Method for removing endotoxin from pullulan and use thereof

Abstract

The application discloses a method for removing endotoxin in pullulan and application thereof, and mainly comprises the following steps: crude pullulan extract is precipitated and washed by an organic solvent to obtain pullulan; after being dissolved, a surfactant is added into the solution to make endotoxin aggregates depolymerize into monomolecular form; then, the solution is filtered through an ultrafiltration membrane to remove endotoxin and residual small molecular impurities and inorganic salts; finally, the polysaccharide solution is precipitated by ethanol, dried and crushed to obtain refined pullulan; the endotoxin removal method adopted by the prepared pullulan has little influence on the molecular weight of the pullulan, the endotoxin content of the obtained pullulan is not higher than 0.05 EU / mg, the prepared pullulan meets the medical standard, has high biological safety, the operation steps are simple, the pullulan is easy to realize large-scale production, and the pullulan has the advantages of rapidness, simplicity, low cost and industrialization; and the technical scheme of the application is favorable to promoting the application of the pullulan in the field of tissue engineering.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, more particularly to a method for removing endotoxin in pullulan and application thereof. BACKGROUND

[0002] Pullulan is a water-soluble glucan, which can be produced by aerobic fermentation of Aureobacidium pullulans. It is a regular repeating copolymer, in which glucose is first connected by α-(1, 4)-glycosidic bond to form maltotriose, and then the two ends are connected to another maltotriose by α-(1→6) glycosidic bond to form a linear polysaccharide. This structure gives pullulan structural flexibility and strong water solubility, and the unique connection mode makes pullulan have unique adhesive properties and fiber-forming properties and film-forming ability. In addition, the pullulan chain has a large number of hydroxyl groups, which is easy to realize chemical modification, such as adjusting its solubility or providing more active groups by chemical derivatization. Therefore, pullulan and its derivatives have great potential in food, pharmaceutical and other industrial applications.

[0003] At present, pullulan has attracted a lot of attention in the fields of drug carriers and tissue engineering due to its good biocompatibility, biodegradability and no immune rejection. For example, researchers found that pullulan has certain biological adhesion, which can fix chondrocytes on the surface of articular cartilage and promote the osteogenic differentiation of chondrocytes. As a medical material, it has high requirements for mechanical properties and degradation performance, therefore, high molecular weight pullulan has wide application prospects in the field of tissue engineering and other medical fields.

[0004] However, during the production and purification process of pullulan, bacterial contamination will be introduced to some extent. The introduction and death of gram-negative bacteria will lead to the release of endotoxin in pullulan, and the high content of endotoxin in the product will cause health problems such as body fever, microcirculation disorder and endotoxin shock, therefore, the content of endotoxin in the product needs to be strictly controlled to ensure the safety in use. Medical materials have very strict requirements for the residual amount of endotoxin due to their special use, and the national standard limits the amount of endotoxin in tissue engineering sodium hyaluronate to ≤0.05 EU / mg. In addition, the medium and high molecular weight pullulan forms a transparent gelatinous liquid after dissolving in water, which has high viscosity and poor flowability. The ultrafiltration process has poor effect on removing endotoxin, and the sample loss is large, which cannot realize large-scale production.

[0005] In view of the above problems, the present application provides a method for removing endotoxin from pullulan, which uses pullulan crude extract as raw material, and obtains pullulan by preliminary purification and washing. The polysaccharide is dissolved again, treated with a surfactant, so that endotoxin aggregates in the solution are depolymerized into monomolecular form, and then the endotoxin is removed by ultrafiltration membrane filtration, and then the refined pullulan with endotoxin removed is obtained by precipitation and drying again and crushing. The endotoxin removal method used in the present application has little effect on the molecular weight of pullulan, and the obtained pullulan has an endotoxin content of not higher than 0.05 EU / mg, which meets the medical standard, has high biological safety, and has simple operation steps, easy to realize scale-up production, and is conducive to promoting the application of pullulan in the field of tissue engineering. SUMMARY

[0006] The present application aims at the deficiencies in the prior art, and provides a method for removing endotoxin from pullulan, which has simple operation steps, is easy to realize scale-up production, and can effectively remove endotoxin from pullulan.

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] 1. A method for removing endotoxin from pullulan, comprising the following steps:

[0009] 1) Using Aureobasidium pullulans fermentation to obtain pullulan fermentation broth, and then centrifuging to obtain pullulan crude liquid;

[0010] 2) Precipitating, washing and drying the pullulan crude extract with an organic solvent to obtain a solid;

[0011] 3) Dissolving the solid obtained in step 2), adding a surfactant of a certain concentration, stirring thoroughly, reacting, ultrafiltrating, and precipitating and drying under clean conditions (thirty million level cleanliness) with an organic solvent to obtain pullulan with endotoxin removed.

[0012] Further, the pullulan crude extract in step 1) has a molecular weight of more than 2560 kDa, and the crude extract is removed of impurities such as proteins.

[0013] Further, the organic solvent includes anhydrous ethanol, acetone and methanol; the final concentration of the organic solvent used for precipitation is 40-85%; and the organic precipitation concentration used for washing is 90-100%.

[0014] The concentration percentage of the organic solvent is all volume concentration percentage.

[0015] Further, the surfactant in step 3) includes one or a combination of two or more of Tween-20, Tween-60, sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, sodium dioctyl succinimide sulfonate, and sodium lauryl sulfate.

[0016] Further, the specific treatment method of the surfactant in step 3) is as follows:

[0017] (1) The surfactant is dissolved in the pullulan solution to a final concentration of 1-10%; the surfactant concentration is a weight / volume percentage;

[0018] (2) The temperature of the reaction system is adjusted to 25-50°C, and the reaction is carried out under stirring for 0.5-5h;

[0019] (3) The solution is ultrafiltered to obtain a pullulan solution under a clean environment, and the ultrafiltration membrane has a molecular weight cut-off of ≤10kD.

[0020] Further, the drying method is drying under atmospheric pressure at 40-80°C, drying under reduced pressure, vacuum freeze drying, and spray drying after re-dissolving the precipitate.

[0021] Further, the water used in the pullulan treatment process is all high-temperature treated pyrogen-free water.

[0022] Further, the containers used in the treatment process are all heat source-free containers treated by 0.1% NaOH soaking or high-temperature treatment.

[0023] A pullulan with a low endotoxin content.

[0024] The application of a pullulan with a low endotoxin content in the medical gel, tissue engineering, wound repair material, and other clinical medical fields.

[0025] The beneficial effects of the present application are as follows: by utilizing the hydrophobic and negative charge properties of endotoxin, the characteristics that endotoxin molecules in the solution will aggregate into macromolecules due to hydrophobic interaction, and by treating the endotoxin with a surfactant, the endotoxin micelles can be depolymerized to form a single molecular state, according to the difference in molecular weight between endotoxin and pullulan, the endotoxin can be efficiently removed by ultrafiltration, the preparation process is simple, the endotoxin content in the obtained pullulan is very low, and the method has no obvious effect on the molecular weight of the pullulan. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0027] Figure 1 Figure is the comparison of the molecular weight of pullulan before and after removing endotoxin and the pullulan molecular weight standard. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0029] Embodiment 1

[0030] A method for removing endotoxin from pullulan and a detection method, the specific operation steps are as follows:

[0031] (1) 1 L of pullulan crude extract solution is measured, anhydrous ethanol is slowly added to a final concentration of 60%, and after white flocculent precipitate is precipitated, the precipitate is collected by centrifugation;

[0032] (2) The precipitate is sequentially washed with 75%, 80%, 90% and 100% ethanol, and after washing, the pullulan is obtained by drying at 50°C under normal pressure;

[0033] (3) 1 g of pullulan is accurately weighed and added to 40 mL of water without pyrogen, and the solution is left to stand overnight at room temperature to form a uniform solution;

[0034] (4) 2 mL of Tween-20 is measured and added dropwise to the pullulan solution, and the reaction is carried out at 30°C for 2 h;

[0035] (5) After the temperature of the solution decreases to room temperature, the solution is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10 kD;

[0036] (6) The polysaccharide solution after ultrafiltration is collected, anhydrous ethanol is added again to a final concentration of 90%, and the precipitate is separated by centrifugation;

[0037] (7) The precipitate is redissolved in water without pyrogen to a concentration of 10%, and the endotoxin-removed pullulan is obtained by spray drying;

[0038] (8) Detection of endotoxin content in pullulan before and after treatment by using limulus reagent method. The endotoxin standard solution of 10 EU / ml was diluted into four concentrations of 2λ, λ, 0.5λ and 0.25λ, and the concentration of λ was 0.0125. In addition, 10 mg of pullulan powder before and after treatment was taken and dissolved in pyrogen-free water to prepare a 5 mg / mL solution, which was then diluted into different concentrations. 0.1 mL of limulus reagent was taken and added to the endotoxin standard solution and the pullulan solution with different concentrations, respectively. The negative control was pyrogen-free water. The reaction solution was incubated at 37℃±1℃ for 60 min, and then the gel formation state was observed, and the endotoxin content in the sample was calculated. The results showed that the residual amount of endotoxin in the untreated pullulan was ≤1.25 EU / mg, and the residual amount of endotoxin in the treated pullulan was ≤0.0125 EU / mg. The removal rate of endotoxin content reached 99%, and the removal effect was very obvious.

[0039] (9) Chromatographic analysis of the molecular weight of pullulan showed that the molecular weight of the obtained pullulan was more than 2560 kDa, and there was no significant difference in the molecular weight and distribution of pullulan before and after treatment, which proved that the treatment did not obviously cause the degradation of pullulan.

[0040] (10) The recovery rate of pullulan: the weight of pullulan after removing endotoxin was weighed and compared with that before treatment. The results showed that the recovery rate of pullulan could reach 98.2%, which indicated that there was no obvious loss of pullulan during the treatment process. The specific results are shown in Table 1.

[0041] Example 2

[0042] A method for removing endotoxin from pullulan, the specific operation steps are as follows:

[0043] (1) 1L of pullulan crude extract solution was taken and anhydrous ethanol was slowly added to a final concentration of 70%. After white flocculent precipitate was precipitated, the precipitate was collected by centrifugation;

[0044] (2) The precipitate was washed with 80%, 90%, 95% and anhydrous ethanol in turn, and then dried at 40℃ under normal pressure to obtain pullulan;

[0045] (3) 5g of pullulan was accurately weighed and added to 50mL of pyrogen-free water. The solution was left to stand at room temperature overnight to form a uniform solution;

[0046] (4) 2.5mL of Tween-20 was taken and added dropwise to the above pullulan solution, and the reaction was carried out at 30℃ water bath for 2h;

[0047] (5) After the solution temperature drops to room temperature, the solution is subjected to ultrafiltration through an ultrafiltration membrane with a molecular weight cut-off of 10 kD;

[0048] (6) The polysaccharide solution after ultrafiltration is collected, and anhydrous ethanol is added again to a final concentration of 90%, and the precipitate is separated by centrifugation;

[0049] (7) The precipitate is redissolved in pyrogen-free water to a concentration of 10%, and the endotoxin-removed pullulan is obtained by spray drying; after treatment, the endotoxin content in the pullulan is less than 0.0125 EU / mg, and the pullulan recovery rate reaches 97.5%, indicating that the increase in the concentration of pullulan within the range will not have a significant difference in the treatment results, and the specific results are shown in Table 1.

[0050] Example 3

[0051] A method for removing endotoxin from pullulan, the specific operation steps are as follows:

[0052] (1) 1 L of crude pullulan solution is measured, and anhydrous ethanol is slowly added to a final concentration of 80%, and after white flocculent precipitate is precipitated, the precipitate is collected by centrifugation;

[0053] (2) The precipitate is sequentially washed with 80%, 90% and 100% ethanol, and the pullulan is obtained by drying at 40°C under normal pressure after washing;

[0054] (3) 1 g of pullulan is accurately weighed and added to 40 mL of pyrogen-free water, and the solution is allowed to stand overnight at room temperature to form a uniform solution;

[0055] (4) 1 mL of Tween-60 is measured and added dropwise to the above pullulan solution, and the reaction is carried out at 400°C water bath for 1 h;

[0056] (5) After the solution temperature drops to room temperature, the solution is subjected to ultrafiltration through an ultrafiltration membrane with a molecular weight cut-off of 5 kD;

[0057] (6) The polysaccharide solution after ultrafiltration is collected, and anhydrous ethanol is added again to a final concentration of 95%, and the precipitate is separated by centrifugation;

[0058] (7) The precipitate is redissolved in pyrogen-free water to a concentration of 10%, and the endotoxin-removed pullulan is obtained by spray drying; after treatment, the endotoxin content in the pullulan is less than 0.025 EU / mg, and the pullulan recovery rate reaches 98%, indicating that the use of the surfactant can make the endotoxin content of the pullulan meet the medical grade standard requirements, and the specific results are shown in Table 1.

[0059] Example 4

[0060] A method for removing endotoxin from pullulan, the specific operation steps are as follows:

[0061] (1) Take 1 L of crude pullulan extract, slowly add anhydrous ethanol to a final concentration of 75%, and after the white flocculent precipitates, centrifugal collection of the precipitate;

[0062] (2) The precipitate is sequentially washed with 90%, 95%, and 100% ethanol, and after washing, the pullulan is obtained by drying at 50°C under normal pressure;

[0063] (3) Accurately weigh 2 g of pullulan and add to 20 mL of water without pyrogens, and let it stand overnight at room temperature to form a uniform solution;

[0064] (4) Take 0.2 g of sodium lauryl sulfate and add to the pullulan solution, and react at 60°C for 1 h;

[0065] (5) After the solution temperature drops to room temperature, the solution is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10 kD;

[0066] (6) Collect the polysaccharide solution after ultrafiltration, and add anhydrous ethanol to a final concentration of 95% and centrifugal separation of the precipitate;

[0067] (7) The precipitate is dried under reduced pressure at 30°C, crushed, and the endotoxin-removed pullulan is obtained. After treatment, the endotoxin content in the pullulan is less than 0.05 EU / mg, and the pullulan recovery rate reaches 98.6%, indicating that the use of the surfactant can make the endotoxin content of the pullulan meet the medical grade standard requirements, and the specific results are shown in Table 1.

[0068] Comparative Example 1

[0069] A method for removing endotoxin from pullulan, the specific operation steps are as follows:

[0070] (1) Take 1 L of crude pullulan extract, slowly add anhydrous ethanol to a final concentration of 60%, and after the white flocculent precipitates, centrifugal collection of the precipitate;

[0071] (2) The precipitate is sequentially washed with 75%, 80%, 90%, and 100% ethanol, and after washing, the pullulan is obtained by drying at 50°C under normal pressure;

[0072] (3) Accurately weigh 1 g of pullulan and add to 100 mL of water without pyrogens, and let it stand overnight at room temperature to form a uniform solution;

[0073] (4) Add polystyrene-divinylbenzene-based macroporous adsorption resin to the pullulan solution, adsorb for 8 h, and centrifugal collection of the supernatant;

[0074] (5) Add anhydrous ethanol to a final concentration of 90% and centrifugal separation of the precipitate;

[0075] (6) The endotoxin-removed pullulan was obtained by dissolving the precipitate in pyrogen-free water to form a solution with a concentration of 10% and then spray drying; the endotoxin content in the pullulan after treatment was lower than 0.1 EU / mg, and the recovery rate of the pullulan was 90.4%, and the specific results are shown in Table 1.

[0076] Table 1

[0077]

[0078]

[0079] The various embodiments described in the specification are progressive in nature, and each embodiment highlights the differences from other embodiments. The same or similar parts between the embodiments can be mutually referred to. For the device disclosed in the embodiments, since it corresponds to the method disclosed in the embodiments, the description is relatively simple, and the relevant parts can be referred to the method part.

[0080] The above description of disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to the embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for removing endotoxin from pullulan, characterized by, The method comprises the following steps: 1) obtaining a pullulan fermentation liquor by fermentation of Aureobasidium pullulans, and then centrifuging to obtain a pullulan crude extract; 2) precipitating the pullulan crude extract with an organic solvent, washing, and then drying to obtain a solid; 3) dissolving the solid to obtain a pullulan solution, adding a surfactant, stirring sufficiently, ultrafiltrating after reaction, and precipitating with an organic solvent and drying under clean conditions to obtain a pullulan with removed endotoxin; In step 1), the molecular weight of the pullulan in the pullulan crude extract is greater than 2560 kDa; In steps 2) and 3), the organic solvent comprises one of anhydrous ethanol, acetone, and methanol; the final concentration of the organic solvent used for precipitation is 40-85%, and the concentration of the organic solvent used for washing is 90-100%; The concentration of the organic solvent is a volume percentage; In step 3), the surfactant comprises one or more than two of Tween-20, Tween-60, and sodium lauryl sulfate; In step 3), the surfactant is dissolved in the pullulan solution, and the final concentration is 1-10%; the reaction is carried out at 25-50℃ for 0.5-5 h under stirring; and after the reaction, the pullulan solution with removed endotoxin is obtained by ultrafiltration under a clean environment. The concentration of the surfactant is a weight / volume percentage; The ultrafiltration membrane has a molecular weight cut-off of ≤10 kD.

2. The method for removing endotoxin from pullulan according to claim 1, characterized in that: In steps 2) and 3), the drying method is any one of the following: oven drying at 40-80℃ under normal pressure, drying under reduced pressure, vacuum freeze drying, and spray drying after redissolving the precipitate.

3. The method for removing endotoxin from pullulan according to claim 1, characterized by: In the process of treating the pullulan in claim 1, the water used is all pyrogen-free water treated at high temperature.

4. The method for removing endotoxin from pullulan according to claim 1, characterized by: In claim 1, the containers used are all heat source-free containers treated by 0.1% NaOH soaking or high temperature treatment.

5. Pullulan according to any one of claims 1 to 4, characterized in that The endotoxin content is ≤0.05 EU / mg.

6. Use of the pullulan obtained by any one of the methods in claims 1-4 in preparation of a medical gel or a wound repair material.

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