A method for preparing nanofiber membranes using a bacterial cell lysate

Abstract

The present application belongs to the field of fermentation engineering and biomass resource utilization, and particularly relates to preparation of a composite nanofiber membrane by utilizing fermentation bacteria after fermentation production is completed. In the present application, the fermentation bacteria are subjected to chemical lysis to release biological macromolecules. The lysis solution of the bacteria after lysis contains biological macromolecules such as proteins, nucleic acids, peptidoglycan and flocculating agent chitosan. Then, the bacteria lysis solution is blended with starch and PEO, and a composite nanofiber is prepared by electrospinning, so as to realize productized application of the waste fermentation bacteria.

Description

Technical fields:

[0001] This invention belongs to the fields of fermentation engineering and biomass resource utilization, specifically relating to composite nanofiber membranes prepared by the resource utilization of fermentation cells after fermentation production. Background technology:

[0002] With the rapid development of science and technology, microbial methods are increasingly replacing traditional chemical methods in industry. However, with the widespread application of microbial methods in industry, a large amount of fermentation waste microbial bodies are generated. For example, in the production of glutamic acid using Corynebacterium glutamicum fermentation, it is estimated that 30-50 kg of waste microbial bodies are generated for every ton of glutamic acid produced, while the annual production of glutamic acid is as high as millions of tons (Zhou Dawei, Xiao Dongguang, Guo Xuewu, Lü Hongyan. Food Research and Development, 2012). my country's annual L-tryptophan production is about 10,000 tons, and about 1.3-1.5 tons of waste microbial bodies are generated for every ton of L-tryptophan produced (Qingyang Xu, Fang Bai, Ning Chen, and Gang Bai. Bioengineered, 2019). Moreover, my country's annual citric acid production reaches 500,000 tons, producing more than 80,000 tons of Aspergillus niger dried mycelium (Jiang Deyuan, Lü Mengyuan, Shi Jiaxian. Hubei Agricultural Sciences, 2014).

[0003] Taking Gram-negative bacteria as an example, their cell membranes are mainly composed of proteins, lipids, and small amounts of carbohydrates. These substances can provide the functional groups such as carboxyl, hydroxyl, phosphate, sulfate, and amino groups needed for the binding of metal ions. Therefore, some researchers have tried to use waste organisms such as filamentous fungi, yeast, and bacteria to remove some heavy metal ions from water. Other recycling methods include preparing waste microbial cells into high-purity protein feed products (Chinese patent application CN107011409A; Chinese patent application CN107418897A); and using fermented microbial cells as a nitrogen source for further fermentation (Chinese patent application CN115029389A), etc. Although the current recycling methods for fermented microbial cells have achieved certain economic benefits, they have not fully explored their application value, resulting in serious waste of resources. Most fermented microbial cells have not yet been "turned from waste into treasure."

[0004] In recent years, the preparation of nanofibers using electrospinning technology has become a research hotspot. The raw materials used for electrospinning are mainly synthetic polymers and natural polymers, with natural polymers primarily including protein and polysaccharide compounds. However, it is difficult to spin proteins directly, so it is necessary to blend the proteins with some easily spinnable polymers, such as polyvinyl alcohol, polyethylene oxide, and polycaprolactone.

[0005] Starch is a biodegradable polymer and the most abundant and inexpensive natural polysaccharide. Because of its multiple hydroxyl groups, starch can crosslink some materials, but its viscosity is unsuitable for spinning (Jing Xiang, Tan Ying, Xu Kun, New Chemical Materials, 2016). Therefore, starch needs to be modified for spinning, or blended with other polymers for this purpose. Polyethylene oxide (PEO) is a water-soluble polymer that exhibits good biocompatibility and spinnability as a biomaterial. Summary of the Invention:

[0006] The purpose of this invention is to release biomacromolecules from fermented bacterial cells under the action of a chemical lysis agent. The lysate contains biomacromolecules such as proteins, nucleic acids, peptidoglycans, and the flocculant chitosan. The lysate is then blended with starch and PEO, and composite nanofibers are prepared by electrospinning, enabling the commercial application of waste fermented bacterial cells.

[0007] One of the technical solutions provided by this invention is a method for preparing composite nanofiber membranes by blending fermented bacterial cells with starch and PEO, comprising the following steps:

[0008] (1) Collect the bacterial cells before or after the extraction of the target product from the microbial fermentation broth;

[0009] Furthermore, a flocculant is added to the fermentation broth to collect the bacterial cells;

[0010] Furthermore, the flocculation conditions are: stirring speed of 50-500 r / min, flocculation temperature of 45-85℃, stirring time of 1-30 min, and pH adjustment to 6-11;

[0011] Furthermore, the flocculant is chitosan, and the addition amount is 0.01% to 1% (w / v);

[0012] Furthermore, the amount of chitosan added is 0.04% to 0.6%, and after reacting for 1-30 minutes, the pH is adjusted to 7-9 with calcium hydroxide;

[0013] Furthermore, the bacterial cells are collected using plate and frame filtration, membrane filtration, or centrifugation;

[0014] Furthermore, the fermentation broth is a lactic acid fermentation broth using Escherichia coli as the fermentation strain;

[0015] (2) The collected bacterial cells were resuspended and treated with lysing enzymes and / or chemical lysing agents to prepare bacterial cell lysate;

[0016] Furthermore, the cell disruption rate in the lysis buffer is 30%–55%;

[0017] Furthermore, the lysin is at least one of lysozyme, snail enzyme, cellulase, or cellolytic enzyme;

[0018] Furthermore, the chemical pyrolysis agents include, but are not limited to, NaOH, KOH, SDS, Triton X-100, etc.

[0019] Furthermore, the pyrolysis conditions were: 20℃~70℃, pyrolysis for 5~30 min;

[0020] Furthermore, lysis was carried out at 20℃~70℃ and pH 7~14 for 5-30 min;

[0021] Further, the collected bacterial resuspended the bacterial cells and added sodium hydroxide to a final concentration of 0.25–1 M and 0.25%–0.5% SDS (w / v) as lysis agents.

[0022] (3) Preparation of starch / PEO blend solution

[0023] A PEO / starch blend solution was prepared by mixing a PEO solution with a starch solution.

[0024] Furthermore, the mixing ratio of PEO solution to starch solution is 4:1 to 1:4 (v / v);

[0025] Furthermore, PEO was dissolved in distilled water to prepare PEO solutions of different concentrations;

[0026] Furthermore, the PEO solution concentration is 1%-10% (w / v);

[0027] Furthermore, starch is gelatinized in a water bath to prepare starch solution;

[0028] Furthermore, the starch includes corn starch, wheat starch, tapioca starch, and potato starch;

[0029] Further, after preparing a starch solution with a mass fraction of 5-30% (w / v), gelatinize it at a gelatinization temperature of 60-90℃ and a stirring time of 10-60 min.

[0030] (4) Preparation of nanofiber membranes from bacterial cell lysis filtrate / starch / PEO spinning solution

[0031] Composite nanofiber membranes were prepared by mixing bacterial cell lysates with starch / PEO mixtures to prepare spinning solutions and then electrospinning them.

[0032] Furthermore, the blending ratio of the pyrolysis solution to the starch / PEO mixture is 9:1-1:9 (v / v);

[0033] Furthermore, the electrospinning voltage is 10-30KV, the distance between the spinneret and the receiving screen is 10-25cm, the spinning flow rate is 0.1-2mL / h, and the spinning temperature is 20-50℃.

[0034] The second technical solution provided by the present invention is a composite nanofiber membrane prepared by the above method.

[0035] The third technical solution mentioned in this invention is the application of the above-mentioned composite nanofiber membrane, especially in the fields of preparing nonwoven fabrics, hygiene products, and filter media.

[0036] The beneficial effects achieved by this invention are as follows:

[0037] This invention enables the efficient collection and appropriate lysis of microbial cells in fermentation broth for the preparation of composite nanofibers, achieving simultaneous production of fermentation products and composite nanofibers from fermented microbial cells. This completely solves the problem of fermentation waste formation in existing industrial production systems. Consequently, it significantly improves the economic efficiency and environmental benefits of fermentation production while significantly reducing overall production costs.

[0038] This invention can also be applied to the utilization of waste microorganisms generated during the production of other organic acids such as citric acid, malic acid, succinic acid, etc., or amino acids such as lysine, glutamic acid, threonine, alanine, etc.

[0039] This invention utilizes chitosan as a bioflocculant during cell collection at the end of lactic acid fermentation, maintaining a specific temperature and pH. The flocculant is collected entirely along with the cells, without affecting subsequent lactic acid separation and purification. The collected cells undergo simple lysis, and the lysate is blended with starch and PEO to prepare composite nanofibers via electrospinning. Key aspects of the process include controlling the degree of cell lysis, the concentrations of the starch and PEO solutions, the blending ratio, and the electrospinning conditions. The flocculant, starch, and PEO are all inexpensive raw materials. Detailed implementation method:

[0040] To make the objectives, technical solutions, and advantages of this patent clearer, the following detailed description is provided in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of this patent and are not intended to limit the scope of the invention.

[0041] This invention takes the fermentation broth for producing lactic acid by the calcium salt method as an example. After the lactic acid fermentation is completed, the fermentation broth is heated to 45℃~85℃, and the bio-flocculating agent chitosan is added and stirred to maintain the reaction for 1-30 minutes. Then, solid-liquid separation is performed by filtration. The solid part is collected as bacterial cells, and the liquid part is collected as free lactic acid liquid containing lactic acid monomers. The obtained bacterial cells can be used for the subsequent preparation of composite nanofibers. The obtained free lactic acid liquid is filtered and concentrated to obtain crude lactic acid product, which can be used for subsequent purification such as nanofiltration, decolorization, and ion exchange to obtain high-purity lactic acid monomers.

[0042] The main experimental methods used in this invention are as follows:

[0043] (1) Preparation of fermentation broth - collection of microbial cells (flocculation). The preparation of calcium salt lactic acid fermentation broth was carried out according to the method of the invention patent (Wang Zhengxiang et al., ZL201580000781.7). The fermentation strains were CGMCC 11059 or CGMCC 11060, of which CGMCC 11059 was used for the fermentation production of D-lactic acid and CGMCC 11060 was used for the fermentation production of L-lactic acid (Wang Zhengxiang et al., ZL201580000781.7). At the beginning of fermentation, glucose was added to the basic fermentation medium to a final concentration of 10–50 g / L. The culture was carried out at 30–37°C, pH 5.5–7.5, aeration of 0.1–2.0 vvm, and stirring at 100–1000 r / min. The culture time was 5–15 h, and the cell count reached 10–50 OD. The aeration was then turned off, the stirring speed was reduced to 0–300 r / min, the fermentation temperature was increased to 37–50°C, and a glucose solution with a final concentration of 16%–25% was added. The feed rate was controlled at 3 g / (L h)–25 g / (L h), and 5%–35% calcium hydroxide was added simultaneously. The fermentation pH was controlled between 5.0 and 8.0. Flocculation involves adding 0.1-10 g / L of the bioflocculant chitosan to the fermentation broth, stirring for 1-30 minutes at a stirring speed of 50-500 r / min, and at a flocculation temperature of 45-85℃. The pH of the fermentation broth is then adjusted to 7.0-9.0 using calcium hydroxide, followed by filtration and collection of the bacterial cells.

[0044] (2) Cell lysis - Preparation of cell lysis buffer. Escherichia coli was subjected to cell disruption treatment using NaOH and SDS solutions. The final concentration of NaOH was 0.25-1M and the final concentration of SDS was 0.25-0.5% (w / v). The temperature was 20℃-70℃, the pH was 7-14, and the treatment time was 5-30 min.

[0045] (3) Preparation of starch / PEO blend. Starch is dissolved in distilled water to obtain a starch solution with a mass fraction of 5-30%, which is then gelatinized at a temperature of 60-90℃ for 10-60 min. PEO powder is dissolved in distilled water at 85℃ to obtain a PEO solution with a mass fraction of 1-10%. The starch solution and PEO solution are blended at a volume ratio of 1:4-4:1 to obtain a starch / PEO blend.

[0046] (4) Preparation and electrospinning of bacterial lysate / starch / PEO spinning solution. Bacterial lysate and starch / PEO blend were mixed at a volume ratio of 1:9-9:1 to obtain the bacterial lysate / starch / PEO spinning solution. The spinning solution was transferred to a 10ml syringe. The electrospinning voltage was 10-30KV, the distance between the spinneret and the receiving screen was 10-25cm, the spinning flow rate was 0.1-2mL / h, and the spinning temperature was 20-50℃.

[0047] The present invention will be further explained and illustrated below through specific embodiments.

[0048] Example 1: A method for D-lactic acid fermentation

[0049] Cryopreserved glycerol tubes of the D-lactic acid producing strain CGMCC 11059 were inoculated into 50 mL of LB liquid medium and cultured at 37°C and 200 rpm for 12 h as the primary seed culture. The primary seed culture was then inoculated into 5 L of M9 liquid medium with glucose as the carbon source, with an initial sugar concentration of 0.5%, and cultured at 37°C and 200 rpm for 10 h as the secondary seed culture. The secondary seed culture was then inoculated into a fermenter containing M9 liquid medium at an initial OD value of 0.3. After inoculation, the culture was cultured for 5 m... 3 The initial volume of the fermentation tank is 2.5m³. 3 The initial addition of invert sugar syrup was 3%, initiating the fermentation production of lactic acid monomers. The fermentation start temperature was controlled at 37℃, and the pH was maintained at 6.5 using ammonia. During cell growth, the aeration rate was adjusted to 1.5 vvm, and the stirring speed was 600 r / min. When the cell concentration reached OD... 600 After 30 minutes, turn off the ventilation, control the fermentation temperature at 40℃, adjust the stirring speed to 200r / min, add 25% calcium hydroxide suspension to maintain pH at 7.0, add a total of 6.0kg of glucose, and the fermentation ends when the residual sugar concentration is below 0.5g / L.

[0050] M9 medium composition: NaCl 0.5 g / L, NH4Cl 1 g / L, KH2PO4 3 g / L, Na2HPO4·12H2O 15.1 g / L, the remainder is water.

[0051] Using the obtained lactic acid fermentation broth as raw material, it was heated to 60℃ in a reaction vessel and maintained at this temperature. Chitosan solution (2 g / L) was added while stirring at 100 rpm. After all the chitosan solution was added, the reaction was maintained for 15 minutes. Then, 20% calcium hydroxide was added to adjust the pH of the fermentation broth to 7.5. After the reaction, solid-liquid separation of the reaction solution was performed using a plate and frame filter with a 6 μm pore size. The collected solid fraction was the bacterial cells.

[0052] Example 2: PEO electrospinning

[0053] PEO preparation: Dissolve 5g of PEO powder in 100mL of distilled water to prepare a 5% PEO solution (w / v).

[0054] A 5% PEO solution was transferred into a 10 mL syringe. The electrospinning voltage was fixed at 15 kV, the spinning distance at 15 cm, the syringe flow rate at 0.5 mL / h, and the spinning temperature at 25 °C. PEO nanofiber membranes were obtained.

[0055] Example 3: Starch electrospinning

[0056] Starch solution preparation: Dissolve 20g of corn starch in 100mL of distilled water to prepare a starch solution with a mass fraction of 20% (w / v), and gelatinize it in an 80℃ water bath for 20min.

[0057] The starch gelatinized solution was transferred to a 10mL syringe. The electrospinning voltage was fixed at 15KV, the spinning distance at 15cm, the syringe flow rate at 0.5mL / h, and the spinning temperature at 25℃. During the electrospinning process, it was found that the starch gelatinized solution could not be spun into fibers.

[0058] Example 4: PEO + starch electrospinning

[0059] A starch / PEO blend was obtained by mixing 5% PEO solution and 20% corn starch solution (prepared in Example 3) at a volume ratio of 4:1.

[0060] The starch / PEO blend spinning solution was poured into a 10 mL syringe. The electrospinning voltage was fixed at 15 kV, the spinning distance at 15 cm, the syringe flow rate at 0.5 mL / h, and the spinning temperature at 25 °C. A starch / PEO composite nanofiber membrane was obtained.

[0061] Example 5: Electrospinning of PEO + bovine serum albumin

[0062] Weigh 0.38g of bovine serum albumin and add distilled water to make up to 10g. Stir with a magnetic stirrer at 90℃ to prepare a bovine serum albumin solution (w / v) of 38mg / g.

[0063] A bovine serum albumin / PEO blend was prepared by mixing 5% PEO solution and 38 mg / g bovine serum albumin at a volume ratio of 4:1.

[0064] The bovine serum albumin / PEO blend was transferred into a 10 mL syringe. The electrospinning voltage was fixed at 15 kV, the spinning distance at 15 cm, the syringe flow rate at 0.5 mL / h, and the spinning temperature at 25 °C. This yielded a bovine serum albumin / PEO composite nanofiber membrane.

[0065] Example 6: PEO + bovine serum albumin + chitosan

[0066] Weigh 9.62g of 0.25% chitosan solution into a 25mL container, add 0.38g of bovine serum albumin, and stir at 90℃ with a magnetic stirrer to dissolve into a clear solution, so that the final concentration of bovine serum albumin is 38mg / g, thus obtaining bovine serum albumin / chitosan solution.

[0067] A bovine serum albumin / chitosan / PEO spinning solution was obtained by blending 5% PEO solution and bovine serum albumin / chitosan solution at a volume ratio of 4:1.

[0068] The bovine serum albumin / chitosan / PEO blend spinning solution was poured into a 10 mL syringe. The electrospinning voltage was fixed at 15 kV, the spinning distance at 15 cm, the syringe flow rate at 0.5 mL / h, and the spinning temperature at 25 °C. This yielded a bovine serum albumin / chitosan / PEO composite nanofiber membrane.

[0069] Example 7: Cell lysis under different conditions and electrospinning of cell / starch / PEO

[0070] The bacterial cells obtained in Example 1 were resuspended in water at a ratio of 1:4 (w / v), and then a certain final concentration of NaOH and SDS solution was added for cell disruption treatment at a certain temperature for a certain period of time. The cell breakage rate after lysis is shown in the table below. The obtained bacterial cell lysate was mixed with the starch / PEO blend solution in Example 4 at a ratio of 1:1 to obtain the bacterial cell lysate / starch / PEO spinning solution.

[0071] The spinning solution was transferred to a 10 mL syringe. The electrospinning voltage was fixed at 15 KV, the spinning distance at 15 cm, the syringe flow rate at 0.5 mL / h, and the spinning temperature at 25 °C.

[0072]

[0073] Through the above experiments, it was determined that the cell breakage rate is an important factor affecting electrospinning. Based on the above experiments, the inventors conducted a large number of experiments to verify the results and finally determined that a cell breakage rate of 30% to 55% in the bacterial lysate is the best condition for electrospinning.

[0074] Example 8: Electrospinning of bacterial cell lysate / starch / PEO spinning solution

[0075] A starch / PEO blend was obtained by mixing 1% PEO solution and 5% starch solution (the starch solution was prepared in the same way as in Example 3) at a volume ratio of 4:1. Then, the bacterial lysate obtained in Example 7, with lysis conditions of 0.25M NaOH, 0.5% SDS, 50°C, and 20 min, was mixed with the starch / PEO blend at a volume ratio of 9:1 to obtain a bacterial lysate / starch / PEO spinning solution.

[0076] The spinning solution was transferred to a 10 mL syringe. The electrospinning voltage was fixed at 10 KV, the spinning distance at 10 cm, the syringe flow rate at 0.1 mL / h, and the spinning temperature at 20 °C.

[0077] Example 9: Electrospinning of bacterial cell lysate / starch / PEO spinning solution

[0078] A starch / PEO blend was prepared by mixing 5% PEO solution and 20% starch solution (the starch solution was prepared in the same way as in Example 3) at a volume ratio of 4:1. Then, the bacterial cell lysis buffer (protein concentration of 38 mg / g in the lysis buffer) prepared in Example 7 under the conditions of 0.5 M NaOH, 0.5% SDS, 50°C, and 20 min was mixed with the starch / PEO blend at a volume ratio of 8:2 to obtain a bacterial cell lysis buffer / starch / PEO spinning solution.

[0079] The spinning solution was transferred to a 10 mL syringe. The electrospinning voltage was fixed at 15 kV, the spinning distance at 15 cm, the syringe flow rate at 0.5 mL / h, and the spinning temperature at 25 °C. During the electrospinning process, a large number of filaments were observed on the receiving screen, resulting in a composite nanofiber membrane.

[0080] Example 10: Electrospinning of bacterial cell lysate / starch / PEO spinning solution

[0081] A starch / PEO blend was prepared by mixing 10% PEO solution and 30% starch solution (the starch solution was prepared in the same way as in Example 3) at a volume ratio of 4:1. Then, the bacterial lysate obtained in Example 7, with lysis conditions of 0.5M NaOH, 0.5% SDS, 60°C, and 15 min, was mixed with the starch / PEO blend at a volume ratio of 7:3 to obtain a bacterial lysate / starch / PEO spinning solution.

[0082] The spinning solution was transferred to a 10 mL syringe. The electrospinning voltage was fixed at 20 kV, the spinning distance at 20 cm, the syringe flow rate at 2 mL / h, and the spinning temperature at 50 °C. During the electrospinning process, a large number of filaments were observed on the receiving screen, resulting in a composite nanofiber membrane.

[0083] Example 11: Diameter of the composite nanofibers of the present invention

[0084] Nanofiber membranes are composed of a large number of individual nanofibers. Nanofibers are used as samples during the preparation of nanofiber membranes. The nanofiber diameter data obtained in this embodiment of the invention are shown in the table below:

[0085]

[0086] The addition of starch and bacterial lysate increased the diameter of the nanofibers, from 248 nm to 335 nm and 407 nm, respectively. As can be seen from Example 12, the increased nanofiber diameter enhanced the mechanical properties of the nanofiber membrane.

[0087] Example 12: Mechanical property parameters of the composite nanofiber membrane of the present invention

[0088] The mechanical property data of the spun products obtained in the embodiments of the present invention are shown in the table below:

[0089] Table 2 Mechanical properties of nanofiber membranes

[0090]

[0091] Nanofiber membranes were obtained by electrospinning PEO spinning solution, starch / PEO spinning solution, bovine serum albumin / PEO spinning solution, and bacterial lysate / starch / PEO spinning solution, respectively. The mechanical properties of the nanofiber membranes were measured using a universal tensile testing machine. The tensile strength and elongation at break of the PEO nanofiber membrane were 2.27 MPa and 25.67%, respectively. The tensile strength and elongation at break of the starch / PEO composite nanofiber membrane were increased to 2.64 MPa and 28.79%, respectively. The tensile strength and elongation at break of the bacterial lysate / starch / PEO composite nanofiber membrane were increased to 3.13 MPa and 33.42%, respectively.

[0092] The addition of bacterial lysis buffer improved the tensile strength and elongation at break of the nanofiber membrane, greatly enhancing its extensibility and toughness, and making it less prone to breakage.

Claims

1. A method for preparing a composite nanofiber membrane, characterized in that, Includes the following steps: (1) Before or after the extraction of the target product from the microbial fermentation broth, chitosan is added as a flocculant to collect the bacterial cells; the flocculation conditions are: stirring speed 50-500 r / min, flocculation temperature 45-85℃, stirring time 1-30 min, and pH adjusted to 6-11; the amount of chitosan added is 0.01%-1%; (2) The collected bacterial cells were resuspended and treated with sodium hydroxide at a final concentration of 0.25-1 M and SDS at 0.25%-0.5% as lysis agents to prepare a bacterial lysis buffer; the cell disruption rate in the lysis buffer was 30%-55%; The pyrolysis conditions are: 20℃~70℃, pyrolysis for 5~30 min; (3) Preparation of starch / PEO blend solution: PEO solution and starch solution are mixed to prepare PEO / starch blend solution; (4) Preparation of nanofiber membrane by bacterial lysate / starch / PEO spinning solution: After preparing the spinning solution by mixing bacterial lysate with starch / PEO mixture, electrospinning was performed to prepare composite nanofiber membrane.

2. The method for preparing a composite nanofiber membrane as described in claim 1, characterized in that, The PEO solution concentration is 1%-10%; starch is prepared into a starch solution with a mass fraction of 5-30% and then gelatinized at a gelatinization temperature of 60-90℃ and a stirring time of 10-60 min; the blending ratio of PEO solution to starch solution is 4:1-1:

4.

3. The method for preparing a composite nanofiber membrane as described in claim 1, characterized in that, The ratio of bacterial cell lysis broth to starch / PEO mixture is 9:1 to 1:

9.

4. The method for preparing a composite nanofiber membrane as described in claim 1, characterized in that, The electrospinning voltage is 10-30 KV, the distance between the spinneret and the receiving screen is 10-25 cm, the spinning flow rate is 0.1-2 mL / h, and the spinning temperature is 20-50℃.

5. A composite nanofiber membrane prepared by the method according to any one of claims 1-4.

6. The application of the composite nanofiber membrane according to claim 5, characterized in that, It is used in the preparation of nonwoven fabrics, hygiene products, filter media and other fields.

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