A method and apparatus for preparing small molecule ribonucleic acid by low-temperature enzymatic hydrolysis coupled with directional ultrafiltration.

Abstract

This invention discloses a method for preparing small-molecule ribonucleic acid (RNA) using low-temperature enzymatic hydrolysis coupled with directional ultrafiltration, belonging to the field of ribonucleic acid preparation technology. The method includes the following steps: S1: Food-grade dry yeast is added to pure water and stirred to suspend it. After centrifugation, the supernatant is collected; S2: The supernatant is placed in a constant-temperature reaction vessel. In this invention, the entire process only requires standard equipment commonly used in biological raw material plants, such as a stirred reaction vessel, a low-speed centrifuge, a cold water circulating reaction vessel, a spiral wound ultrafiltration unit, a sterile filter, and a vacuum concentration tank. By integrating and continuously operating the five major processes of pretreatment, low-temperature enzymatic hydrolysis, enzyme inactivation and coarse filtration, ultrafiltration classification, and product concentration, the single-batch production cycle is shortened by approximately 54% compared to existing segmented processes, and fixed asset investment is reduced by approximately 65%. Small and medium-sized raw material plants can stably achieve continuous supply of hundreds of kilograms of small-molecule ribonucleic acid using the aforementioned general-purpose equipment, significantly lowering the industrial-scale production threshold for small-molecule ribonucleic acid active raw materials.

Description

Technical Field

[0001] This invention belongs to the field of ribonucleic acid preparation technology, specifically a method and apparatus for preparing small molecule ribonucleic acid by low-temperature enzymatic hydrolysis coupled with directional ultrafiltration. Background Technology

[0002] Ribonucleic acid (RNA) is a class of functional biomolecules widely found in biological cells. It is composed of nucleotides polymerized through phosphodiester bonds. In recent years, with the explosive growth in demand for skin barrier repair and post-cosmetic surgery care, RNA-based active ingredients have gradually become a research hotspot in the field of functional skin care and wound repair due to their unique advantages in promoting cell regeneration, inhibiting the release of inflammatory factors, and repairing damaged tissues. According to differences in molecular weight and chain length, RNA can be divided into macromolecular RNA (long chain) and small molecular RNA (50-200bp fragments). Among them, small molecular RNA, due to its small molecular size, can effectively penetrate the stratum corneum barrier of the skin and penetrate into the deep epidermis and even the dermis to exert a repairing effect from the root. Therefore, it has broad application prospects in high-end functional skin care products and Class II medical device dressings.

[0003] Currently, the mainstream RNA active raw materials on the market are mainly divided into two categories: salmon-derived PDRN extract and conventional yeast crude RNA extract. However, both types of raw materials and their preparation processes have significant technical defects. Salmon PDRN raw materials rely on imported aquatic resources, resulting in an unstable supply chain and high procurement costs. Its extraction process uses a high-temperature alkaline lysis process above 70°C, which not only leads to a significant degradation of RNA activity but also results in fragments that are mainly ultra-long chain macromolecules with poor penetration, posing a significant risk of irritation to hormone-dependent thin skin and minimally invasive surgical wounds. Conventional yeast RNA crude extraction processes only involve simple centrifugation to remove impurities, lacking low-temperature targeted enzymatic digestion and precise molecular weight fractionation. The resulting RNA fragments are of mixed lengths, and the macromolecular components can only form a temporary moisturizing film on the epidermis, unable to penetrate into the dermis to repair the immune and nerve barriers, resulting in short repair time and limited effects.

[0004] Furthermore, existing publicly available yeast RNA preparation processes generally suffer from the following industrialization drawbacks: First, the enzymatic hydrolysis process often uses room temperature or high temperature systems, resulting in uncontrollable reactions and the dissolution of large amounts of impurities such as proteins and pigments. Expensive chromatography equipment is required for deep purification, making mass production difficult for small and medium-sized enterprises. Second, the lack of a fixed molecular weight cutoff ultrafiltration fractionation design prevents the targeted enrichment of small-molecule functional RNA (50-200 bp), resulting in weak barrier repair capabilities. Third, the fermentation, enzymatic hydrolysis, and purification processes are operated independently in separate stages, leading to lengthy production cycles and high overall unit production costs. Fourth, existing processes have not been specifically optimized for safety in minimally invasive cosmetic procedures and for damaged skin caused by hormone-induced dermatitis. The products contain residual sensitizing macromolecular impurities, resulting in high irritation levels and making them unsuitable for direct use in post-operative high-activity repair dressings. Therefore, the industry urgently needs an integrated preparation process that can be put into production using only general-purpose equipment, is gentle and controllable throughout the process, can target the enrichment of small-molecule RNA, and is suitable for skin with triple barrier damage. Summary of the Invention

[0005] The purpose of this invention is to provide a method and apparatus for preparing small molecule ribonucleic acid by low-temperature enzymatic hydrolysis coupled with directional ultrafiltration, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing small molecule ribonucleic acid by low-temperature enzymatic hydrolysis coupled with directional ultrafiltration, comprising the following steps:

[0007] S1: Add food-grade dry yeast to pure water, stir to suspend, centrifuge, and collect the supernatant;

[0008] S2: Place the supernatant in a constant temperature reactor, add the complex nucleic acid hydrolase, and stir and hydrolyze at 35-40℃ for 2-3 hours to obtain the hydrolysate;

[0009] S3: Heat the enzymatic hydrolysate to 55°C and inactivate the hydrolytic enzyme, then filter it through a filter screen and collect the coarse filtrate;

[0010] S4: The coarse filtrate is passed through an ultrafiltration device with a molecular weight cutoff of 3000 Da for ultrafiltration, the permeate is collected, and small molecule ribonucleic acid is enriched.

[0011] S5: After sterilization filtration, the permeate is concentrated under low temperature and reduced pressure to obtain small molecule ribonucleic acid stock solution;

[0012] As a further preferred embodiment of this technical solution: the food-grade dry yeast in S1 is food-grade high-activity dry yeast, the pure water is room temperature pure water, and the stirring and suspension time is 20-40 min;

[0013] As a further preferred embodiment of this technical solution: the constant temperature reactor mentioned in S2 is a cold water circulating constant temperature reactor, the enzymatic hydrolysis temperature is 38℃, and the enzymatic hydrolysis time is 2.5h;

[0014] As a further preferred embodiment of this technical solution: the inactivation time in S3 is 15-30 minutes, and the filter screen is an 80-mesh single-layer filter screen;

[0015] As a further preferred embodiment of this technical solution: the ultrafiltration device mentioned in S4 is a spiral wound ultrafiltration device, which operates using a low-pressure circulating separation method;

[0016] As a further preferred embodiment of this technical solution: the sterilization filtration in S5 uses a 0.22μm precision filter membrane, and the temperature of the low-temperature vacuum concentration is 35-45℃;

[0017] As a further preferred embodiment of this technical solution: the proportion of 50-200bp small molecule ribonucleic acid in the small molecule ribonucleic acid stock solution is ≥70% of the total ribonucleic acid mass;

[0018] As a further preferred embodiment of this technical solution: the maximum material processing temperature throughout the entire process does not exceed 55°C, and strong acids or alkalis are not used to adjust the pH of the system throughout the entire process;

[0019] As a further preferred embodiment of this technical solution, it also includes step six, drying and powdering: the small molecule ribonucleic acid stock solution is subjected to low-temperature drying treatment to obtain small molecule ribonucleic acid powder;

[0020] As a further preferred embodiment of this technical solution: the method uses general-purpose basic equipment throughout, including: a stirred suspension reactor, a low-speed centrifuge, a cold water circulating constant temperature reactor, a 3000Da spiral wound ultrafiltration unit, a 0.22μm sterilization filter, and a vacuum low-temperature concentration tank.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. In this invention, the entire process only requires standard equipment commonly used in biological raw material plants, such as stirred reactors, low-speed centrifuges, cold water circulating reactors, spiral wound ultrafiltration units, sterile filters, and vacuum concentration tanks. By integrating and continuously operating the five major processes of pretreatment, low-temperature enzymatic hydrolysis, enzyme inactivation and coarse filtration, ultrafiltration classification, and product concentration, the production cycle of a single batch is shortened by about 54% compared to the existing segmented process, and the fixed asset investment is reduced by about 65%. Small and medium-sized raw material plants can rely on the above-mentioned general equipment to stably achieve continuous supply of hundreds of kilograms of small molecule ribonucleic acid, which significantly reduces the threshold for industrial mass production of small molecule ribonucleic acid active raw materials.

[0023] 2. In this invention, through the synergistic effect of gradient low-temperature enzymatic hydrolysis coupled with 3000Da molecular weight cutoff spiral ultrafiltration at 35-40℃, it is possible to accurately retain large molecular weight impurities and ultra-long chain ribonucleic acid, and directionally enrich small molecular weight ribonucleic acid fragments of 50-200bp. The proportion of small molecular weight ribonucleic acid in the finished product reaches more than 70% of the total ribonucleic acid mass. The resulting small molecular weight ribonucleic acid has a small molecular weight, which can effectively penetrate the damaged stratum corneum to the deep epidermis and superficial dermis, and simultaneously repair the physical barrier of the stratum corneum, the skin immune barrier and the peripheral nerve barrier. Unlike commercially available large molecular weight ribonucleic acid, which can only provide temporary surface relief, this invention achieves a root-cause long-term repair effect for hormone-dependent thin skin and skin damaged after minimally invasive surgery.

[0024] 3. In this invention, the highest processing temperature of materials throughout the entire process is only 55℃. Strong acids and alkalis are not used to adjust the pH of the system, and there are no high-temperature pyrolysis or extreme chemical degradation operations. This maximizes the preservation of the natural biological activity of ribonucleic acid, with an activity retention rate of up to 91%. At the same time, the 3000Da ultrafiltration process effectively removes allergenic macromolecular proteins from yeast. The product has been verified by human skin patch tests to have no redness, stinging or other irritation reactions, and has high safety. It can be added at a high concentration to professional-grade hyaluronic acid serums, home repair essences and Class II medical aesthetic wound repair dressings, thus broadening the application range of ribonucleic acid active raw materials in sensitive skin and open minimally invasive scenarios. Attached Figure Description

[0025] Figure 1 This is a flowchart of a method and apparatus for preparing small molecule ribonucleic acid by low-temperature enzymatic hydrolysis coupled with directional ultrafiltration according to the present invention. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1:

[0028] Please see Figure 1 As shown, the present invention provides a technical solution: a method for preparing small molecule ribonucleic acid by low-temperature enzymatic hydrolysis coupled with directional ultrafiltration, comprising the following steps:

[0029] Step 1: Raw material pretreatment: Weigh 10kg of food-grade high-activity dry yeast, add 100L of room temperature pure water, put it into a stirred suspension reactor, stir and suspend at 60r / min for 30min to fully disperse the yeast cells, transfer the suspension to a low-speed centrifuge, centrifuge at 3000r / min for 15min to remove the yeast coarse cell wall residue and insoluble large particulate impurities that settle at the bottom, and collect the upper milky white yeast supernatant for later use;

[0030] Step 2: Low-temperature enzymatic hydrolysis: Pump the supernatant obtained in step (1) into a constant temperature reactor equipped with a cold water circulation jacket, turn on the circulating cooling water, and when the temperature of the liquid drops to and stabilizes at 38°C, add the complex nucleic acid hydrolase (the amount added is 0.5% of the volume of the supernatant), maintain the temperature inside the reactor at 38°C, and stir at a constant temperature of 100r / min for 2.5h to hydrolyze the long chain ribonucleic acid (RNA) into small molecular fragments.

[0031] Step 3: Enzyme inactivation and coarse filtration: After the enzymatic hydrolysis is completed, turn off the cooling water and heat the hydrolysate to 55°C by jacket steam heating. Keep it at this temperature for 20 minutes to completely denature and inactivate the complex nucleic acid hydrolysate. Then, coarsely filter the inactivated solution through an 80-mesh single-layer stainless steel filter screen to remove incompletely hydrolyzed cell wall fragments and a small amount of flocculent matter. Collect the clear coarse filtrate.

[0032] Step 4: Ultrafiltration fractionation and enrichment: The coarse filtrate obtained in step (3) is passed into a spiral wound low-pressure ultrafiltration unit with a molecular weight cutoff of 3000 Da. Under the conditions of operating pressure of 0.3~0.5 MPa and circulation flow rate of 2.5 m³ / h, circulation ultrafiltration separation is carried out. During the ultrafiltration process, large molecular weight impurities with a molecular weight greater than 3000 Da and long chain RNA that has not been completely enzymatically hydrolyzed are retained at the reflux end, while the feed liquid rich in small molecular RNA of 50~200 bp permeates through the ultrafiltration membrane to form permeate. The permeate is continuously collected.

[0033] Step 5: Sterilization and low-temperature concentration: The ultrafiltration permeate collected in step (4) is filtered through a 0.22μm precision sterilization filter to remove residual microorganisms and endotoxins. The sterilized permeate is then transferred into a vacuum low-temperature concentration tank. The vacuum degree inside the tank is controlled at -0.085MPa and the temperature at 40℃. The solution is concentrated under reduced pressure until the solid content of the liquid is 12%, thus obtaining the small molecule ribonucleic acid stock solution.

[0034] Step 6: Finished product testing: The distribution of RNA fragments in the stock solution was detected by capillary electrophoresis. The results showed that small molecule ribonucleic acid of 50-200bp accounted for 76% of the total ribonucleic acid mass. In accordance with the human skin patch test method of the "Cosmetic Safety Technical Specification", 30 subjects were tested with a 24-hour closed patch test. The results were all negative (no irritation reactions such as erythema, edema, stinging).

[0035] Example 2 (lower limit of parameter range)

[0036] The difference between this embodiment and embodiment 1 is that in step (2), the low-temperature enzymatic hydrolysis temperature is set to 35°C and the enzymatic hydrolysis time is set to 2h. The remaining steps and parameters are exactly the same as in embodiment 1.

[0037] Testing revealed that in the small molecule ribonucleic acid stock solution obtained in this embodiment, small molecule RNA of 50–200 bp accounted for 71% of the total ribonucleic acid mass.

[0038] Human skin patch test results showed no skin irritation.

[0039] Example 3 (Upper Limit of Parameter Range)

[0040] The difference between this embodiment and embodiment 1 is that in step (2), the low-temperature enzymatic hydrolysis temperature is set to 40°C and the enzymatic hydrolysis time is set to 3h. The remaining steps and parameters are exactly the same as in embodiment 1.

[0041] Testing revealed that in the small molecule ribonucleic acid stock solution obtained in this embodiment, small molecule RNA of 50–200 bp accounted for 73% of the total ribonucleic acid mass.

[0042] Human skin patch test results showed no skin irritation.

[0043] Comparative Example 1 (High-Temperature Enzymatic Hydrolysis Comparison)

[0044] This comparative example aims to simulate the high-temperature pyrolysis conditions of the existing salmon PDRN extraction process. The difference from Example 1 is that in step (2), the enzymatic hydrolysis temperature is set to 70℃ (higher than the upper limit of 55℃ specified in this invention), the enzymatic hydrolysis time is still 2.5h, and the remaining steps and parameters are exactly the same as in Example 1.

[0045] Testing revealed that small RNA molecules of 50–200 bp accounted for only 21% of the total ribonucleic acid mass in the product obtained in this comparative example. A large amount of RNA underwent depurination and backbone breakage degradation at high temperatures, losing its original biological activity. At the same time, the high temperature caused the yeast protein to denature and produce pyrogenic sensitizing components. In the human skin patch test, 30% of the subjects showed grade II erythema reaction (moderate irritation), indicating that the product obtained by this high-temperature process is not suitable for minimally invasive and sensitive skin repair.

[0046] Comparative Example 2 (Comparison without Ultrafiltration)

[0047] This comparative example aims to simulate the conventional yeast crude RNA extraction process. The difference from Example 1 is that the 3000Da spiral ultrafiltration fractionation enrichment step (4) is cancelled. That is, after the coarse filtration in step (3), the coarse filtrate is directly subjected to 0.22μm sterilization and low temperature concentration in step (5). The remaining steps and parameters are exactly the same as in Example 1.

[0048] Testing revealed that small RNA molecules of 50–200 bp accounted for only 36% of the total ribonucleic acid mass in the product obtained in this comparative example. Furthermore, the product contained a large amount of residual proteins and polysaccharides with molecular weights greater than 50 kDa. The patch test on human skin showed slight redness, but due to the lack of targeted enrichment, the large RNA molecules could not penetrate the stratum corneum and only formed a moisturizing film on the epidermis. In human testing, the transepidermal water loss (TEWL) of the skin returned to the level before use after 3 days of discontinuation, indicating that it did not have a long-lasting barrier repair effect on the dermis.

[0049] A comparison of the data from Examples 1-3 and Comparative Examples 1-2 shows that, by strictly limiting the synergistic effect of low-temperature enzymatic hydrolysis at 35-40℃ and directional ultrafiltration at 3000Da, the present invention can only achieve its intended effect by simultaneously meeting the following two core conditions:

[0050] Excessive enzymatic hydrolysis temperature (Comparative Example 1) can destroy RNA activity and introduce irritating impurities;

[0051] Without ultrafiltration fractionation (Comparative Example 2), it is impossible to selectively retain large molecular allergens or efficiently enrich small molecule RNAs with dermal permeability of 50-200bp.

[0052] Only under the mild conditions of the entire process specified in this invention can we stably obtain ribonucleic acid raw materials with a small molecule RNA content of ≥70%, low irritation, and triple barrier repair function, achieving unexpected technical results.

[0053] The comparative test data is shown in the table below:

[0054]

[0055] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing small molecule ribonucleic acid by low-temperature enzymatic hydrolysis coupled with directional ultrafiltration, characterized in that, Includes the following steps: S1: Add food-grade dry yeast to pure water, stir to suspend, centrifuge, and collect the supernatant; S2: Place the supernatant in a constant temperature reactor, add the complex nucleic acid hydrolase, and stir and hydrolyze at 35-40℃ for 2-3 hours to obtain the hydrolysate; S3: Heat the enzymatic hydrolysate to 55°C and inactivate the hydrolytic enzyme, then filter it through a filter screen and collect the coarse filtrate; S4: The coarse filtrate is passed through an ultrafiltration device with a molecular weight cutoff of 3000 Da for ultrafiltration, the permeate is collected, and small molecule ribonucleic acid is enriched. S5: After sterilization filtration, the permeate is concentrated under low temperature and reduced pressure to obtain small molecule ribonucleic acid stock solution.

2. The method according to claim 1, characterized in that: The food-grade dry yeast mentioned in S1 is food-grade high-activity dry yeast, the pure water is room temperature pure water, and the stirring and suspension time is 20-40 min. The centrifugation process is carried out at a speed of 2500–3500 r / min for a time of 10–20 min.

3. The method according to claim 1, characterized in that: The constant temperature reactor described in S2 is a cold water circulating constant temperature reactor with an enzymatic hydrolysis temperature of 38℃ and an enzymatic hydrolysis time of 2.5h.

4. The method according to claim 1, characterized in that: The inactivation time in S3 is 15-30 minutes, and the filter screen is an 80-mesh single-layer filter screen.

5. The method according to claim 1, characterized in that: The ultrafiltration equipment described in S4 is a spiral wound ultrafiltration equipment, which operates using a low-pressure circulating separation method.

6. The method according to claim 1, characterized in that: The sterilization filtration described in S5 uses a 0.22μm precision filter membrane, and the temperature for the low-temperature vacuum concentration is 35-45℃.

7. The method according to claim 1, characterized in that: The proportion of 50-200bp small molecule ribonucleic acid in the stock solution is ≥70% of the total ribonucleic acid mass.

8. The method according to claim 1, characterized in that: The method describes a process where the maximum material processing temperature does not exceed 55°C throughout the entire process, and strong acids or alkalis are not used to adjust the pH of the system.

9. The method according to claim 1, characterized in that: It also includes step six, drying and powdering: the small molecule ribonucleic acid stock solution is dried at low temperature to obtain small molecule ribonucleic acid powder.

10. The method according to claim 1, characterized in that: The method uses general-purpose basic equipment throughout, including: a stirred suspension reactor, a low-speed centrifuge, a cold water circulating constant temperature reactor, a 3000Da spiral wound ultrafiltration unit, a 0.22μm sterilization filter, and a vacuum low-temperature concentration tank.

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