Pulullan refining method

Abstract

The invention provides a pulullan refining method, which belongs to the technical field of biology, and specifically comprises the following steps: fermenting a microbial strain for producing pulullan to obtain fermentation liquor, and sterilizing and decoloring the fermentation liquor to obtain a crude pure extracting solution; carrying out a membrane process and a resin process on the obtained crude pure extracting solution to obtain a refined pure solution; the pore diameter of the membrane material is 500-1000Da; according to the method disclosed by the invention, it is found for the first time that the pullulan is extracted and purified by using a small-aperture nanofiltration membrane and is matched with resin adsorption, so that the purification effect can be achieved, the purity of the product is improved, and the yield of the product is also obviously improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for purifying pullulan. Background Technology

[0002] Pullulan is a natural extracellular polysaccharide secreted by *Brachystomata buddingae*. Its molecular structure consists of maltotriose units repeatedly polymerized via α-1,6-glycosidic bonds. This unique linear structure endows pullulan with excellent physicochemical properties, including high water solubility, film-forming ability, plasticity, thermal stability, and biocompatibility. The traditional pullulan extraction process involves centrifuging or plate-and-frame extraction to remove bacterial cells from the fermentation broth, followed by multiple alcohol precipitations to remove impurities, and finally drying to obtain pure pullulan. Traditional alcohol precipitation consumes large amounts of organic solvents, resulting in high costs and the risk of solvent residue.

[0003] Currently, many research institutions and research institutes have carried out research on alternatives to pullulan polysaccharide precipitation processes, but their scale remains at the laboratory level, and almost none have achieved large-scale production.

[0004] Nanofiltration technology, due to its low molecular weight cutoff and small membrane pore size, is mainly used for water purification and the purification of small molecules, and is basically not used for the purification of large molecules.

[0005] Pullulan, as a multifunctional biopolymer, continues to see its application value and market demand grow. Improving the extraction process to enhance product quality and yield remains key to its industrialization. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for purifying pullulan.

[0007] The purpose of this invention is to overcome the problem of the traditional pullulan production process using alcohol and other cumbersome non-alcoholic process steps, and to provide a pullulan purification process that does not use organic solvents and is simple in process.

[0008] The present invention adopts the following technical solution: A method for purifying pullulan includes the following steps: (1) Fermentation broth was obtained by fermenting the microbial strain that produces pullulan polysaccharide. The fermentation broth was sterilized and decolorized to obtain crude pure extract. (2) The crude extract obtained in step (1) is processed through membrane technology and resin technology to obtain a refined extract.

[0009] Preferably, in step (1), the microbial strain that produces pullulan is *Brucea buddingis*.

[0010] More preferably, the budding short-stemmed mold is budding short-stemmed mold BL80ΔPMAs.

[0011] Preferably, in step (1), the sterilization method is sterilization by centrifugation or filtration; the decolorization method is decolorization by using activated carbon.

[0012] Preferably, in step (2), the membrane process is as follows: the feed temperature is ≤30℃, the transmembrane pressure difference is ≤4MPa, and deionized water is added for dialysis until the conductivity is ≤1000μs / cm.

[0013] Preferably, the membrane material has a pore size of 500-1000 Da, and the membrane material is one or both of organic and inorganic membranes.

[0014] More preferably, the organic membrane includes one or more of the following: polysulfone, polyvinylidene fluoride, polyethersulfone composite membrane, polypropylene, and aromatic polyamide composite membrane; the inorganic membrane is a ceramic membrane.

[0015] Preferably, in step (2), the resin process is as follows: liquid temperature 20-30℃, pH 6.0-9.0, chromatography linear velocity 1-5m / h, and feed pressure ≤2MPa.

[0016] Preferably, the resin is one or more of anionic, cationic, or mixed resins.

[0017] Preferably, the ion exchange resin is in the form of a gel or macroporous resin.

[0018] More preferably, the anion exchange resin has a weak or strong base functional group; The cationic resin has strong acid or weak acid functional groups.

[0019] More preferably, the anion exchange resin includes one or two of D201 resin and D301 resin; the cation exchange resin includes one or more of 001*10 resin, 101*7 resin, 001*8 resin, and 001*7 resin; and the mixed resin includes one or two of MX-1 resin and MX-21 resin.

[0020] More preferably, the anion exchange resin, cationic resin, and mixed resin can be used individually or in series.

[0021] Preferably, in step (2), the purified liquid is dried by drum drying to obtain pullulan polysaccharide.

[0022] Further optimized, the obtained pullulan polysaccharide has a purity of ≥97%.

[0023] Beneficial effects of the present invention This invention is the first to discover that using a nanofiltration membrane with a small pore size to extract and purify pullulan polysaccharide, combined with resin adsorption, can achieve a high degree of purification, improve the purity of the product, and significantly increase the product yield. Attached Figure Description

[0024] Figure 1 This is a liquid-phase detection image of a nanofiltration membrane with a molecular weight cutoff of 1000 Da.

[0025] Figure 2 This is a liquid-phase detection image of the permeate from an ultrafiltration membrane with a molecular weight cutoff of 10000 Da. Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0027] Experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions.

[0028] Unless otherwise specified, all materials and reagents used in the embodiments are commercially available.

[0029] Example 1 A method for purifying pullulan includes the following steps: (1) Fermentation broth was obtained by fermenting *Brachystomata buddingis* BL80ΔPMAs (the strain BL80ΔPMAs has been disclosed in patent literature, publication number CN 115197857 A, application number 202211106612.X) in a 60T tank; the culture medium was: glucose 160g / L, corn steep liquor powder 20g / L, potassium chloride 4g / L, sodium dihydrogen phosphate 4g / L, magnesium chloride 0.4g / L; the fermentation conditions were: rotation speed 110rpm, aeration 1450m 3 Fermentation broth was obtained by incubation at pH 6, temperature 30℃ for 132 hours; diatomaceous earth (3% by mass) was added to the fermentation broth, and the mixture was sterilized by plate and frame filtration. Then, activated carbon (1% by mass) was added for decolorization to obtain crude pure broth.

[0030] (2) A polyethersulfone composite membrane (Hefei Xinda Membrane Technology Co., Ltd.) with a molecular weight cutoff of 500 Da was selected for membrane dialysis filtration. The temperature of the crude pure liquid was controlled at 25℃ through a heat exchanger, and the pressure difference across the membrane was controlled at 3 MPa. Deionized water was added for dialysis until the conductivity of the feed solution was 961 μs / cm. Membrane filtration was then completed to obtain pure liquid 1.

[0031] (3) The purified solution 1 was subjected to cation exchange resin adsorption, as follows: Adsorption was performed using 001*10 (Shandong Dechuan Chemical Technology Co., Ltd.). The pH of the purified solution 1 was adjusted to 6.2. The feed temperature was controlled at 20℃ using a heat exchanger, the resin inlet pressure was controlled at 0.5 MPa, and the chromatography linear velocity was 1.2 m / h. Purified solution 2 was obtained after adsorption using cation exchange resin.

[0032] (4) Pure solution 2 is subjected to anion exchange resin adsorption. D201 (Shandong Dechuan Chemical Technology Co., Ltd.) was selected for adsorption. The pH of the purified solution 2 was adjusted to 8.0, and the temperature of the purified solution 2 was controlled at 25℃ via a heat exchanger. The resin inlet pressure was 1.5 MPa, and the chromatography linear velocity was 4.3 m / h. After adsorption by the anion exchange resin, purified solution 3 was obtained.

[0033] (5) The purified solution 3 is dried in a drum at 150°C to obtain pullulan polysaccharide product.

[0034] Since pullulan is a mixture containing components of different molecular weights, there is currently no direct method to detect its purity. This invention uses the difference method (methods for detecting individual components, GB / 28402-2012) to detect the purity of pullulan.

[0035] Pullulan purity = 100% - mono- and di-oligosaccharide content - moisture content; The content of monosaccharides, disaccharides, and oligosaccharides refers to the content of monosaccharides, disaccharides, and oligosaccharides.

[0036] The purity of pullulan was determined to be 98.5%.

[0037] Pullulose yield was 94%.

[0038] ; P: Concentration of pullulan polysaccharide in crude pure solution; V: Volume of crude pure liquid; P3: Concentration of pullulan polysaccharide in purified solution 3; V3: 3 volumes of pure liquid.

[0039] Example 2 (1) Prepare crude pure liquid according to the method in Example 1.

[0040] (2) Select a polypropylene membrane (Hefei Xinda Membrane Technology Co., Ltd.) with a molecular weight cutoff of 1000 Da for membrane dialysis filtration. Control the temperature of the crude pure liquid at 30℃ through a heat exchanger, control the pressure difference across the membrane at 2 MPa, add deionizers for dialysis until the conductivity of the feed solution is 790 μs / cm, end the membrane filtration, and obtain pure liquid 1.

[0041] (3) Pure solution 1 is subjected to cation exchange resin adsorption. Adsorption was performed using 101*7 (Shandong Dechuan Chemical Technology Co., Ltd.). The pH of the purified solution 1 was adjusted to 7.5. The feed temperature was controlled at 20℃ using a heat exchanger, the resin inlet pressure was controlled at 1 MPa, and the chromatography linear velocity was 2.6 m / h. Purified solution 2 was obtained after adsorption using cation exchange resin.

[0042] (4) Pure solution 2 is subjected to anion exchange resin adsorption. D301 (Shandong Dechuan Chemical Technology Co., Ltd.) was selected for adsorption. The pH of the purified solution 2 was adjusted to 6, and the temperature of the purified solution 2 was controlled at 20℃ via a heat exchanger. The resin inlet pressure was 2 MPa, and the chromatography linear velocity was 4.9 m / h. After adsorption by the anion exchange resin, purified solution 3 was obtained.

[0043] (5) The purified solution 3 was dried in a drum at 140°C to obtain pullulan polysaccharide product.

[0044] The purity of pullulan was 98.1%, and the yield of pullulan was 95%.

[0045] Example 3 (1) Prepare crude pure liquid according to the method in Example 1.

[0046] (2) Select an aromatic polyamide composite membrane (Hefei Xinda Membrane Technology Co., Ltd.) with a molecular weight cutoff of 1000 Da for membrane dialysis filtration. Control the temperature of the crude pure liquid at 20℃ through a heat exchanger, control the pressure difference across the membrane at 1 MPa, add deionized water for dialysis until the conductivity of the feed solution is 586 μs / cm, end the membrane filtration, and obtain pure liquid 1.

[0047] (3) Pure solution 1 is subjected to cation exchange resin adsorption. Adsorption was performed using 001*8 (Shandong Dechuan Chemical Technology Co., Ltd.). The pH of the purified solution 1 was adjusted to 7.0. The feed temperature was controlled at 25℃ using a heat exchanger, the resin inlet pressure was controlled at 1.6 MPa, and the chromatography linear velocity was 3.3 m / h. Purified solution 2 was obtained after adsorption using cation exchange resin.

[0048] (4) The purified solution 2 was subjected to adsorption by a mixed macroporous ion exchange resin. Adsorption was performed using MX-1 (Shandong Dechuan Chemical Technology Co., Ltd.). The pH of the purified solution 2 was adjusted to 9.0, and the temperature of the purified solution 2 was controlled at 30℃ via a heat exchanger. The resin inlet pressure was 0.5 MPa, and the chromatography linear velocity was 1.8 m / h. After passing through the mixed resin, purified solution 3 was obtained.

[0049] (5) The purified solution 3 is dried in a drum at 160°C to obtain pullulan polysaccharide product.

[0050] The purity of pullulan was 97.9%, and the yield of pullulan was 95%.

[0051] Example 4 (1) Prepare crude pure liquid according to the method in Example 1 (2) Select a ceramic membrane (Hefei Xinda Membrane Technology Co., Ltd.) with a molecular weight cutoff of 1000 Da for membrane dialysis filtration. Control the temperature of the crude pure liquid at 30℃ through a heat exchanger, control the pressure difference across the membrane at 4 MPa, add deionized water for dialysis until the conductivity of the feed solution is 953 μs / cm, end the membrane filtration, and obtain pure liquid 1.

[0052] (3) Pure solution 1 was subjected to adsorption by a mixed macroporous ion exchange resin. MX-21 (Shandong Dechuan Chemical Technology Co., Ltd.) was selected for adsorption. The pH of the purified solution 2 was adjusted to 8.5, and the temperature of the purified solution 1 was controlled at 30℃ via a heat exchanger. The resin inlet pressure was 1.0 MPa, and the chromatography linear velocity was 3.1 m / h. After adsorption by the mixed resin, purified solution 2 was obtained.

[0053] (4) Pure solution 2 is subjected to cation exchange resin adsorption. Adsorption was performed using 001*7 (Shandong Dechuan Chemical Technology Co., Ltd.). The pH of the purified solution was adjusted to 6.5. The feed temperature was controlled at 25℃ using a heat exchanger, the resin inlet pressure was controlled at 1.5 MPa, and the chromatography linear velocity was 2 m / h. After adsorption by the cation exchanger, purified solution 3 was obtained.

[0054] (5) The purified solution 3 is dried in a drum at 130°C to obtain pullulan polysaccharide product.

[0055] The purity of pullulan was 98.3%, and the pullulan yield was 94%.

[0056] Comparative Example 1 The difference from Example 2 is that: the membrane material is a polypropylene membrane with a molecular weight cutoff of 10000 Da, the amount of liquid is 200 L of the crude pure liquid in Example 2, and other conditions remain unchanged, and a small-scale comparative experiment is carried out.

[0057] Pullulan purity was 93.2% (moisture content was higher in small-scale drum drying experiments), and pullulan yield was 73%.

[0058] The permeate from the membrane-treated samples of Example 2 and Comparative Example 1 was analyzed by liquid chromatography (LC) according to size exclusion chromatography (General Chapter 0514, Chinese Pharmacopoeia 2020). A GMPW gel permeate column was used. XL The mobile phase was 2% sodium nitrite solution (water as solvent), the column temperature was 30℃, the flow rate was 0.5 mL per minute, and the detection was performed using a differential refractive index detector. The detection results are shown in [Figure number missing]. Figure 1 and Figure 2 .

[0059] Figure 1 The molecular weight was 651 Da at the elution time of 21.456 min and 262 Da at the elution time of 22.310 min.

[0060] Figure 2 The molecular weight at the peak elution time of 15.932 min is 233151 Da. The molecular weight at the peak elution time of 21.463 min is 647 Da, and the molecular weight at the peak elution time of 22.317 min is 261 Da.

[0061] The two permeate solutions show that the nanofiltration membrane with a molecular weight cutoff of 1000 Da can retain all macromolecules; while the ultrafiltration membrane with a molecular weight cutoff of 10000 Da has a loss of 233151 Da molecules, which means there is a loss of macromolecules.

[0062] The pullulan purification method provided by this invention produces pullulan products with high purity and significantly improved product yield, which is beneficial for the industrial production of pullulan.

Claims

1. A method for purifying pullulan, characterized in that, Includes the following steps: (1) Fermentation broth was obtained by fermenting the microbial strain that produces pullulan polysaccharide. The fermentation broth was sterilized and decolorized to obtain crude pure extract. (2) The crude extract obtained in step (1) is processed through membrane technology and resin technology to obtain a refined extract.

2. The purification method as described in claim 1, characterized in that, In step (1), the microbial strain that produces pullulan is *Brucea buddingis*.

3. The purification method as described in claim 2, characterized in that, The budding short-stemmed mold is budding short-stemmed mold BL80ΔPMAs.

4. The purification method as described in claim 1, characterized in that, In step (1), the sterilization method is to use centrifugation or filtration; the decolorization method is to use activated carbon for decolorization.

5. The purification method as described in claim 1, characterized in that, In step (2), the membrane process is as follows: feed temperature ≤30℃, transmembrane pressure difference ≤4MPa, and deionized water is added for dialysis until the conductivity ≤1000μs / cm.

6. The purification method as described in claim 1, characterized in that, In step (2), the pore size of the membrane material is 500-1000 Da; the membrane material is one or both of organic and inorganic membranes. Preferably, the organic membrane includes one or more of the following: polysulfone, polyvinylidene fluoride, polyethersulfone composite membrane, polypropylene, and aromatic polyamide composite membrane; the inorganic membrane is a ceramic membrane.

7. The purification method as described in claim 1, characterized in that, In step (2), the resin process is as follows: liquid temperature 20-30℃, pH 6.0-9.0, chromatography linear velocity 1-5m / h, and feed pressure ≤2MPa.

8. The purification method as described in claim 1, characterized in that, The resin is one or more of anionic, cationic, or mixed resins; Preferably, the ion exchange resin is in the form of a gel or macroporous resin; Preferably, the anion exchange resin has a weak or strong base functional group. The cation exchange resin has strong acid or weak acid functional groups. Preferably, the anion exchange resin includes one or two of D201 resin and D301 resin; the cationic resin includes one or more of 001*10 resin, 101*7 resin, 001*8 resin, and 001*7 resin; and the mixed resin includes one or two of MX-1 resin and MX-21 resin. Preferably, the anion exchange resin, cationic resin, and mixed resin can be used individually or in series.

9. The purification method as described in claim 1, characterized in that, In step (2), the purified liquid is dried by drum drying to obtain pullulan polysaccharide.

10. The purification method as described in claim 9, characterized in that, The purity of the obtained pullulan polysaccharide is ≥97%.

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