A rapid preparation method of pancreatin without exogenous activation

Abstract

This invention discloses a rapid method for preparing pancreatic enzymes without exogenous activation. This invention relates to the field of bioenzyme preparation technology and includes the following steps: S1: Crush pancreatic raw materials to prepare pancreatic slurry; S2: Add purified water, cold acetone, and trypsin to the pancreatic slurry, mix well, and control the initial temperature of the system to 0-2℃; S3: Perform gradient-controlled low-temperature autolysis under continuous stirring, controlling the temperature gradient of the system to rise to 10-14℃; S4: Filter to remove connective tissue and collect the pancreatic enzyme slurry; S5: Add cold acetone to the pancreatic enzyme slurry for precipitation, layering, defatting, and dehydration, and collect the precipitate; S6: Perform low-temperature vacuum drying on the precipitate to obtain the finished pancreatic enzyme product. This invention achieves the purpose of shortening the preparation cycle and improving the recovery rate of trypsin, pancreatic amylase, and pancreatic lipase activities in the finished pancreatic enzyme product by combining low-temperature gradient-controlled autolysis with cold acetone to inhibit excessive hydrolysis of proteases.

Description

Technical Field

[0001] This invention relates to the field of biological enzyme preparation technology, specifically to a rapid method for preparing pancreatic enzymes without exogenous activation. Background Technology

[0002] Pancreatic enzymes are complex enzyme preparations extracted from the pancreas of animals such as pigs and cattle. Their core active components include trypsin, pancreatic amylase, and pancreatic lipase, and they have wide applications in the pharmaceutical, feed, and food processing fields. As a key raw material for digestive drugs, the quality of pancreatic enzyme preparations directly depends on the synergistic activity and recovery rate of these three enzymes. Currently, industrial-scale pancreatic enzyme preparation generally adopts a technical route of pancreatic crushing – exogenous activation – autolysis extraction – separation and purification. This involves adding metal salts such as calcium chloride and magnesium chloride, or exogenous bioactivators such as enterokinase and duodenal extract, to the system after crushing the pancreatic tissue. The mixture is then incubated at a constant temperature of 20–35°C for 8–48 hours to convert endogenous pancreatic trypsinogen into catalytically active trypsin. This activation step is the core of the entire process chain, directly determining the subsequent autolysis efficiency and the enzyme activity level of the final product.

[0003] Among representative existing technologies, Chinese patent CN115216466A uses CaCl2 in combination with exogenous trypsin for activation at 20-25℃ for 1-2 hours followed by cold acetone precipitation, relying entirely on calcium salt as the exogenous activator. Chinese patent CN101805728A uses CaCl2 / MgCl2 for activation at 15-35℃ for 8-48 hours, reflecting the industry's general understanding of long-term activation at medium to high temperatures to achieve full activation of the zymogen. While US patent US5861291A introduces acetone during the autolysis stage, it is only used as an auxiliary means of reducing viscosity and inhibiting bacteria, and does not constitute a technical approach to using acetone to regulate protease hydrolysis behavior and protect amylase and lipase activity. These technical routes have been validated through years of industrial practice and have formed relatively fixed process models.

[0004] However, the existing technical approach has revealed multiple shortcomings in long-term application. First, the exogenous activation step is extremely time-consuming, extending the overall production cycle by more than 8 hours, resulting in low equipment turnover efficiency and a significant increase in energy costs, making it difficult to meet the requirements of modern biomanufacturing for efficient and continuous production. Second, the introduction of exogenous materials such as calcium salts, intestinal fluid, and bile introduces a large amount of inorganic salt ions and impurities into the system, not only increasing the load on subsequent purification processes but also leading to low purity of the finished product, affecting the safety and stability of the formulation. More critically, under the high-temperature activation conditions of 20-35°C, activated trypsin lacks effective inhibition mechanisms, continuously undergoing self-hydrolysis and degradation of other enzyme components, resulting in the loss of pancreatic amylase and pancreatic lipase activity, and generally low recovery rates of the three enzymes. Simultaneously, prolonged exposure to medium-high temperatures significantly increases the risk of microbial growth, raising the probability of exceeding microbial limits in the product.

[0005] For a long time, a technical bias has existed in the field that trypsinogen must rely on exogenous metal ions or enterokinase for activation and can only achieve efficient activation in the 20-35°C range. It is believed that endogenous zymogens cannot spontaneously transform under low-temperature conditions, and that low temperatures significantly reduce enzyme activity. This entrenched understanding has prevented current technologies from overcoming the dual dependence on exogenous activators and high-temperature conditions. No solution has yet been found that can simultaneously achieve process integration that eliminates the need for exogenous activators, allows for controlled autolysis at low temperatures, and allows for the gentle regulation of protease hydrolysis behavior by acetone. This remains a technical challenge that needs to be addressed in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid preparation method for trypsin without exogenous activation. This method uses low-temperature gradient temperature-controlled autolysis combined with cold acetone to inhibit excessive hydrolysis of the protease, thereby solving the technical problems of traditional processes that rely on exogenous activators, have long activation times, and low recovery rates of the three enzyme activities.

[0007] To address the aforementioned technical problems, embodiments of the present invention provide a rapid method for preparing trypsin without exogenous activation, comprising the following steps: S1: The raw pancreas is crushed to produce pancreatic syrup; S2: Add purified water, cold acetone and trypsin to the pancreatic syrup, mix well and control the initial temperature of the system to 0~2℃; S3: Under continuous stirring, perform gradient temperature-controlled low-temperature autolysis, controlling the temperature gradient of the system to rise to 10~14℃. S4: Filter to remove connective tissue and collect pancreatic enzyme slurry; S5: Add cold acetone to the pancreatic enzyme slurry for precipitation, separation, defatting, and dehydration, and collect the precipitate; S6: The precipitate is dried under low temperature and vacuum to obtain the pancreatic enzyme product.

[0008] As an optional implementation, in step S3, the heating rate of the gradient temperature-controlled low-temperature autolysis is 1~3℃ / h.

[0009] As an optional implementation, based on 1 kg of pancreatic raw material, the amount of cold acetone added in step S2 is 200~500 mL, and the temperature of the cold acetone is -15℃~-10℃.

[0010] As an optional implementation, the amount of trypsin added in step S2 is 1~10g, based on 1kg of pancreatic raw material.

[0011] As an optional implementation, the amount of purified water added in step S2 is 300~900g, based on 1kg of pancreatic raw material.

[0012] As an optional implementation, in step S1, the crushing process sequentially includes slicing, coarse crushing with a meat grinder, and fine grinding with a pulping machine, and removing free fat clumps from the surface.

[0013] As an optional implementation method, based on 1 kg of pancreatic raw material, the amount of cold acetone added in step S5 is 5-7 L. After standing at low temperature and separating into layers, the upper organic waste liquid is discarded, and the resulting precipitate is then repeatedly degreased and dehydrated by adding 2-3 L of cold acetone.

[0014] As an optional implementation, in step S5, the temperature of the cold acetone is -15°C to -10°C.

[0015] As an optional implementation, in step S6, the low-temperature vacuum drying is carried out under low-temperature negative pressure conditions to remove organic solvents; The drying temperature is 20~30℃, the drying time is 12~16h, and the vacuum degree is 600~750mmHg.

[0016] As an optional implementation, in step S1, the pancreatic raw material is porcine pancreas and / or bovine pancreas, and the particle size of the homogenized pancreatic slurry is 5-15 μm.

[0017] Compared with the prior art, the embodiments of the present invention have the following advantages and beneficial effects: 1. In this embodiment of the invention, pancreatic raw material is crushed into pancreatic paste. Purified water, cold acetone, and trypsin are added to the pancreatic paste and mixed thoroughly. The initial temperature of the system is controlled at 0-2°C. Under continuous stirring, a gradient temperature-controlled low-temperature autolysis is performed to raise the system temperature gradient to 10-14°C. The low-temperature environment slows down the hydrolysis rate of the protease, while relying on a trace amount of trypsin to initiate a cascade activation to achieve the autonomous conversion of endogenous trypsinogen into active trypsin. Enzyme activation can be achieved without the intervention of exogenous activators such as calcium salts, magnesium salts, enterokinase, or duodenal extract. The addition of cold acetone slightly changes the hydrophobic conformation of the protease surface by reducing the dielectric constant of the aqueous phase, and gently inhibits the excessive hydrolytic activity of the activated trypsin during the low-temperature autolysis process, avoiding its impact on the spatial structure of pancreatic amylase and pancreatic lipase. The gradient temperature control mechanism matches the zymogen activation rate with the protease hydrolysis rate, preventing the degradation of complex enzymes caused by a sudden burst of large amounts of active protease. After autolysis, the connective tissue is removed by filtration and the pancreatic enzyme slurry is collected. Then, it undergoes cold acetone precipitation and separation, as well as defatting and dehydration to remove lipid-soluble impurities and residual organic solvents. Finally, the organic solvent is removed by low-temperature vacuum drying to obtain the finished pancreatic enzyme. The overall process eliminates the more than 8-hour exogenous activation step in the traditional process, avoids the introduction of exogenous metal ions and contaminating proteins, and reduces the subsequent purification load. At the same time, the low-temperature short-cycle operation effectively inhibits the growth of contaminating bacteria, so that the three core active components of pancreatic enzyme, pancreatic amylase and pancreatic lipase are synergistically protected during autolysis and separation purification, realizing the efficient preparation of pancreatic enzyme without exogenous activators.

[0018] 2. In this embodiment of the invention, the heating rate of the gradient temperature-controlled low-temperature autolysis is limited to 1~3℃ / h, so that the system temperature slowly rises from 0~2℃ to 10~14℃ at a controllable rate. This heating rate matches the spontaneous activation kinetics of the zymogen, avoiding excessively rapid heating that would cause a large amount of trypsinogen to be converted into active trypsin in a short time, thus preventing concentrated burst hydrolysis. At the same time, it prevents excessively slow heating that would cause the autolysis period to be excessively prolonged. Thus, at the kinetic level, the zymogen activation efficiency and the protease hydrolysis inhibition requirements are balanced, minimizing the loss of activity of trypsin, pancreatic amylase and pancreatic lipase, and achieving efficient retention of the synergistic activity of the three enzymes.

[0019] 3. In this embodiment of the invention, by limiting the amount of cold acetone added in step S2 to 200~500mL (based on 1kg of pancreatic raw material) and controlling the temperature at -15~-10℃, the cold acetone is introduced into the system at the initial stage of autolysis to exert a mild inhibitory effect. This dosage range ensures that acetone molecules are fully distributed in the aqueous phase to effectively reduce the dielectric constant. If the dosage is below this range, the inhibition will be insufficient, leading to excessive hydrolysis of the protease and severe degradation of the three enzymes. If the dosage is above this range, it will easily cause irreversible denaturation and inactivation of the protease. At the same time, the introduction of low-temperature acetone does not destroy the spatial conformational stability of amylase and lipase, thereby forming a synergistic mechanism of cascade activation initiated by a trace amount of trypsin and rate-controlled inhibition by cold acetone in the autolysis stage, achieving the dual technical effects of controllable activation of zymogen and protection of complex enzymes.

[0020] 4. In this embodiment of the invention, the amount of cold acetone added in step S5 is limited to 5-7L (based on 1kg of pancreatic raw material) and its temperature is controlled at -15 to -10℃. After low-temperature settling and separation, the upper organic waste liquid is discarded. The resulting precipitate is then repeatedly degreased and dehydrated by adding 2-3L of cold acetone. By utilizing the high efficiency of low-temperature acetone in extracting fat-soluble impurities and its separation characteristics with water, selective precipitation of pancreatic enzyme proteins and simultaneous removal of fat impurities are achieved during the precipitation stage. At the same time, the low-temperature environment maintains the conformational stability of the enzyme protein, avoiding the loss of protease activity during traditional high-temperature or room-temperature precipitation. Thus, efficient degreasing and dehydration are completed without introducing exogenous salts, improving the purity of the finished product and reducing the ash content. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings: Figure 1 This is a schematic diagram of a gradient heating curve provided in an embodiment of the present invention; Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0023] Therefore, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0024] This invention provides a rapid method for preparing trypsin without exogenous activation, comprising the following: (1) Crushing pretreatment Pancreatic raw materials are crushed and pretreated to produce pancreatic paste; specifically, pig pancreas and / or bovine pancreas are sequentially sliced, coarsely crushed and finely ground, and free fat blocks on the surface are removed to produce homogeneous pancreatic paste with a particle size of 5-15 μm, so as to ensure the uniformity of prozymogen release during subsequent autolysis. (2) Low-temperature batching Add purified water, cold acetone, and trypsin to the pancreatic slurry, mix well, and control the initial temperature of the system to 0-2℃. Specifically, based on 1 kg of pancreatic raw material, the amount of purified water added is 300-900 g, the amount of cold acetone added is 200-500 mL at a temperature of -15 to -10℃, and the amount of trypsin added is 1-10 g. The mechanism of action of cold acetone at this stage is to reduce the dielectric constant of the aqueous phase and slightly alter the hydrophobic conformation of the protease surface, thereby gently inhibiting the excessive hydrolytic activity of trypsin, while not destroying the spatial structural stability of pancreatic amylase and pancreatic lipase, achieving synergistic protection through low-temperature autolysis activation and acetone-controlled hydrolysis. (3) Gradient temperature control low-temperature autolysis Gradient-controlled low-temperature autolysis was performed under continuous stirring, with the temperature gradient controlled to rise to 10–14 °C at a rate of 1–3 °C / h (refer to...). Figure 1 (As shown). This gradient temperature control mechanism matches the zymogen activation rate with the protease hydrolysis rate, avoiding a sudden burst of large amounts of active protease, reducing the degradation of complex enzymes from a kinetic perspective, while the low-temperature environment effectively inhibits the growth of miscellaneous bacteria; (4) Separation and impurity removal After autolysis is complete, filter to remove connective tissue and collect pancreatic enzyme slurry; (5) Cold acetone precipitation and layering Cold acetone was added to the pancreatic enzyme slurry for precipitation, separation, defatting, and dehydration, and the precipitate was collected. Specifically, based on 1 kg of pancreatic raw material, the amount of cold acetone added was 5-7 L and the temperature was -15 to -10℃. After low-temperature standing and separation, the upper organic waste liquid was discarded. The obtained precipitate was then repeatedly defatted and dehydrated with 2-3 L of cold acetone to fully remove fat-soluble impurities and reduce the ash content of the finished product. (6) Low-temperature vacuum drying Finally, the above precipitate is subjected to low-temperature vacuum drying to remove organic solvents, yielding the finished trypsin product. The drying temperature is 20-30℃, the drying time is 12-16 hours, and the vacuum degree is 600-750 mmHg. The low-temperature negative pressure conditions effectively prevent thermal denaturation of the enzyme protein, ensuring the activity of the finished product.

[0025] In summary, this invention provides a rapid method for preparing pancreatic enzymes without exogenous activation. After homogenizing porcine and / or bovine pancreatic slurry, 300-900g of purified water, 200-500mL of cold acetone at -15 to -10°C, and 1-10g of trypsin are added. The initial temperature is 0-2°C, and the temperature is gradually increased at a rate of 1-3°C / h to 10-14°C for low-temperature autolysis. Spontaneous cascade activation is achieved by endogenous pancreatic trypsinogen induced by a trace amount of trypsin. The entire process requires no CaCl2, MgCl2, enterokinase, or duodenal immersion. No exogenous activators such as effluent are used; during the autolysis stage, a quantitative amount of cold acetone is used to reduce the dielectric constant of the aqueous phase and slightly change the hydrophobic conformation of the protease surface, thus gently inhibiting the excessive hydrolytic activity of trypsin, while maintaining the spatial structural stability of pancreatic amylase and pancreatic lipase, achieving synergistic protection of the complex enzymes; after autolysis, the connective tissue is removed by slurry filtration, and then 5~7L of cold acetone at -15~-10℃ is added to the pancreatic enzyme slurry for precipitation and separation. After discarding the upper organic waste liquid, the precipitate is repeatedly degreased and dehydrated with 2~3L of cold acetone, and finally the organic solvent is removed by low-temperature vacuum drying to obtain the finished pancreatic enzyme product.

[0026] The core innovation of this invention, which differs from the prior art, lies in the following: Unlike CN115216466A and CN101805728A, this invention completely eliminates the use of exogenous activators such as CaCl2 metal salts and abandons the 20-35℃ high-temperature activation step. Instead, it employs a gradient low-temperature autolysis process with an initial temperature of 0-2℃, an final temperature of 10-14℃, and a heating rate of 1-3℃ / h. This allows endogenous pancreatic trypsinogen to achieve autonomous cascade activation under the initiation of trace amounts of trypsin, completing the zymogen conversion without calcium ion mediation. Unlike US5861291A, in this invention, acetone is not merely used for viscosity reduction and antibacterial purposes. Instead, it is introduced into the autolysis system at a precise dosage of 200-500 mL (per 1 kg of pancreatic raw material) and a low-temperature condition of -15 to -10℃, forming a synergistic technical concept of low-temperature autolysis activation + acetone-controlled hydrolysis. This technology is not found in the prior art.

[0027] The combined effect of temperature control rate and limited acetone usage breaks the industry's technical prejudice that it must rely on high-temperature exogenous activation, eliminating the need for an exogenous activation process of more than 8 hours, shortening the overall preparation cycle by more than 40%, and improving equipment utilization. Without the introduction of exogenous proteins such as calcium salts and intestinal fluid, the ash and protein content of the finished product are reduced by more than 30%. The total activity recovery rate of trypsin, pancreatic amylase, and pancreatic lipase is increased to over 90%, which is 15-22% higher than the traditional CaCl2 high-temperature activation process. The elimination of activator procurement costs and the lack of a significant increase in total organic solvent consumption result in a reduction of approximately 18% in overall manufacturing costs. The low-temperature, short-cycle process reduces microbial growth, and the microbial limit index of the finished product is superior to that of the traditional high-temperature activation process. The process is simple, cost-controllable, and suitable for continuous industrial production.

[0028] To better demonstrate the significant effects of the embodiments of the present invention, specific experimental examples will be set up below to verify the effects.

[0029] Example 1: This embodiment of the invention provides a rapid preparation method for trypsin without exogenous activation, comprising the following: (1) Crushing pretreatment The pig pancreas was processed by slicing, coarse crushing and fine grinding, and the free fat blocks on the surface were removed to make a homogeneous pancreatic paste with a particle size of 10 μm.

[0030] (2) Low-temperature batching Add 500g of purified water, 350mL of cold acetone at -10℃ and 5g of trypsin to the homogenized pancreatic syrup, stir at low speed to mix well, and control the initial temperature of the system to 0℃.

[0031] (3) Gradient temperature control low-temperature autolysis Continue stirring and control the heating rate at 2℃ / h to allow the system temperature to rise uniformly from 0℃ to 12℃, thus completing autolysis.

[0032] (4) Separation and impurity removal The separator filters out connective fibrous tissue and collects the clear pancreatic enzyme slurry.

[0033] (5) Cold acetone precipitation and layering Add 6L of cold acetone at -10℃ to the above pancreatic enzyme slurry, let it stand at low temperature to separate into layers, and discard the upper organic waste liquid; add 2.5L of cold acetone to the obtained precipitate for repeated degreasing and dehydration, and collect the precipitate.

[0034] (6) Low-temperature vacuum drying The above precipitate was placed under low temperature and negative pressure conditions for vacuum drying. The drying temperature was 25℃, the drying time was 14h, and the vacuum degree was 675 mmHg. After removing the organic solvent, the pancreatic enzyme product was obtained.

[0035] Example 2: This embodiment of the invention provides a rapid preparation method for trypsin without exogenous activation, comprising the following: (1) Crushing pretreatment The pig pancreas was processed by slicing, coarse crushing and fine grinding, and the free fat blocks on the surface were removed to make a homogeneous pancreatic paste with a particle size of 5 μm.

[0036] (2) Low-temperature batching Add 300g of purified water, 200mL of cold acetone at -15℃ and 1g of trypsin to the homogenized pancreatic syrup, stir at low speed to mix, and control the initial temperature of the system to 1℃.

[0037] (3) Gradient temperature control low-temperature autolysis Continue stirring and control the heating rate at 1℃ / h to make the system temperature rise uniformly from 1℃ to 10℃, thus completing autolysis.

[0038] (4) Separation and impurity removal The separator filters out connective fibrous tissue and collects the clear pancreatic enzyme slurry.

[0039] (5) Cold acetone precipitation and layering Add 5L of cold acetone at -15℃ to the above pancreatic enzyme slurry, let it stand at low temperature to separate into layers, and discard the upper organic waste liquid; add 2L of cold acetone to the obtained precipitate for repeated degreasing and dehydration, and collect the precipitate.

[0040] (6) Low-temperature vacuum drying The above precipitate was placed under low temperature and negative pressure conditions for vacuum drying. The drying temperature was 20℃, the drying time was 12h, and the vacuum degree was 600 mmHg. After removing the organic solvent, the pancreatic enzyme product was obtained.

[0041] Example 3: This embodiment of the invention provides a rapid preparation method for trypsin without exogenous activation, comprising the following: (1) Crushing pretreatment Bovine pancreas was processed by slicing, coarse crushing and fine grinding, and free fat blocks on the surface were removed to produce homogeneous pancreatic paste with a particle size of 15 μm.

[0042] (2) Low-temperature batching Add 900g of purified water, 500mL of cold acetone at -12℃ and 10g of trypsin to the homogenized pancreatic syrup, stir at low speed to mix, and control the initial temperature of the system to 2℃.

[0043] (3) Gradient temperature control low-temperature autolysis Continue stirring and control the heating rate at 3℃ / h to make the system temperature rise uniformly from 2℃ to 14℃, thus completing autolysis.

[0044] (4) Separation and impurity removal The separator filters out connective fibrous tissue and collects the clear pancreatic enzyme slurry.

[0045] (5) Cold acetone precipitation and layering Add 7L of cold acetone at -12℃ to the above pancreatic enzyme slurry, let it stand at low temperature to separate into layers, and discard the upper organic waste liquid; add 3L of cold acetone to the obtained precipitate for repeated degreasing and dehydration, and collect the precipitate.

[0046] (6) Low-temperature vacuum drying The above precipitate was placed under low temperature and negative pressure conditions for vacuum drying. The drying temperature was 30℃, the drying time was 16h, and the vacuum degree was 750mmHg. After removing the organic solvent, the pancreatic enzyme product was obtained.

[0047] Example 4: This embodiment of the invention provides a rapid preparation method of trypsin without exogenous activation. The difference from Example 1 is that the heating rate in step (3) is 0.5℃ / h, while the other steps remain unchanged.

[0048] Example 5: This embodiment of the invention provides a rapid preparation method of trypsin without exogenous activation. The difference from Example 1 is that the heating rate in step (3) is 4℃ / h, while the other steps remain unchanged.

[0049] Example 6: This embodiment of the invention provides a rapid preparation method of trypsin without exogenous activation. The difference from Example 1 is that in step (2), 180 mL of cold acetone at -10°C is used, while the other steps remain unchanged.

[0050] Example 7: This embodiment of the invention provides a rapid preparation method of trypsin without exogenous activation. The difference from Example 1 is that in step (2), 600 mL of cold acetone at -10°C is used, while the other steps remain unchanged.

[0051] Example 8: This embodiment of the invention provides a rapid preparation method of trypsin without exogenous activation. The difference from Example 1 is that the temperature of the cold acetone in step (2) is -8°C, while the other steps remain unchanged.

[0052] Example 9: This embodiment of the invention provides a rapid preparation method of trypsin without exogenous activation. The difference from Example 1 is that the amount of trypsin added in step (2) is 0.5g, while the other steps remain unchanged.

[0053] Example 10: This embodiment of the invention provides a rapid preparation method of trypsin without exogenous activation. The difference from Example 1 is that the amount of trypsin added in step (2) is 11g, while the other steps remain unchanged.

[0054] Example 11: This embodiment of the invention provides a rapid preparation method of trypsin without exogenous activation. The difference from Example 1 is that the amount of purified water added in step (2) is 200g, while the other steps remain unchanged.

[0055] Example 12: This embodiment of the invention provides a rapid preparation method of trypsin without exogenous activation. The difference from Example 1 is that the amount of purified water added in step (2) is 1000g, while the other steps remain unchanged.

[0056] Comparative Example 1: Using the traditional CaCl2 high-temperature activation process, referencing patent publication number CN103343114A, Example 1 of this patent was adopted. The complete process steps are as follows: 1) Fresh pig pancreas was taken, and fat and connective tissue were removed. After coarse crushing in a meat grinder, it was circulated and ground twice in a colloid mill. The median particle size D50 of the homogenate was 10-30 μm; 2) 0.15 mol / L dilute sulfuric acid was added at a mass ratio of 1:4 between pancreatic slurry and extract. The mixture was stirred and extracted at 4°C for 20 h. The supernatant was collected by plate and frame filtration; 3) Anhydrous CaCl2 was added to the supernatant to a final mass fraction of 2%. The pH of the system was adjusted to 7.5-7.8 using a dilute disodium hydrogen phosphate buffer system. The mixture was stirred and activated at 28°C for 8 h. Calcium ions stabilized the enzyme protein and completed the activation of propancreatin; 4) Ammonium sulfate was used for fractional salting out. Impurities were removed at 28% saturation, and crude pancreatin was precipitated at 70% saturation. The precipitate was collected by centrifugation; 5) 95% Degrease twice with acetone at 0-10℃ for 30 min each time, then centrifuge to drain the solvent; 6) Remove organic solvent by low-temperature vacuum drying at 30-38℃, vacuum degree 600 mmHg, drying time 10 h, and then pulverize through an 80-mesh sieve to obtain pancreatic enzyme powder using traditional process.

[0057] Comparative Example 2: The difference from Example 1 is that cold acetone at -10°C is not added in step (2), autolysis is performed directly at 25°C for 8 hours in step (3), and cold acetone is not added in step (5). The remaining steps remain unchanged.

[0058] Comparative Example 3: The difference from Example 1 is that cold acetone at -10°C is not added in step (2), while the other steps remain unchanged.

[0059] Comparative Example 4: The difference from Example 1 is that trypsin is not added in step (2), while the other steps remain unchanged.

[0060] Comparative Example 5: The difference from Example 1 is that in step (3), the system temperature is raised from 0°C to 16°C at a constant rate, while the other steps remain unchanged.

[0061] Comparative Example 6: The difference from Example 1 is that in step (3), the system temperature is raised from 0°C to 8°C at a constant rate, while the other steps remain unchanged.

[0062] Comparative Example 7: The difference from Example 1 is that step (3) does not involve gradient heating, but directly adjusts the temperature to 12°C to complete autolysis, while the remaining steps remain unchanged.

[0063] The pancreatic enzyme products prepared in Examples 1-12 and Comparative Examples 1-6 were subjected to the following performance tests: (1) Enzyme activity recovery rate: according to the method for determining pancreatic enzyme activity in the Chinese Pharmacopoeia, the activities of trypsin, pancreatic amylase and pancreatic lipase were detected respectively. Enzyme activity recovery rate = (Total enzyme activity of finished product / Total enzyme activity of raw pancreatic plasma) × 100%; (2) Autolysis time (h); (3) Ash content of finished product (%): The test standard is based on the Ash Content Determination Method in Section 2302 of the General Chapter of Part IV of the 2025 Edition of the Pharmacopoeia of the People's Republic of China; Test procedure: Take 2-3g of pancreatic enzyme product, crush it through a No. 2 sieve, accurately weigh it into a crucible that has been ignited to constant weight, slowly carbonize it at low temperature until there is no black smoke, heat it to 500-600℃ and ignite it until it is completely ashed, cool it in a desiccator and weigh it, ignite it repeatedly until constant weight, and calculate the total ash content using the formula: Total ash content (%) = (ash constant weight mass / sample mass of test sample) × 100%; (4) Microbial limits: total number of aerobic bacteria (CFU / g), total number of molds and yeasts (CFU / g), control bacteria (Escherichia coli, Salmonella); the detection standards are based on the 2025 edition of the Pharmacopoeia of the People's Republic of China, Part IV, General Chapter 1105 Microbial limit test for non-sterile products: microbial counting method, and General Chapter 1106 Microbial limit test for non-sterile products: control bacteria test method; detection steps: take 1g of pancreatic enzyme product to prepare a 1:10 test solution, after confirming that there is no matrix interference through the method suitability test, count aerobic bacteria, molds and yeasts using tryptic soy agar and Sabouraud dextrose agar plates respectively; simultaneously inoculate enrichment medium to check for Escherichia coli and Salmonella, and interpret the detection status of target control bacteria according to the pharmacopoeia.

[0064] The test results are shown in Tables 1 and 2 below: Table 1

[0065] Table 2

[0066] As shown in Tables 1 and 2, compared with Comparative Example 1, Examples 1-12 of the present invention eliminate the exogenous activation process of more than 8 hours, shortening the overall preparation cycle by more than 40% and improving equipment utilization; without the introduction of exogenous proteins such as calcium salts and intestinal fluid, the ash and protein content of the finished product are reduced by more than 30%, and the total activity recovery rate of the three enzymes is increased to more than 90%, which is 15-22% higher than the traditional CaCl2 high-temperature activation process; the cost of activator procurement is eliminated, the total consumption of organic solvents does not increase significantly, and the overall manufacturing cost decreases by about 18%; the low-temperature short-cycle process reduces microbial growth, and the microbial limit index of the finished product is better than that of the traditional high-temperature activation process.

[0067] As can be seen from the comparison between Examples 4 and 5 and Example 1, when the heating rate in step (3) is less than 1℃ / h, the slow heating rate will lead to an excessively long autolysis cycle. Although the enzyme activity recovery rate decreases slightly, it still remains above 87%, and the production efficiency is significantly reduced. When the heating rate in step (3) is greater than 3℃ / h, the rapid heating will cause trypsinogen to be converted into a large amount of active trypsin in a short time, triggering concentrated burst hydrolysis, resulting in the loss of activity of pancreatic amylase and pancreatic lipase. The recovery rates of the three enzymes all drop to below 83%, and the autolysis time is shortened but the enzyme activity decreases by more than 10%, resulting in a worse overall benefit.

[0068] As can be seen from the comparison between Examples 6 and 7 and Example 1, when the amount of cold acetone added in step (2) is less than 200 mL, the amount of acetone is insufficient and cannot fully exert the mild inhibitory effect on the excessive hydrolysis of protease, resulting in the continuous degradation of amylase and lipase by the activated trypsin, and the recovery rate of the three enzymes drops to below 81%; when the amount of cold acetone added in step (2) is greater than 500 mL, the excess acetone will cause irreversible denaturation and inactivation of protease, and at the same time destroy the spatial conformational stability of amylase and lipase, the recovery rate of the three enzymes drops significantly to below 78%, and the ash content increases slightly, and the purity of the finished product decreases.

[0069] As can be seen from the comparison between Example 8 and Example 1, when the temperature of cold acetone in step (2) is greater than -10℃, the increase in acetone temperature will reduce its ability to regulate the conformation of protease, weaken the effect of inhibiting hydrolysis, and lead to the intensification of degradation of complex enzyme during autolysis. The recovery rates of the three enzymes all show varying degrees of decline, and the overall performance is inferior to the -10℃ cold acetone system.

[0070] As can be seen from the comparison between Examples 9, 10 and Example 1, when the amount of trypsin added in step (2) is less than 1g, the amount of initiating enzyme that triggers cascade activation is insufficient, resulting in the inability of endogenous trypsinogen to be fully converted into active trypsin, the autolysis activation efficiency is greatly reduced, the recovery rate of the three enzymes is reduced to below 80%, and the autolysis time is extended to more than 10h; when the amount of trypsin added in step (2) is greater than 10g, the excessive amount of initiating enzyme will cause the enzymeogen activation rate to be too fast, and a large amount of active trypsin will be generated instantaneously, causing explosive hydrolysis, resulting in severe degradation of amylase and lipase, a significant decrease in the recovery rate of the three enzymes, and a deterioration in the quality of the finished product.

[0071] Comparing Examples 11 and 12 with Example 1, it can be seen that when the amount of purified water added in step (2) is less than 300g, the solid-liquid ratio of the system is too high, the pancreatic slurry concentration is too high, resulting in a decrease in mass transfer efficiency, insufficient activation of zymogen and autolysis reaction, and the recovery rate of the three enzymes all drops below 82%, and the ash content increases; when the amount of purified water added in step (2) is greater than 900g, the system is over-diluted, the enzyme concentration decreases, resulting in a slower autolysis reaction rate, a longer autolysis time, and a slight decrease in the recovery rate of the three enzymes, thus reducing production efficiency.

[0072] As can be seen from the comparison between Comparative Example 2 and Example 1, when only high-temperature autolysis is used without the inhibitor acetone, the trypsin activated at 25°C lacks effective inhibition and will continue to undergo self-hydrolysis and degradation of other enzyme components. At the same time, the high temperature exacerbates the growth of miscellaneous bacteria, ultimately resulting in the recovery rate of the three enzymes being less than 71%, the microbial limit being significantly increased, and the quality of the finished product being greatly reduced.

[0073] As can be seen from the comparison between Comparative Example 3 and Example 1, when cold acetone is not added in step (2) and acetone is only added during precipitation, there is no hydrolysis inhibition effect of acetone in the autolysis stage. Excessive hydrolysis of protease leads to severe degradation of the complex enzyme. The recovery rate of the three enzymes is less than 76%, which is only slightly better than the traditional calcium salt process and cannot achieve the high recovery rate effect of the present invention.

[0074] As can be seen from the comparison between Comparative Example 4 and Example 1, when trypsin is not added in step (2), there is no initiating enzyme to trigger cascade activation, the endogenous trypsinogen in the pancreas cannot be converted into active trypsin on its own, the autolysis process can hardly be carried out, and the final recovery rate of the three enzymes is less than 35%, which is completely unable to meet the needs of industrial production.

[0075] Comparing Comparative Examples 5 and 6 with Example 1, it can be seen that when the temperature of low-temperature autolysis is higher than 14°C, the temperature increase leads to a significant increase in the hydrolysis rate of the protease, exceeding the activation rate of the zymogen, resulting in severe degradation of the complex enzyme. The recovery rates of the three enzymes are all below 75%, and the risk of microbial proliferation increases. When the temperature of low-temperature autolysis is lower than 10°C, the temperature is too low, which leads to a significant decrease in the spontaneous activation kinetic rate of the zymogen, resulting in insufficient activation efficiency. The autolysis time is extended to more than 11 hours, and the recovery rates of the three enzymes are all below 69%. The production efficiency and product quality cannot meet the standards.

[0076] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A rapid method for preparing trypsin without exogenous activation, characterized in that, Includes the following steps: S1: The raw pancreas is crushed to produce pancreatic syrup; S2: Add purified water, cold acetone and trypsin to the pancreatic syrup, mix well and control the initial temperature of the system to 0 ~ 2℃; S3: Under continuous stirring, perform gradient temperature-controlled low-temperature autolysis, controlling the temperature gradient of the system to rise to 10~14℃. S4: Filter to remove connective tissue and collect pancreatic enzyme slurry; S5: Add cold acetone to the pancreatic enzyme slurry for precipitation, separation, defatting, and dehydration, and collect the precipitate; S6: The precipitate is dried under low temperature and vacuum to obtain the pancreatic enzyme product.

2. The rapid preparation method of trypsin without exogenous activation according to claim 1, characterized in that, In step S3, the heating rate of the gradient temperature-controlled low-temperature autolysis is 1~3℃ / h.

3. The rapid preparation method of trypsin without exogenous activation according to claim 2, characterized in that, Based on 1 kg of pancreatic raw material, the amount of cold acetone added in step S2 is 200~500 mL, and the temperature of the cold acetone is -15℃~-10℃.

4. The rapid preparation method of trypsin without exogenous activation according to claim 3, characterized in that, Based on 1 kg of pancreatic raw material, the amount of trypsin added in step S2 is 1~10 g.

5. The rapid preparation method of trypsin without exogenous activation according to claim 4, characterized in that, Based on 1 kg of pancreatic raw material, the amount of purified water added in step S2 is 300~900 g.

6. The rapid preparation method of trypsin without exogenous activation according to claim 1, characterized in that, In step S1, the crushing process includes slicing, coarse crushing with a meat grinder, and fine grinding with a pulping machine, and the removal of free fat lumps from the surface.

7. The rapid preparation method of trypsin without exogenous activation according to claim 1, characterized in that, Based on 1 kg of pancreatic raw material, the amount of cold acetone added in step S5 is 5-7 L. After standing at low temperature and separating into layers, the upper organic waste liquid is discarded, and the resulting precipitate is then repeatedly degreased and dehydrated by adding 2-3 L of cold acetone.

8. The rapid preparation method of trypsin without exogenous activation according to claim 7, characterized in that, In step S5, the temperature of the cold acetone is -15 to -10°C.

9. The rapid preparation method of trypsin without exogenous activation according to claim 1, characterized in that, In step S6, the low-temperature vacuum drying is carried out under low-temperature negative pressure conditions to remove organic solvents; The drying temperature is 20~30℃, the drying time is 12h~16h, and the vacuum degree is 600~750mmHg.

10. A rapid preparation method for trypsin without exogenous activation according to claim 6, characterized in that, In step S1, the pancreatic raw material is porcine pancreas and / or bovine pancreas, and the particle size of the homogenized pancreatic slurry is 5~15 μm.

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