Preparation method of low-endotoxin epsilon-polylysine
Through single ultrafiltration and pH adjustment methods, the problem of endotoxin removal in ε-polylysine is solved, and the efficient preparation of low endotoxin products is achieved, reducing production costs and broadening the application scope.
Patent Information
- Application Number
- CN202510776864.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-11
- Publication Date
- 2025-07-22
AI Technical Summary
The prior art is difficult to effectively remove endotoxins in ε-polylysine, resulting in limited application in the medical device field, especially in the three types of medical devices that are exposed to blood. In addition, traditional processes have problems such as high loss rate, complex processes and high acid and alkali consumption.
Single ultrafiltration method was used to utilize the molecular weight difference between ε-polylysine and endotoxin, and use an ultrafiltration membrane of specific materials for separation, and pH adjustment and drying treatment were used to obtain low endotoxin ε-PL products.
Significantly reduce endotoxin content, reduce production costs, improve product purity and adapt to different application scenarios, broaden product uses, and meet high-end application needs.
Smart Images
Figure CN120349506A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and particularly to a method for preparing low-endotoxin ε-polylysine. Background Art
[0002] The great potential shown by the application of ε-polylysine in the biomedical field has greatly expanded the market and application prospects. However, due to the limitations of its process and the strong endotoxin adsorption ability of ε-polylysine itself, ε-polylysine produced by the microbial fermentation method often has extremely high endotoxin. The application of these ε-polylysines is not restricted in the food field, but in the application in the medical device field, especially in the field of class III blood-contact medical devices, the high endotoxin causes usage restrictions. Therefore, it is particularly important to develop a method for preparing low-endotoxin ε-polylysine.
[0003] Existing endotoxin removal technologies have the following defects: Activated carbon adsorption method: The loss rate of ε-PL is as high as 15%, and the decolorization effect is unstable. Ion exchange resin method: Multiple-stage treatment is required, the process is complex and the consumption of acid and alkali is large. Traditional ultrafiltration method: It is not designed according to the molecular weight difference between ε-PL and endotoxin, and it is difficult to separate efficiently (the molecular weight of endotoxin is usually >100 kDa, while the molecular weight of ε-PL is 3.6 - 4.3 kDa).
[0004] We innovatively utilize the difference in the molecular weights of ε-polylysine and endotoxin, use ultrafiltration technology, and can effectively remove endotoxin in ε-polylysine through single ultrafiltration. The ultrafiltered ε-polylysine solution can be directly dried and used in biomedicine or medical devices, or can be dried after adjusting the pH of the solution to obtain ε-polylysine products with different pH values. This greatly facilitates the subsequent use of ε-polylysine and has excellent flexibility and variability. Summary of the Invention
[0005] The present invention provides a method for preparing low-endotoxin ε-polylysine, which solves the above problems existing in the prior art during use.
[0006] The technical solution of the present invention is realized as follows:
[0007] A method for preparing low-endotoxin ε-polylysine, comprising
[0008] a) Dissolving the crude ε-PL product produced by the microbial fermentation method in pure water to form a solution;
[0009] b) Performing single ultrafiltration using an ultrafiltration membrane and collecting the permeate;
[0010] c) After adjusting the pH value of the permeate to the target range, dry it to obtain a low endotoxin ε-PL product.
[0011] Preferably, the solution concentration is 5%-50%.
[0012] Preferably, the ultrafiltration membrane material is any one of polyethersulfone (PES), regenerated cellulose (RC), polyacrylonitrile (PAN), polysulfone (PSF), polyvinylidene fluoride (PVDF), and polyvinyl chloride (PVC), with a molecular weight cut-off of 5-50 kDa and a transmembrane pressure of 0.2-0.4 MPa.
[0013] Preferably, the pH regulator is hydrochloric acid, sodium hydroxide, triethanolamine, or sodium bicarbonate.
[0014] Preferably, the pH value of the permeate is 3-10, which is respectively used for medical device applications in acidic or neutral environments.
[0015] Preferably, the drying method is freeze-drying or spray-drying.
[0016] Preferably, for the prepared ε-PL product, the endotoxin content is ≤0.30 EU / mg.
[0017] The present invention reduces the consumption of acids and bases and wastewater discharge, and reduces production costs. The present invention can be adapted to different application scenarios (such as acidic / neutral environments) through pH regulation, broadening the product uses.
[0018] In summary, the beneficial effects of the present invention are as follows:
[0019] A preparation method of low endotoxin ε-polylysine disclosed by the present invention:
[0020] First of all, it can efficiently reduce the endotoxin content of ε-polylysine. Single ultrafiltration can reduce the endotoxin content from 5000 EU / mg to 0.2 EU / mg, which is significantly better than the traditional multi-stage process, thus providing a reliable technical means for producing high-purity and low-endotoxin ε-polylysine products and meeting the high-end application requirements for strict endotoxin control, such as in fields with extremely high safety requirements such as medicine and food.
[0021] Secondly, by optimizing parameters such as the ultrafiltration membrane material, the molecular weight cut-off, and the transmembrane pressure, this method achieves efficient separation and purification effects. At the same time, it reduces the consumption of acids and alkalis and wastewater discharge, lowers production costs, improves production efficiency, and has significant economic and environmental benefits, contributing to the sustainable development of the ε-polylysine industry. In addition, through pH regulation, the product can be adapted to different application scenarios, such as acidic and neutral environments, broadening the scope of product uses, enhancing its competitiveness in the market, and providing the possibility for the application of ε-polylysine in more fields. For example, its applications in food preservation, cosmetics anti-corrosion, pharmaceutical preparations, etc. can better meet the needs of different customers and promote the development of related industries. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.
[0023] Figure 1 It is a schematic diagram of obtaining an ε-PL product by freeze-drying.
[0024] Figure 2 It is a schematic diagram of obtaining an ε-PL product by spray drying. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0025] The following will combine the drawings in the embodiments of the present invention Figure 1-2 to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.
[0026] Example 1
[0027] As Figures 1 to 2 shown, this embodiment discloses a method for preparing low-endotoxin ε-polylysine, including
[0028] a) Dissolving the crude ε-PL product produced by the microbial fermentation method in pure water to form a solution;
[0029] b) Performing single ultrafiltration using an ultrafiltration membrane and collecting the permeate;
[0030] c) Adjusting the pH value of the permeate to the target range and then drying to obtain a low-endotoxin ε-PL product.
[0031] 2. The preparation method of a low endotoxin ε-polylysine according to claim 1, wherein the solution concentration is 5%-50%.
[0032] Preferably, the ultrafiltration membrane material is any one of polyethersulfone (PES), regenerated cellulose (RC), polyacrylonitrile (PAN), polysulfone (PSF), polyvinylidene fluoride (PVDF), and polyvinyl chloride (PVC), the molecular weight cut-off is 5-50 kDa, and the transmembrane pressure is 0.2-0.4 MPa.
[0033] Preferably, the pH regulator is hydrochloric acid, sodium hydroxide, triethanolamine, or sodium bicarbonate.
[0034] Preferably, the pH value of the permeate is 3-10, which is respectively used for medical device applications in acidic or neutral environments.
[0035] Preferably, the drying method is freeze-drying or spray drying.
[0036] Preferably, for the prepared ε-PL product, the endotoxin content is ≤0.30 EU / mg.
[0037] The present invention reduces the consumption of acids and bases and wastewater discharge, and reduces production costs. The present invention can be adapted to different application scenarios (such as acidic / neutral environments) through pH regulation, and broadens the uses of the product.
[0038] Ultrafiltration process optimization
[0039] Experimental conditions: 100 grams of crude ε-PL (endotoxin 5000 EU / mg) was dissolved in pure water to prepare filtrates with different concentrations, and single ultrafiltration was carried out using ultrafiltration membranes of different materials, and the permeate was collected;
[0040] Results:
[0041]
[0042] Example 2: pH regulation and drying
[0043] 100 grams of crude ε-PL (endotoxin 5000 EU / mg) was dissolved in pure water to prepare a 10% filtrate, and single ultrafiltration was carried out using a polyethersulfone ultrafiltration membrane with a molecular weight cut-off of 10 kDa under a transmembrane pressure of 1 MPa, and the permeate was collected. The ultrafiltered ε-PL solution was adjusted to the target pH and dried to obtain a powdery ε-PL product, as Figure 1 (ε-PL product obtained by freeze-drying) Figure 2 (ε-PL product obtained by spray drying) shown. The product was redissolved to a 10% concentration, and its redissolution solubility and the difference between the dissolved pH and the target pH were measured.
[0044] Results:
[0045]
[0046] Example 3: Verification of Medical Device Application
[0047] The low endotoxin ε-PL was combined with 4-arm PEG-SG to form a covalently bonded hydrogel. After testing according to the ISO 10993 standard, the biocompatibility met the standard.
[0048] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
Claims
1. A method for preparing low-endotoxin ε-polylysine, comprising a) dissolving the crude ε-PL produced by microbial fermentation in pure water to form a solution; b) performing single ultrafiltration using an ultrafiltration membrane and collecting the permeate; c) adjusting the pH value of the permeate to the target range and then drying to obtain a low-endotoxin ε-PL product.
2. The preparation method of a low endotoxin ε-polylysine according to claim 1, wherein: The concentration of the solution is 5%-50%.
3. The preparation method of a low endotoxin ε-polylysine according to claim 1, characterized in that: The ultrafiltration membrane material in step b) is any one of polyethersulfone (PES), regenerated cellulose (RC), polyacrylonitrile (PAN), polysulfone (PSF), polyvinylidene fluoride (PVDF), and polyvinyl chloride (PVC), the molecular weight cut-off is 5-50 kDa, and the transmembrane pressure is 0.2-0.4 MPa.
4. The preparation method of a low endotoxin ε-polylysine according to claim 1 or 2 or 3, characterized in that: The pH regulator is hydrochloric acid, sodium hydroxide, triethanolamine, or sodium bicarbonate.
5. The preparation method of a low endotoxin ε-polylysine according to claim 1 or 3, characterized in that: The pH value of the permeate in step b) is adjusted to 3-10, which is used for medical device applications in acidic or neutral environments respectively.
6. The preparation method of a low endotoxin ε-polylysine according to claim 1, characterized in that: The drying method in step c) is freeze drying or spray drying.
7. The preparation method of a low endotoxin ε-polylysine according to claim 1, characterized in that: For the prepared ε-PL product, the endotoxin content is ≤0.30 EU / mg.
8. The preparation method of a low endotoxin ε-polylysine according to claim 1, characterized in that: The single ultrafiltration can reduce the endotoxin content from 5000 EU / mg to 0.2 EU / mg.