, all embodiments and optional embodiments of the present application can be combined with each other to form new technical solutions.
[0033] If not specifically stated, all technical features and optional technical features of the present application can be combined with each other to form new technical solutions.
[0034] If not specifically stated, the "includes" and "contains" mentioned in the present application means open, and can also be closed. For example, the "includes" and "contains" can mean that other substances not listed can also be included or contained, or only the listed substances can be included or contained.
[0035] If not otherwise specified, all steps of the present application can be carried out in sequence or randomly, preferably in sequence. For example, the method comprising steps (a) and (b) means that the method can comprise steps (a) and (b) in sequence, or steps (b) and (a) in sequence. For example, the method mentioned above can further comprise step (c), which means that step (c) can be added to the method in any order, for example, the method can comprise steps (a), (b) and (c), or steps (a), (c) and (b), or steps (c), (a) and (b), etc.
[0036] The technical solutions of the present application are further described in detail below in combination with examples.
[0037] It should be noted that the experimental methods used in the examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, which can be obtained by commercial channels by those skilled in the art.
[0038] Example 1 A preparation method of high-activity pancreatin, comprising the following steps.
[0039] Step 1: raw material selection and pre-cooling treatment Select 1 kg of quarantine qualified live pig pancreas as raw material, and freeze at -20℃ within 2 hours after slaughter for preservation, and maintain cold chain (-18℃±2℃) during transportation. The thawing of the raw material adopts gradient temperature rising method: 0℃~4℃ cold storage thawing for 12~16 hours, and the final temperature of thawing is ≤8℃, so as to avoid cell rupture caused by ice crystal recrystallization.
[0040] Step 2: fine grinding process The thawed pancreas is washed with sterile water to remove surface bloodstains, and is broken by a double helix mincer. Primary crushing: 6 mm screen coarse crushing, rotation speed 500 rpm~800 rpm, time consumption 5~10 minutes. Secondary fine grinding: change 2 mm screen, rotation speed is increased to 1000 rpm~1200 rpm. Time consumption 5~10 minutes, and get paste slurry.
[0041] Step 3: construction of composite enzyme system Add custom composite enzyme solution according to the weight of the paste slurry, and the amount of the enzyme solution is 0.5% (w / w) based on the weight of the paste slurry. Lipase: the amount of addition is 0.5% (w / w), and the enzyme activity is ≥10000 U / g. Its specificity catalyzes the hydrolysis of triglyceride into free fatty acid (FFA) and monoglyceride, and destroys the structure of fat cells; Phospholipase A2: Addition amount 0.2% (w / w), enzyme activity ≥5000 U / g, selectively hydrolyzes the ester bond at the sn-2 position of phospholipids to generate lysophospholipids, thereby increasing cell membrane permeability; Co-buffering system: 10% (w / w) Tris-HCl buffer (pH 6.8, 0.1 M; containing 5 mM Ca) was used. 2+ It dissolves lipase and phospholipase A2, maintaining the optimal pH of the enzyme and stabilizing the enzyme conformation through calcium ions.
[0042] Step 4: Optimization of the enzymatic hydrolysis process After mixing the slurry and the compound enzymatic hydrolysate, the mixture was transferred to a jacketed reactor and enzymatically hydrolyzed at a constant temperature of 35°C for 3 hours with stirring. The key technical parameters are as follows: Temperature control: The temperature fluctuation is maintained within ±0.5℃ through a PID temperature control system; Oxygen content control: Nitrogen gas is introduced to maintain dissolved oxygen ≤0.5 mg / L; Real-time monitoring: Samples are taken every 30 minutes to detect the amount of FFA generated (GB 5009.168-2016 method). The reaction is terminated when the amount of FFA released reaches 80% to 85% of the theoretical value.
[0043] Step 5: Centrifugal degreasing and oil recovery The enzymatically hydrolyzed material is then separated and purified using a tubular centrifuge: Primary separation: The upper free oil (accounting for about 68% of the total lipids) is removed at 4℃. The collected crude oil is purified by molecular distillation (120℃ / 0.1 Pa). The acid value (AV) is ≤1.5 mg KOH / g, which can be used as a cosmetic raw material. Secondary separation: Adjust the pH to 5.0 to precipitate undegraded lipoproteins, and centrifuge to remove the intermediate flocculent layer; Three-stage refining: A clear enzymatic hydrolysate is obtained by filtration through a 0.22μm membrane, with a final defatting rate of ≥94% and a residual fat content of ≤1.5% (the residual fat content of the traditional acetone method is 4.5%±0.3%).
[0044] Step 6: Gradient Activation First stage: Add a compound activator (CaCl2 0.3% + MgCl2 0.1% + glutathione 0.05%) to defatted pancreatic syrup, stir at 4℃ for 1 h to activate trypsinogen; Second stage: Heat to 15°C, add bile salts (0.2%) and trehalose (1%), stir for 1 hour to activate lipase and stabilize amylase activity.
[0045] Step 7: Step-by-step extraction (adapting to ELP tags) (1) Adjusting ionic strength: Add 40% (v / w, based on the weight of the raw material) of pH 6.5 phosphate buffer containing 1 M NaCl to the defatted pancreatic slurry of step 6, stir at 4°C for 30 min, and collect the supernatant by centrifugation (4°C, 8000 rpm x 10 min); (2) Medium temperature extraction of lipase: Adjust the pH of the supernatant in step 7(1) to 7.0, add 0.1% (w / v, based on the volume of the supernatant in step 7(1)) of ELP fusion lipase capture agent, stir at 25°C for 30 min, filter, and reserve the supernatant, and collect the magnetic bead-lipase complex precipitate; (3) Lipase purification Add dissociation buffer (0.1 M glycine-HCl, pH 2.5) to the magnetic bead-lipase complex precipitate of step 7(2), stir at 25°C for 5 min, separate the magnetic beads in the solution with a magnet, and collect the supernatant (i.e., purified lipase solution).
[0046] Step 8: Phase transition precipitation of trypsin (1) Take the supernatant of step 7(2), warm to 37°C, stand for 60 min, low-speed centrifugation (4°C, 4000 rpm x 5 min), reserve the supernatant, and collect the ELP-trypsin complex precipitate.
[0047] (2) Dissolve the ELP-trypsin complex precipitate with 4°C TEC buffer (50 mM Tris, 1 mM EDTA, 5 mM CaCl2, pH 8.0), filter with 0.22 μm, collect the filtrate (i.e., purified trypsin solution), and collect the membrane surface intercept for ELP tag regeneration (containing ELP polymer).
[0048] Step 9: Amylase extraction Adjust the pH of the supernatant of step 8(1) to 6.5, add 15% PEG 6000, stir at 25°C for 30 min, collect the precipitate by centrifugation (8000 rpm x 10 min), dissolve the precipitate with pH 7.0 PBS, and collect the filtrate (i.e., purified pancreatic amylase solution) by centrifugation.
[0049] Step 10: Drying Combine the supernatant of step 7(3) with the filtrate of step 8(2) and the filtrate of step 9, vacuum freeze-dry, crush through a 100-mesh sieve, and obtain the finished product of pancreatin.
[0050] Example 2 The difference between this example and Example 1 is that the pH adjustment for the secondary separation in step 5 is 4.5.
[0051] Example 3 The difference between this example and Example 1 is that the amount of phospholipase A2 added in step 3 is increased to 0.6% (w / w).
[0052] Comparative Example 1 The difference between this comparative example and Example 1 is that the enzyme system in step 3 is replaced by lipase + buffer, i.e. phospholipase A2 is removed.
[0053] Comparative Example 2 The difference between this comparative example and Example 1 is that nitrogen is not introduced during the enzymatic process in step 4.
[0054] Comparative Example 3 The difference between this comparative example and Example 1 is that step 6 is not gradient activated, but only the first stage of activation is performed.
[0055] Comparative Example 4 The difference between this comparative example and Example 1 is that step 6 is not gradient activated, but only the first stage of activation is performed.
[0056] Comparative Example 5 The difference between this comparative example and Example 1 is that step 6 is not gradient activated, but both activators are added to the defatted pancreatic juice at the same time, and stirred at 15°C for 1 h.
[0057] Comparative Example 6 The difference between this comparative example and Example 1 is that steps 7-9 are not ELP precipitation extraction, i.e. replaced by: (1) Precipitant preparation: 0.5% chitosan + 0.3% sodium alginate is dissolved in 0.1 M acetic acid buffer (pH 5.0); (2) Precipitation operation: add the precipitant (volume ratio 1:4) to the defatted pancreatic juice of step 6, stir at 4°C for 1 h, centrifuge (8000 rpm x 10 min), and wash the precipitate with deionized water twice; (3) Dissociation and purification: add 0.1 M phosphate buffer (pH 7.5) containing 0.5 M NaCl to the precipitate, stir at 4°C for 30 min, and collect the supernatant by centrifugation.
[0058] Comparative Example 7 The difference between this comparative example and Example 1 is that step 5 uses the traditional acetone method for defatting, with the following specific operation: Add acetone to the material after enzymatic hydrolysis until the specific gravity of the mixture is 0.830-0.840 (20-25°C), stir for 30 minutes, stand for 30 minutes, siphon the upper liquid, and retain the precipitate; repeat the above operation once.
[0059] The defatting rate and residual fat amount of the materials treated in step 5 of the above Example 1 and Comparative Example 7 are detected, and the data are shown in Table 1.
[0060] Table 1. The defatting rate and residual fat amount data of Example 1 and Comparative Example 7 after step 5 treatment
[0061] From the data in Table 1, it can be seen that by using the three-stage defatting treatment method of the present application, the defatting rate can reach 94%, and the residual fat amount is only 1.5%, while the defatting rate of the traditional acetone method is lower, only 72%, and the residual fat content is higher, 4.6%.
[0062] The finished pancreatin prepared in the above examples and comparative examples was detected, and the pancreatin activity and yield data are shown in Table 2.
[0063] Table 2, detection data of the finished pancreatin prepared in the examples and comparative examples
[0064] From the data in Table 2, it can be seen that: Comparative Example 1 does not use a composite enzymatic hydrolysis system, and only uses lipase for enzymatic hydrolysis, resulting in a very low enzyme release rate, which proves that the composite enzymatic hydrolysis system has a promoting effect on improving the activity and yield of the enzyme; Comparative Example 2 does not pass nitrogen protection during enzymatic hydrolysis, and the activity of each enzyme is also greatly reduced, which proves that nitrogen protection can effectively improve the activity of the enzyme and increase the yield of pancreatin.
[0065] Comparative Examples 3-4 use a single activation method, and the activity of the enzyme and the yield of pancreatin are greatly reduced. Although Comparative Example 5 uses two activators, it does not use gradient activation, and the activity of the enzyme and the yield of pancreatin are also significantly reduced.
[0066] Comparative Example 6 does not use ELP precipitation separation to extract pancreatin, and the activity and yield of the product are greatly reduced.
[0067] Finally, it should be noted that: the above specific examples are only used to explain the purpose, technical solutions and beneficial effects of the present application, and it should be understood that the above description is only a specific embodiment of the present application and does not limit the protection scope of the present application; although the present application has been described in detail with reference to the foregoing specific embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent replacement, improvement, etc. to part or all of the technical features; and these modifications, equivalent replacement, improvement, etc. do not make the essence of the corresponding technical solution deviate from the scope of the technical solutions of the embodiments of the present application, and they should be covered in the scope of the claims and description of the present application.
Claims
1. A method for preparing a high activity pancreatin, characterized by, The method comprises the following steps: The pig pancreas is broken, enzymatically hydrolyzed, and defatted to obtain defatted pancreas pulp; A first activator CaCl2+MgCl2+glutathione is added to the defatted pancreas pulp, and a first-stage activation is performed at 3-5℃, and then the temperature is raised to 13-17℃, and a second activator bile salt+trehalose is added to perform a second-stage activation; An ELP fusion lipase capture agent is added to the activated pancreas pulp to obtain a magnetic bead-lipase complex precipitate and a first supernatant; The magnetic bead-lipase complex precipitate is removed to obtain lipase. The ELP-pancreatin complex precipitate is removed to obtain pancreatin. PEG 6000 is added to the second supernatant to obtain amylase. The enzymatic hydrolysis refers to adding a complex enzymatic hydrolysis system to the broken pulp, and the complex enzymatic hydrolysis system comprises lipase and phospholipase A2, and the addition amounts are 0.5% to 1.0% and 0.2% to 1.5% of the weight of the pulp, respectively.
2. A process for the preparation of high activity pancreatin as claimed in claim 1 wherein, The enzymatic hydrolysis is performed for 2 to 5 hours in a nitrogen protection environment.
3. The method of claim 1, wherein the high activity pancreatin is prepared by the steps of: The defatting comprises three-stage treatment:
4. The method of claim 1, wherein the pancreatin is prepared by the steps of: First-stage centrifugation: removing free oil at the top under the condition of 3-5℃; Second-stage centrifugation: adjusting the pH to 4.5 to 6.0 to precipitate undegraded lipoprotein, and removing the intermediate layer of flocculation by centrifugation; Third-stage refining: using membrane filtration to obtain clear enzymatic hydrolysate. The addition amounts of CaCl2, MgCl2, and glutathione in the first-stage activator formula are 0.3% to 1.0%, 0.1% to 0.5%, and 0.05% to 0.2% of the weight of the defatted pancreas pulp, respectively.
5. The method of claim 1, wherein the pancreatin is prepared by the steps of: The addition amounts of bile salt and trehalose in the second-stage activator formula are 0.2% to 1.0% and 1.0% to 2.0% of the weight of the defatted pancreas pulp, respectively.
6. The process for the preparation of high activity pancreatin as claimed in claim 1 wherein, Further comprising adding 30% to 50% (v / w, based on the weight of the pig pancreas) of phosphate buffer to the activated pancreas pulp for extraction, centrifugation, collection of the third supernatant, and then adding 0.1% to 0.2% (w / v, based on the volume of the third supernatant) of ELP fusion lipase capture agent.
7. The method for preparing a highly active trypsin according to claim 1, characterized in that, The specific method for obtaining lipase after removing the magnetic beads from the magnetic bead-lipase complex precipitate is as follows:
8. The method for preparing a highly active trypsin according to claim 1, characterized in that, Glycine-HCl buffer is added to the magnetic bead-lipase complex precipitate, and the mixture is stirred uniformly at 22-28℃, the magnetic beads in the solution are separated out by using a magnet, the supernatant is collected, and freeze-drying is performed to obtain finished lipase. The specific method for obtaining pancreatin by removing ELP from the ELP-pancreatin complex precipitate is as follows:
9. The method for preparing a highly active trypsin according to claim 1, characterized in that, The ELP-pancreatin complex precipitate is dissolved in TEC buffer, membrane filtration is performed, the filtrate is collected, and freeze-drying is performed to obtain finished pancreatin. The specific method for obtaining amylase by adding PEG 6000 to the second supernatant is as follows:
10. The method for preparing a highly active trypsin according to claim 1, characterized in that, The pH of the second supernatant is adjusted to 6-7, PEG 6000 is added, the mixture is stirred and reacted at 22-28℃, the precipitate is collected after centrifugation, the precipitate is dissolved in PBS, the filtrate is collected after centrifugation again, and freeze-drying is performed on the filtrate to obtain finished amylase.
Citation Information
Patent Citations
Methods for preparing pancreatic enzymes
CN107338235B