Treatment unit and sterilization method for removing microorganisms, especially bacillus cereus, without affecting pancreatin activity

Abstract

The invention discloses a treatment unit and a sterilization method for removing microorganisms, especially bacillus cereus, without affecting pancreatin activity, the treatment unit comprises a double-layer solid filter element to terminate the sterilization effect of a sterilization solution and stabilize the pH value of a system, and the sterilization method comprises the steps of microorganism inactivation, treatment by the treatment unit and drying by a forced air drying process; according to the treatment unit, through the synergistic effect of the upper-layer solid filter element and the lower-layer solid filter element, the stable PH value is maintained while the sterilization effect of the sterilization liquid is terminated, and the pancreatin solution which is free of oxidability, moderate in pH and capable of achieving the microorganism standard is obtained. In addition, through the specific working concentration of the non-compound PAA stock solution, the specific treatment unit composition and the forced air drying process and parameters, the problem that the existing sterilization method cannot give consideration to both the bacillus cereus removal effect and the pancreatin activity retention is solved.

Description

Technical Field

[0001] This invention relates to the field of pancreatic enzyme sterilization technology, and more specifically to a sterilization method for removing microorganisms, particularly Bacillus cereus, without affecting pancreatic enzyme activity. Background Technology

[0002] Pancreatic enzymes are a mixture extracted from the pancreas of animals (usually pigs or cows) and contain a variety of powerful digestive enzymes. Pancreatic enzymes mainly consist of three core digestive enzymes, each responsible for breaking down the three major nutrients: The functions of trypsin and chymotrypsin: They are responsible for breaking down proteins into smaller peptide chains and amino acids. They are secreted in inactive zymogen form, which is activated in the intestine to prevent the digestion of the pancreas itself.

[0003] The function of pancreatic lipase: It is the most crucial enzyme in breaking down fats. It breaks down fats (triglycerides) into fatty acids and glycerol, which can then be absorbed by the intestines.

[0004] The function of pancreatic amylase: It is responsible for breaking down carbohydrates, especially starch, into smaller sugar molecules such as maltose. It is a concentrated form of a key substance naturally secreted by the pancreas for digesting food.

[0005] In the pharmaceutical field, pancreatic enzymes are primarily used as prescription drugs or over-the-counter supplements to treat pancreatic exocrine insufficiency. In bioscience research, pancreatic enzymes (especially those containing trypsin) are a very common reagent in laboratories, mainly used for cell culture: when cells have reached confluence in a culture dish, researchers use pancreatic enzyme solutions (often called "trypsin-EDTA digestion solutions") to digest and break down intercellular junction proteins (such as cadherins), causing adherent cells to detach from the culture dish surface, forming a single-cell suspension for further amplification in separate flasks.

[0006] Bacillus cereus is a species of Bacillus, a Gram-positive bacterium with relatively smooth ends. Most cells are arranged in chains, are catalase-positive, and are aerobic (or facultative anaerobic). They can survive in environments ranging from 10-50 °C, but growth is slower at lower temperatures. The spores are highly resistant to physical and chemical factors (such as high and low temperatures, dryness, radiation, disinfectants, antibiotics, and other toxins), and can grow under both aerobic and anaerobic conditions. Furthermore, Bacillus cereus is not easily killed by alcohol; they can even multiply in alcoholic products, potentially causing infection.

[0007] Currently, the FDA stipulates that pancreatic enzymes must not contain Bacillus cereus. The currently disclosed processes for removing or inactivating Bacillus cereus in pancreatic enzymes cannot simultaneously meet the two core requirements: completely removing or inactivating Bacillus cereus to meet FDA quality control standards, and maximizing the preservation of the pancreatic enzyme's biological activity (such as amylase and protease activity) and efficacy. The balance between these two aspects remains a technical challenge that has not yet been fully resolved.

[0008] Therefore, this application is hereby submitted. Summary of the Invention

[0009] The technical problem to be solved by the present invention is that existing sterilization methods cannot simultaneously achieve the removal effect of Bacillus cereus and the preservation of pancreatic enzyme activity. The purpose is to provide a sterilization method for removing microorganisms, especially Bacillus cereus, without affecting pancreatic enzyme activity.

[0010] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a treatment unit for microbial inactivation, comprising a double-layer solid filter element, wherein the upper solid filter element is used to decompose the sterilization solution, and the lower solid filter element is used to reduce dynamic pH changes.

[0011] In one specific embodiment, the upper solid filter element is manganese dioxide solid particles.

[0012] In this scheme, manganese dioxide solid particles are a highly efficient and specific catalyst for PAA, and their high catalytic efficiency is the technical basis for ensuring rapid termination.

[0013] In one specific embodiment, the lower solid filter element is a sodium bicarbonate microsphere capsule.

[0014] In this solution, the mild buffering effect of sodium bicarbonate ensures effective pH stability without interfering with sterilization. Simultaneously, it works synergistically with manganese dioxide solid particles to guarantee the compliance of enzyme activity, aerobic bacteria, and Bacillus cereus.

[0015] In one specific embodiment, the capsule material of the sodium bicarbonate microcapsule is formed by cross-linking hydroxypropyl methylcellulose with calcium ions.

[0016] Secondly, the present invention also provides a sterilization method for removing microorganisms, particularly Bacillus cereus, without affecting pancreatic enzyme activity, comprising the following steps: Prepare the sample to be treated and the sterilization solution. The sample to be treated contains Bacillus cereus. The sample to be treated is mixed with 0.3-0.75% sterilization solution and dynamically contacted for a period of time to complete the microbial inactivation. Microbial inactivation is completed using the above-mentioned treatment unit to terminate the sterilization effect of the sterilizing solution and stabilize the pH value of the system. Processed using a forced-air drying process.

[0017] In one specific embodiment, the sample to be treated is a trypsin-isopropanol solution.

[0018] In one specific embodiment, the sterilization solution is a non-compound PAA stock solution with a concentration of 15-20%.

[0019] In this protocol, the non-compound PAA stock solution is a relatively pure peracetic acid solution that contains no hydrogen peroxide or only trace amounts of hydrogen peroxide. This ensures that the inactivation effect is achieved by the non-compound PAA stock solution, unaffected by other substances.

[0020] In one specific embodiment, the sample to be treated and the sterilization solution are uniformly mixed by instantaneous mixing and dynamic contact, with a mixing time not exceeding 5 minutes and a mixing flow rate of 500 mL / min.

[0021] In one specific embodiment, the instantaneous mixing and dynamic contact between the sample to be treated and the sterilization solution is achieved by a T-type mixer, a Y-type mixer, or an online static mixer.

[0022] In one specific embodiment, the specific process of the forced-air drying process is as follows: exhaust air at 25℃ for 6-8 hours until the moisture content is <12%; dry at 30-35℃ for 4 hours, dry at 40-45℃ for 4 hours until the moisture content is <10%; dry at 50-55℃ for 4-6 hours until the moisture content is <5%; dry at 60-65℃ for 3 hours until the moisture content is <3%. That is, there is no final finishing process: drying at 70-75℃ for 2 hours.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. A processing unit for microbial inactivation according to the present invention, through the synergistic effect of an upper solid filter element and a lower solid filter element, maintains a stable pH value while terminating the sterilization effect of the sterilizing solution, resulting in a pancreatic enzyme solution that is non-oxidizing, has a moderate pH, and meets microbial standards. This provides a stable and safe feed for subsequent processes such as forced-air drying.

[0024] 2. The present invention provides a sterilization method for removing microorganisms, particularly Bacillus cereus, without affecting pancreatic enzyme activity. This method combines a dynamic mixing method of the sample and sterilization solution, a specific processing unit composition, a specific working concentration of the non-compound PAA stock solution, and a forced-air drying process to work together to achieve both the removal effect of Bacillus cereus and the retention of pancreatic enzyme activity. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of this invention are only used to explain this invention and are not intended to limit this invention.

[0026] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that these specific details are not necessary to practice the invention. In other embodiments, well-known materials or methods have not been specifically described in order to avoid obscuring the invention.

[0027] Throughout this specification, references to “an embodiment,” “an example,” or “an example” mean that a particular feature, structure, or characteristic described in connection with that embodiment or example is included in at least one embodiment of the invention. Therefore, the phrases “an embodiment,” “an example,” “an example,” or “an example” appearing in various places throughout the specification do not necessarily refer to the same embodiment or example. Furthermore, specific features, structures, or characteristics can be combined in one or more embodiments or examples in any suitable combination and / or sub-combination. The term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0028] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of the particular range. The range defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range.

[0029] In the description of this invention, unless otherwise specified, all embodiments and optional embodiments of this application can be combined with each other to form new technical solutions.

[0030] In the description of this invention, unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form new technical solutions.

[0031] In the description of this invention, unless otherwise specified, the terms "comprising" and "including" as used herein can be open-ended or closed-ended. For example, "comprising" and "including" may mean that other components not listed may also be included, or that only the listed components may be included. Example 1

[0032] This embodiment 1 provides a sterilization method for removing microorganisms, especially Bacillus cereus, without affecting pancreatic enzyme activity, comprising the following steps: Prepare the sample to be treated, which is a trypsin-isopropanol solution, i.e., a mixture of trypsin powder or concentrate with an aqueous solution of isopropanol of a certain concentration. The trypsin is of porcine origin, and isopropanol itself is an effective disinfectant. Isopropanol can disrupt the structure of the lipid bilayer of microbial cell membranes, increasing their permeability.

[0033] The trypsin-isopropanol solution is instantaneously mixed and dynamically contacted with a 0.5% (working concentration) sterilizing solution to perform microbial inactivation for a first predetermined time. Specifically, the mixture is rapidly contacted and uniformly mixed using a mixer at a flow rate of 500 ml / min, ensuring microbial inactivation is completed within 5 minutes. The mixer can be a T-type mixer, a Y-type mixer, or an online static mixer. Experiments have shown that a 0.5% working concentration is the optimal solution in this embodiment.

[0034] The sterilization solution is a non-compound PAA stock solution with a concentration of 15-20% (this concentration is based on the raw material specifications). The non-compound PAA stock solution is a relatively pure peracetic acid solution that does not contain hydrogen peroxide or contains only trace amounts of hydrogen peroxide.

[0035] Specifically, peracetic acid, also known as peracetic acid or peroxyacetic acid, is an organic peroxide. Due to its strong oxidizing power, it is widely considered a highly effective and broad-spectrum disinfectant and sterilizing agent. The bactericidal effect of peracetic acid mainly relies on its strong oxidizing ability: it can penetrate the cell walls of microorganisms, oxidizing and destroying their enzyme systems, proteins, and other key cellular components, leading to cell inactivation and ultimately death. For viruses, it can destroy their nucleic acid and protein capsid.

[0036] After microbial inactivation is completed, the system enters the treatment unit to terminate the sterilization effect of the sterilizing solution and stabilize the pH value of the system. Specifically, the processing unit is a cylindrical device made of stainless steel (316), containing a double-layer solid filter element. The upper layer is composed of manganese dioxide metal particles to catalyze the decomposition of peracetic acid sterilization solution; the lower layer is composed of sodium bicarbonate microcapsules. The capsule material of the sodium bicarbonate microcapsules is formed by cross-linking hydroxypropyl methylcellulose with calcium ions, ensuring that the generation of acetic acid is instantaneous and that its reaction weakens the influence of dynamic pH changes on pancreatic enzymes.

[0037] In this process, the processing unit rapidly and thoroughly decomposes the highly oxidizing PAA solution into oxygen and water through the upper layer filled with manganese dioxide metal particles. This directly removes the "culprit" causing enzyme activity damage at its source. The lower layer is filled with sodium bicarbonate microcapsules, which rupture upon contact with acidic liquids, slowly releasing sodium bicarbonate. This sodium bicarbonate reacts with acetic acid to generate carbon dioxide and water, effectively resisting sudden pH changes and stabilizing the pH within a safe range for pancreatic enzymes.

[0038] As shown in Table 1, a forced-air drying process was used. The forced-air drying process parameters are as follows: Table 1

[0039] Comparative Example 1 Based on Example 1, and referring to the aforementioned steps, the difference between Comparative Example 1 and Example 1 is that: the pancreatic enzyme-isopropanol solution and peracetic acid solution are directly statically soaked. Specifically, the pancreatic enzyme-isopropanol solution and 0.5% of a 15-20% non-compound PAA stock solution are statically reacted in a static mixer for 30 minutes.

[0040] Comparative Example 2 Based on Example 1, and referring to the aforementioned steps, the difference between this comparative example and Example 1 is that: the trypsin-isopropanol solution and 1.0% of the non-compound PAA stock solution with a concentration of 15-20% are rapidly contacted and uniformly mixed through a T-type mixer at a flow rate of 500 mL / min to ensure that microbial inactivation is completed within 5 minutes.

[0041] Comparative Example 3 Based on Example 1, and referring to the aforementioned steps, the difference between this comparative example and Example 1 is that: the trypsin-isopropanol solution and 0.1% of the non-compound PAA stock solution with a concentration of 15-20% are rapidly contacted and uniformly mixed through a T-type mixer at a flow rate of 500 mL / min to ensure that microbial inactivation is completed within 5 minutes.

[0042] Comparative Example 4 Based on Example 1, referring to the aforementioned steps, the difference between this comparative example and Example 1 is that: after mixing and inactivation, the mixed liquid (the solution after non-compound PAA stock solution and pancreatin-isopropanol solution are mixed) immediately flows into the treatment unit. The treatment unit is a cylindrical device made of stainless steel (316) containing a single-layer solid filter element. The solid filter element is manganese dioxide metal particles to catalyze the decomposition of peracetic acid.

[0043] Comparative Example 5 Based on Example 1, referring to the aforementioned steps, the difference between this comparative example and Example 1 is that: after mixing and inactivation, the mixed liquid immediately flows into the processing unit. The processing unit is a cylindrical device made of stainless steel (316), which contains a double-layer solid filter element. The upper layer is manganese dioxide metal particles to catalyze the decomposition of peracetic acid, and the lower layer is sodium hydroxide microcapsules. The capsule material of the sodium hydroxide microcapsules is formed by cross-linking hydroxypropyl methylcellulose with calcium ions to ensure that the acetic acid is generated instantaneously and that its reaction weakens the influence of pH dynamic changes on pancreatic enzymes.

[0044] Comparative Example 6 Based on Example 1, referring to the aforementioned steps, the difference between this comparative example and Example 1 is that: after mixing and inactivation, the mixed liquid immediately flows into the processing unit. The processing unit is a cylindrical device made of stainless steel (316), which contains a double-layer solid filter element. The upper layer is aluminum powder metal to catalyze the decomposition of peracetic acid, and the lower layer is sodium bicarbonate microcapsules. The capsule material of the sodium bicarbonate microcapsules is formed by cross-linking hydroxypropyl methylcellulose with calcium ions to ensure that the acetic acid is generated instantaneously and that its reaction weakens the influence of pH dynamic changes on pancreatic enzymes.

[0045] Comparative Example 7 Based on Example 1 and referring to the aforementioned steps, the difference between this comparative example and Example 1 is that: the specified forced-air drying process is used, with process parameters set as follows: exhaust at 25°C for 6-8 hours until moisture content <12%; dry at 30-35°C for 4 hours, dry at 40-45°C for 4 hours until moisture content <10%; dry at 50-55°C for 4-6 hours until moisture content <5%; and dry at 60-65°C for 3 hours until moisture content <3%. That is, there is no final finishing process: drying at 70-75°C for 2 hours.

[0046] Comparative Example 8 Based on Example 1 and referring to the aforementioned steps, the difference between this comparative example and Example 1 is that: the vacuum drying process is performed using the same process parameters as the forced-air drying process in Example 1, set to exhaust air at 25°C for 6-8 hours until moisture content <12%; drying at 30-35°C for 4 hours, drying at 40-45°C for 4 hours until moisture content <10%; drying at 50-55°C for 4-6 hours until moisture content <5%; and drying at 60-65°C for 3 hours until moisture content <3%. That is, there is no final finishing process: drying at 70-75°C for 2 hours.

[0047] The samples after treatment in Example 1 and Comparative Examples 1-8 were subjected to qualification tests for protease, amylase, lipase, aerobic bacteria and Bacillus cereus. The specific experimental results are shown in Table 2.

[0048] Table 2 Comparison of Experimental Data

[0049]

[0050] Example 1 successfully achieved the required levels of microorganisms, Bacillus cereus, and the activity of all three enzymes. The other comparative examples all had some issues, such as: The experimental results of Comparative Example 1 were as follows: the enzyme activity was qualified, but the aerobic bacteria and Bacillus cereus were both unqualified.

[0051] The experimental results of Comparative Example 2 were as follows: the enzyme activity was unqualified, but the aerobic bacteria and Bacillus cereus were qualified.

[0052] The experimental results of Comparative Example 3 were as follows: the enzyme activity, aerobic bacteria, and Bacillus cereus all failed the test.

[0053] The experimental results of Comparative Example 4 were as follows: the enzyme activity was not up to standard, but the aerobic bacteria and Bacillus cereus were up to standard.

[0054] The experimental results of Comparative Example 5 were as follows: the enzyme activity was qualified, but the aerobic bacteria and Bacillus cereus were both unqualified.

[0055] The experimental results of Comparative Example 6 showed that there were sub-items that failed to meet the standards for enzyme activity, aerobic bacteria, and Bacillus cereus.

[0056] The experimental results of Comparative Example 7 were as follows: enzyme activity and aerobic bacteria were qualified, but Bacillus cereus was not qualified.

[0057] The experimental results of Comparative Example 8 showed that there were sub-items that failed to meet the standards for enzyme activity, aerobic bacteria, and Bacillus cereus.

[0058] Based on the experimental data from Example 1 and Comparative Example 1, even when the exact same sterilizing agent (0.5% non-compound PAA) is used, the experimental results will differ due to the different contact methods (instantaneous mixing and dynamic contact in Example 1, and static immersion in Comparative Example 1). Therefore, extending the PAA contact time does not improve the killing effect on Bacillus cereus. The dynamic mixing method ensures that every microorganism can come into contact with a sufficient amount of PAA instantly, achieving uniform sterilization without dead corners.

[0059] Based on the experimental data from Examples 1, 2, and 3, Comparative Example 2 (1.0% non-compound PAA stock solution) passed sterilization but failed enzyme activity testing. This indicates that increasing the PAA solution concentration causes unacceptable oxidative damage to trypsin. Excessively high concentrations, even within a short time, are sufficient to destroy the key structure of the enzyme protein due to their strong oxidizing power. Comparative Example 3 (0.1% non-compound PAA stock solution) failed both sterilization and enzyme activity testing. This indicates that a PAA solution concentration that is too low cannot effectively kill all microorganisms (especially persistent Bacillus cereus) within 5 minutes. Residual microorganisms consume nutrients and produce metabolic products, similarly leading to unacceptable enzyme activity in the final product. The data from Example 1 demonstrates that only at the concentration of 0.5% non-compound PAA stock solution in this example can the dual goals of thorough sterilization and enzyme activity preservation be achieved.

[0060] Based on Examples 1 and Comparative Examples 4-6, manganese dioxide metal particles are a highly efficient and specific catalyst for PAA (particulate acid oxidative stress), and their high catalytic efficiency is the technical basis for ensuring rapid termination. Furthermore, manganese dioxide metal particles and sodium bicarbonate microcapsules have a synergistic effect, simultaneously addressing both oxidative and acid-induced damage.

[0061] Based on Example 1 and Comparative Examples 1-8, the present invention combines instantaneous mixing with dynamic contact, sets the working concentration of the non-compound PAA stock solution, has a specific processing unit composition, and uses a forced-air drying process and parameters. These parameters and settings are indispensable, working together to balance the removal effect of Bacillus cereus and the retention of trypsin activity.

[0062] 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 treatment unit for microorganism inactivation, characterized in that, The filter cartridge comprises two layers of solid filter, the upper layer of solid filter is used to decompose the sterilization liquid, and the lower layer of solid filter is used to weaken the PH dynamic change.

2. The treatment unit for microorganisms after inactivation according to claim 1, characterized in that, The upper layer of solid filter is manganese dioxide solid particles.

3. The treatment unit for microorganisms after inactivation according to claim 1, characterized in that, The lower layer of solid filter is sodium bicarbonate microsphere capsule.

4. The treatment unit for microorganisms after inactivation according to claim 3, characterized in that, The capsule material of the sodium bicarbonate microsphere capsule is cross-linked by hydroxypropyl methyl cellulose and calcium ions.

5. A sterilization method for removing microorganisms, particularly Bacillus cereus, without affecting the activity of pancreatin, characterized by, The method comprises the following steps: Prepare the sample to be treated and the sterilization liquid, and the sample to be treated contains Bacillus cereus; Mix the sample to be treated with 0.3-0.75% of the sterilization liquid and dynamically contact for a period of time to complete the microbial inactivation; After the microbial inactivation is completed, use the treatment unit according to any one of claims 1-3 to terminate the sterilization effect of the sterilization liquid and stabilize the pH value of the system; Use the air blowing drying process for treatment.

6. The sterilization method for removing microorganisms, particularly Bacillus cereus, without affecting the activity of pancreatin according to claim 5, characterized by, The sample to be treated is trypsin-isopropyl alcohol solution.

7. The sterilization method for removing microorganisms, particularly Bacillus cereus, without affecting the activity of pancreatin according to claim 5, characterized by, The sterilization liquid is non-complex PAA stock solution, and the concentration is 15-20%.

8. The sterilization method for removing microorganisms, particularly Bacillus cereus, without affecting the activity of pancreatin according to claim 5, characterized by, The sample to be treated and the sterilization liquid are uniformly mixed by means of instantaneous mixing and dynamic contact, the mixing time is not more than 5 minutes, and the mixing flow rate is 500 mL / min.

9. The sterilization method for removing microorganisms, particularly Bacillus cereus, without affecting the activity of pancreatin according to claim 5, characterized by, The instantaneous mixing and dynamic contact of the sample to be treated and the sterilization liquid are realized by a T-shaped mixer, a Y-shaped mixer or an online static mixer.

10. The sterilization method for removing microorganisms, particularly Bacillus cereus, without affecting the activity of pancreatin according to claim 5, characterized by, The specific process of the air blowing drying process is as follows: 25℃ exhaust for 6-8h, until the moisture content is less than 12%; 30-35℃ drying for 4h, 40-45℃ drying for 4h, until the moisture content is less than 10%; 50-55℃ drying for 4-6h, until the moisture content is less than 5%; 60-65℃ drying for 3h, until the moisture content is less than 3%; and 70-75℃ drying for 2h. ​

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