CN115613220A

CN115613220A - Method for preparing nanofiber membrane by adopting thallus lysate

Info

Publication number: CN115613220A
Application number: CN202211343190.8A
Authority: CN
Inventors: 田康明; 张德旭
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2022-10-31
Filing date: 2022-10-31
Publication date: 2023-01-17

Abstract

The invention belongs to the field of fermentation engineering and biomass resource utilization, and particularly relates to a method for preparing a composite nanofiber membrane by resource utilization of fermentation thalli after fermentation production is finished. The invention releases biomacromolecules from zymocyte under the action of chemical cracking agent, and the cracked bacterial lysate contains biomacromolecules such as protein, nucleic acid, peptidoglycan, flocculating agent chitosan and the like. And then blending the thallus lysate with starch and PEO, and preparing the composite nanofiber through electrostatic spinning, thereby realizing the commercialized application of the waste fermentation thallus.

Description

Method for preparing nanofiber membrane by adopting thallus lysate

The technical field is as follows:

the invention belongs to the field of fermentation engineering and biomass resource utilization, and particularly relates to a composite nanofiber membrane prepared by resource utilization of fermentation thalli after fermentation production is finished.

Background art:

with the rapid development of scientific technology, microbiological methods are increasingly used in industry instead of traditional chemical methods. However, as the microbiological method is widely used in industry, a large amount of fermentation waste bacteria is produced. For example, in the production of glutamic acid by fermentation of corynebacterium glutamicum, it is statistically estimated that 30-50 kg of waste thallus is produced per ton of glutamic acid produced, and that the annual production of glutamic acid is up to millions of tons (Zhou Dawei, xiao dong, guo Xuewu, lv Hongyan. Food research and development, 2012). The annual L-tryptophan yield in our country is about 10000 tons, and about 1.3 to 1.5 tons of waste bacteria are produced per 1 ton of L-tryptophan produced (Qingyang Xu, fang Bai, ning Chen, and Gang Bai, bioengineered, 2019). And the annual output of citric acid in China reaches 50 ten thousand tons, and the yield of aspergillus niger dry bacteria exceeds 8 ten thousand tons (Jiangduan, lv Mengyuan, dan Jiaxian, hubei agricultural science, 2014).

In the case of gram-negative bacteria, the cell membrane contains proteins, lipids and a small amount of carbohydrates as main components. Since these substances can provide functional groups such as carboxyl, hydroxyl, phosphate, sulfate and amino groups required for binding metal ions, researchers have attempted to remove some heavy metal ions from water by using waste organisms such as filamentous fungi, yeast and bacteria. Other recycling methods comprise preparing the waste thallus into a protein feed product with higher purity (Chinese patent application CN107011409A; chinese patent application CN 107418897A); and (3) treating the fermented bacteria and then fermenting again as a nitrogen source of the culture medium (Chinese patent application CN 115029389A). Although the existing recycling method of the fermentation thalli has certain economic benefit, the application value of the fermentation thalli is not completely exploited to cause serious waste of resources, and most of the fermentation thalli are not changed into valuable.

In recent years, the preparation of nanofibers by using electrospinning technology has been a hot point of research, and the raw materials used for electrospinning are mainly synthetic polymers and natural polymers, and natural polymers mainly include proteins and polysaccharides. However, it is difficult to spin protein directly, and it is necessary to blend protein with some polymers that are easy to spin, such as polyvinyl alcohol, polyethylene oxide, polycaprolactone, etc.

Starch is a biodegradable polymer, is the most abundant biological polysaccharide stored in nature, and is low in price. Starch can cross-link some materials because of its polyhydroxy group, but the viscosity of starch is not suitable for spinning (volvating, tan Ying, xu Kun, new chemical material, 2016). Starch needs to be modified for spinning or blended with some polymers for spinning. Polyethylene oxide (PEO) is a water-soluble polymer having good biocompatibility and spinnability as a biomaterial.

The invention content is as follows:

the invention aims to release biological macromolecules from fermentation thalli under the action of a chemical cracking agent, and cracked bacteria lysate contains biological macromolecules such as protein, nucleic acid, peptidoglycan, flocculating agent chitosan and the like. And then blending the thallus lysate with starch and PEO, and preparing the composite nanofiber through electrostatic spinning, thereby realizing the commercialized application of the waste fermentation thallus.

One of the technical schemes provided by the invention is a method for preparing a composite nanofiber membrane by blending fermentation thalli, starch and PEO, which comprises the following steps:

(1) Before or after the target product is extracted by the microbial fermentation liquor, collecting thalli;

further, adding a flocculating agent into the fermentation liquor to collect thalli;

further, the flocculation conditions were: stirring at 50-500r/min and 45-85 deg.c for 1-30min and regulating pH to 6-11;

further, the flocculant is chitosan, and the addition amount is 0.01-1% (w/v);

furthermore, the addition amount of the chitosan is 0.04-0.6%, and the pH value of calcium hydroxide is adjusted to 7-9 after the reaction is carried out for 1-30 min;

further, collecting the thallus by plate-frame filtration, membrane filtration or centrifugation;

further, the fermentation broth is lactic acid fermentation broth taking escherichia coli as a fermentation strain;

(2) Resuspending the collected thallus, and preparing thallus lysate after treatment by lyase and/or chemical cracking agent;

further, the cell breakage rate in the lysate is 30-55%;

further, the lyase is at least one of lysozyme, helicase, cellulase or lywallzyme;

further, the chemical lysing agents include, but are not limited to, naOH, KOH, SDS, triton X-100, and the like;

further, the cleavage conditions were: cracking for 5-30min at 20-70 ℃;

further, cracking for 5-30min at 20-70 ℃ and pH 7-14;

further, after the collected thalli are resuspended, sodium hydroxide with the final concentration of 0.25-1M and 0.25% -0.5% SDS (w/v) are added as a cracking agent;

(3) starch/PEO blend solution preparation

Mixing a PEO solution and a starch solution to prepare a PEO/starch blending solution;

further, the blending ratio of the PEO solution and the starch solution is 4:1-1:4 (v/v);

further, dissolving PEO in distilled water to prepare PEO solutions with different concentrations;

still further, the PEO solution concentration is 1% to 10% (w/v);

further, gelatinizing starch in a water bath to prepare a starch solution;

further, the starch includes corn starch, wheat starch, tapioca starch, and potato starch;

further, preparing starch solution with the mass fraction of 5-30% (w/v), and gelatinizing at 60-90 deg.C for 10-60min;

(4) Preparation of nanofiber membrane from thallus lysate/starch/PEO spinning solution

Blending thallus lysate with different cracking degrees with starch/PEO mixed solution to prepare spinning solution, and then performing electrostatic spinning to prepare a composite nanofiber membrane;

further, the blending ratio of the lysate to the starch/PEO mixed solution is 9:1-1:9 (v/v);

furthermore, the electrostatic spinning voltage is 10-30KV, the distance between the spinning nozzle and the receiving screen is 10-25cm, the spinning flow is 0.1-2mL/h, and the spinning temperature is 20-50 ℃.

The second technical scheme provided by the invention is the composite nanofiber membrane prepared by the method.

The third technical scheme is the application of the composite nanofiber membrane, in particular to the application in the fields of preparing non-woven fabrics, sanitary products, filter media and the like.

The invention has the following beneficial effects:

the invention can efficiently collect and moderately crack thalli in fermentation feed liquid and use the thalli for preparing the composite nano-fibers, realizes the synchronous production of fermentation products and the composite nano-fibers of the fermentation thalli, and thoroughly solves the problem of the formation of fermentation thalli waste in the existing industrial production system. Therefore, the economic efficiency and the environmental benefit of fermentation production can be obviously improved, and the comprehensive production cost can be obviously reduced.

The invention can also be applied to the utilization of waste thalli generated in the production process of other organic acids such as citric acid, malic acid, succinic acid and the like or amino acids such as lysine, glutamic acid, threonine, alanine and the like.

When the bacteria are collected after lactic acid fermentation is finished, chitosan is used as a bioflocculant and a certain temperature and pH value are maintained, the bioflocculant can be collected together with the bacteria without influencing the subsequent separation and refining of lactic acid, the collected bacteria are subjected to simple cracking, and a bacteria cracking solution, starch and PEO are blended to prepare the composite nanofiber through electrostatic spinning. The key points of the process are the control of the cracking degree of the fermentation thalli, the concentration of the starch solution and the PEO solution, the blending proportion and the electrostatic spinning condition. Wherein, the flocculating agent, the starch, the PEO and other materials are all cheap raw materials.

The specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.

Taking the fermentation liquor for producing lactic acid by a calcium salt method as an example, after the fermentation of the lactic acid is finished, heating the fermentation liquor to 45-85 ℃, adding chitosan serving as a biological flocculant, stirring and maintaining the reaction for 1-30min, then carrying out solid-liquid separation by filtering, collecting a solid part which is thallus, collecting a liquid part which is free lactic acid liquid containing lactic acid monomers, wherein the obtained thallus can be used for preparing the subsequent composite nano-fibers, and the obtained free lactic acid liquid is filtered, concentrated and the like to obtain a crude lactic acid product which can be used for refining such as nano-filtration, decoloration, ion exchange and the like to obtain the high-purity lactic acid monomers.

The main experimental method adopted by the invention is as follows:

(1) Preparation of fermentation liquor-collection of thalli (flocculation). The preparation of the lactic acid fermentation liquor by the calcium salt method is carried out according to the method of the invention patent (Wang Zhengxiang and the like, ZL 201580000781.7) and the fermentation strain is CGMCC 11059 or CGMCC 11060, wherein the strain CGMCC 11059 is used for the fermentation production of D-lactic acid, and the strain CGMCC 11060 is used for the fermentation production of L-lactic acid (Wang Zhengxiang and the like, ZL 201580000781.7). In the initial stage of fermentation, adding glucose into a fermentation minimal medium to a final concentration of 10-50 g/L, and culturing at 30-37 ℃, pH of 5.5-7.5, ventilation of 0.1-2.0 vvm and stirring at 100-1000 r/min; the culture time is 5-15 h, and the bacterial mass reaches 10-50 OD; closing ventilation, reducing the stirring speed to 0-300 r/min, increasing the fermentation temperature to 37-50 ℃, supplementing a glucose solution with the final concentration of 16-25%, controlling the feeding speed to be 3 g/(L h) -25 g/(L h), synchronously feeding 5-35% of calcium hydroxide, and controlling the fermentation pH to be 5.0-8.0. The flocculation is to add 0.1-10g/L of chitosan serving as a biological flocculant into the fermentation liquor, stir for 1-30min at a stirring speed of 50-500r/min and at a flocculation temperature of 45-85 ℃, adjust the pH value of the fermentation liquor to 7.0-9.0 by using calcium hydroxide, filter and collect thalli.

(2) And (3) thallus lysis-thallus lysate preparation. Using NaOH and SDS solution to carry out wall breaking treatment on the escherichia coli, adding NaOH with the final concentration of 0.25-1M, SDS with the final concentration of 0.25-0.5% (w/v), controlling the temperature at 20-70 ℃ and the pH at 7-14,5-30min.

(3) starch/PEO blend preparation. Dissolving starch in distilled water to obtain starch solution with mass fraction of 5-30%, gelatinizing at 60-90 deg.C for 10-60min, and stirring. PEO powder is dissolved in distilled water at 85 ℃ to obtain a PEO solution with the mass fraction of 1-10%. And blending the starch solution and the PEO solution according to the volume ratio of 1:4-4:1 to obtain the starch/PEO blended solution.

(4) Preparing thallus lysate/starch/PEO spinning solution and electrostatic spinning. And blending the thallus lysate and the starch/PEO blended solution according to the volume ratio of 1:9-9:1 to obtain the thallus lysate/starch/PEO spinning solution. Transferring the spinning solution into a 10mL injector, wherein the electrostatic spinning voltage is 10-30KV, the distance between a spinning nozzle and a receiving screen is 10-25cm, the spinning flow is 0.1-2mL/h, and the spinning temperature is 20-50 ℃.

The invention is further illustrated by the following specific examples.

Example 1A D-lactic acid fermentation method

The frozen product of the D-lactic acid production strain CGMCC 11059 is inoculated into 50mL LB liquid culture medium and cultured for 12h at 37 ℃ and 200r/min in a shaking table to serve as first-stage seed liquid. Inoculating the first-stage seed solution into 5L M9 liquid culture medium with glucose as carbon source, wherein the initial sugar concentration is 0.5%, and shake culturing at 37 deg.C and 200r/min for 10 hr to obtain second-stage seed solution. Inoculating the second-stage seed solution into a fermentation tank containing M9 liquid culture medium according to the inoculation amount of initial OD value of 0.3, and inoculating for 5M ³ Initial volume of the fermenter was 2.5m ³ The initial invert syrup addition was 3% and the fermentation production of lactic acid monomer was started. The initial fermentation temperature is controlled at 37 deg.C, pH is maintained at 6.5 with ammonia water, aeration amount is adjusted to 1.5vvm during thallus growth, stirring speed is 600r/min, and when thallus concentration reaches OD ₆₀₀ And after 30, closing ventilation, controlling the fermentation temperature at 40 ℃, adjusting the stirring speed to 200r/min, feeding 25% calcium hydroxide suspension to maintain the pH at 7.0, supplementing total 6.0kg of glucose, and ending fermentation when the concentration of residual sugar is lower than 0.5 g/L.

M9 medium composition: naCl 0.5g/L, NH ₄ Cl 1g/L，KH ₂ PO ₄ 3g/L，Na ₂ HPO ₄ ·12H ₂ O15.1 g/L, and the balance of water.

Heating the obtained lactic acid fermentation liquor as a raw material in a reaction kettle to 60 ℃, maintaining the temperature, adding 2g/L of chitosan solution under stirring at 100r/min, maintaining the reaction for 15min after all the chitosan solution is added, and adding 20% of calcium hydroxide to adjust the pH value of the fermentation liquor to 7.5. After the reaction is finished, solid-liquid separation of the reaction solution is carried out by adopting a plate-frame filter device with the filter aperture of 6 mu m, and the solid part is collected to be the thallus.

Example 2: PEO electrospinning

PEO preparation: 5g of PEO powder was dissolved in 100mL of distilled water to prepare a PEO solution (w/v) with a mass fraction of 5%.

Transferring the 5% PEO solution into a 10mL injector, fixing the electrostatic spinning voltage at 15KV, the spinning distance at 15cm, the injector flow rate at 0.5mL/h, and the spinning temperature at 25 ℃. And obtaining the PEO nanofiber membrane.

Example 3: electrostatic spinning of starch

Preparing starch solution: dissolving 20g of corn starch in 100mL of distilled water to prepare 20% starch solution (w/v), and gelatinizing in 80 ℃ water bath for 20min.

And transferring the starch pasting solution into a 10mL injector, fixing the electrostatic spinning voltage at 15KV, the spinning distance at 15cm, setting the flow rate of the injector at 0.5mL/h and the spinning temperature at 25 ℃. During the electrostatic spinning process, the starch pasting liquid can not be used for spinning.

Example 4: PEO + starch electrospinning

5% of the PEO solution and 20% of the corn starch solution (prepared in example 3) were blended in a volume ratio of 4:1 to give a starch/PEO blend.

And (2) mixing the starch/PEO blended spinning solution into a 10mL injector, fixing the electrostatic spinning voltage to be 15KV, the spinning distance to be 15cm, the flow rate of the injector to be 0.5mL/h, and the spinning temperature to be 25 ℃. Obtaining the starch/PEO composite nanofiber membrane.

Example 5: PEO + bovine serum albumin electrospinning

0.38g of bovine serum albumin was weighed, added with distilled water to 10g, and stirred by a magnetic stirrer at 90 ℃ to prepare a bovine serum albumin solution (w/v) of 38 mg/g.

5% of the PEO solution and 38mg/g of bovine serum albumin were blended in a volume ratio of 4:1 to obtain a bovine serum albumin/PEO blend.

Transferring the bovine serum albumin/PEO blended solution into a 10mL injector, fixing the electrostatic spinning voltage to be 15KV, the spinning distance to be 15cm, the flow rate of the injector to be 0.5mL/h, and the spinning temperature to be 25 ℃. Obtaining the bovine serum albumin/PEO composite nanofiber membrane.

Example 6: PEO + bovine serum albumin + chitosan

Weighing 9.62g of 0.25% chitosan solution in a 25mL container, adding 0.38g of bovine serum albumin, and stirring and dissolving the mixture at 90 ℃ by a magnetic stirrer to obtain a transparent clear solution, wherein the final concentration of the bovine serum albumin is 38mg/g, so as to obtain the bovine serum albumin/chitosan solution.

5 percent of PEO solution and bovine serum albumin/chitosan solution are blended according to the volume ratio of 4:1 to obtain bovine serum albumin/chitosan/PEO spinning solution.

And (2) blending the bovine serum albumin/chitosan/PEO spinning solution into a 10mL injector, fixing the electrostatic spinning voltage at 15KV, the spinning distance at 15cm, the flow velocity of the injector at 0.5mL/h, and the spinning temperature at 25 ℃. Obtaining the bovine serum albumin/chitosan/PEO composite nanofiber membrane.

Example 7: thallus cracking and thallus/starch/PEO electrostatic spinning under different conditions

The bacterial cells obtained in example 1 and water were resuspended at a ratio of 1:4 (w/v), and then a final concentration of NaOH and SDS solution was added to the cells to break the cell walls for a predetermined time at a predetermined temperature, and the cell disruption rate after lysis was as shown in the table below. The obtained bacterial lysate and the starch/PEO blend in example 4 are mixed and dissolved in a ratio of 1:1 to obtain a bacterial lysate/starch/PEO spinning solution.

Transferring the spinning solution into a 10mL injector, fixing the electrostatic spinning voltage at 15KV, the spinning distance at 15cm, the injector flow rate at 0.5mL/h, and the spinning temperature at 25 ℃.

Based on the experiments, the inventor finally determines that the cell breakage rate in the thallus lysate is 30-55% which is the best condition for electrostatic spinning.

Example 8: electrostatic spinning of thallus lysate/starch/PEO spinning solution

1% of the PEO solution and 5% of the starch solution (the starch solution was prepared in the same manner as in example 3) were mixed in a volume ratio of 4:1 to obtain a starch/PEO blend solution. In example 7, the lysate of thallus and the starch/PEO blend at 50 ℃ and 20min under the lysis conditions of 0.25M NaOH, 0.5% SDS and the volume ratio of 9:1 were blended to obtain the lysate/starch/PEO spinning solution.

Transferring the spinning solution into a 10mL injector, fixing electrostatic spinning voltage at 10KV, spinning distance at 10cm, injector flow rate at 0.1mL/h, and spinning temperature at 20 ℃.

Example 9: electrostatic spinning of thallus lysate/starch/PEO spinning solution

5% of the PEO solution and 20% of the starch solution (the starch solution was prepared in the same manner as in example 3) were mixed in a volume ratio of 4:1 to obtain a starch/PEO blend solution. In example 7, the lysate (protein concentration in lysate is 38 mg/g) and starch/PEO blend at a volume ratio of 8:2 were mixed under lysis conditions of 0.5M NaOH, 0.5% SDS, 50 ℃ and 20min to obtain a lysate/starch/PEO spinning solution.

Transferring the spinning solution into a 10mL injector, fixing the electrostatic spinning voltage at 15KV, the spinning distance at 15cm, the flow rate of the injector at 0.5mL/h, and the spinning temperature at 25 ℃. And (3) finding a large number of filaments on the receiving screen in the electrostatic spinning process to obtain the composite nanofiber membrane.

Example 10: electrostatic spinning of thallus lysate/starch/PEO spinning solution

10% of the PEO solution and 30% of the starch solution (the starch solution was prepared in the same manner as in example 3) were mixed in a volume ratio of 4:1 to obtain a starch/PEO blend solution. In example 7, the lysate of thallus and the starch/PEO blend at the lysis conditions of 0.5M NaOH, 0.5% SDS, 60 ℃ and 15min were mixed in the volume ratio of 7:3 to obtain the lysate/starch/PEO spinning solution.

Transferring the spinning solution into a 10mL injector, fixing electrostatic spinning voltage at 20KV, spinning distance at 20cm, injector flow rate at 2mL/h, and spinning temperature at 50 ℃. And (3) finding a large number of filaments on the receiving screen in the electrostatic spinning process to obtain the composite nanofiber membrane.

Example 11: composite nanofiber diameter of the invention

The nanofiber membrane is composed of a large number of single nanofibers, the nanofibers are taken as samples in the process of preparing the nanofiber membrane, and the diameter data of the nanofibers obtained in the embodiment of the invention are as follows:

the addition of starch and thallus lysate raises the diameter of nanometer fiber from 248nm to 335nm and 407nm, respectively. In combination with example 12, it can be seen that the increase in nanofiber diameter increases the mechanical properties of the nanofiber membrane.

Example 12: mechanical property parameters of the composite nanofiber membrane

The spinning product obtained in the embodiment of the invention has the following corresponding mechanical property data:

TABLE 2 nanofiber membrane mechanical properties

Respectively carrying out electrospinning on the PEO spinning solution, the starch/PEO spinning solution, the bovine serum albumin/PEO spinning solution and the thallus lysate/starch/PEO spinning solution to obtain the nanofiber membrane. The mechanical properties of the nanofiber membrane were measured using a universal tensile testing machine, and the tensile strength and elongation at break of the PEO nanofiber membrane were 2.27Mpa and 25.67%, respectively. The tensile strength and the elongation at break of the starch/PEO composite nanofiber membrane are respectively improved to 2.64MPa and 28.79 percent. The tensile strength and the elongation at break of the bacterial lysate/starch/PEO composite nanofiber membrane are respectively improved to 3.13Mpa and 33.42 percent.

The addition of the thallus lysate improves the tensile strength and the elongation at break of the nanofiber membrane, greatly improves the ductility and the toughness of the nanofiber membrane, and is not easy to break.

Claims

1. The preparation method of the composite nanofiber membrane is characterized by comprising the following steps of:

(1) Adding chitosan as flocculant to collect thallus before or after extracting target product with microbial fermented liquid;

(3) Preparation of starch/PEO blend solution: mixing a PEO solution and a starch solution to prepare a PEO/starch blending solution;

(4) Preparing a nanofiber membrane from the thallus lysate/starch/PEO spinning solution: and blending the thallus lysate and the starch/PEO mixed solution to prepare a spinning solution, and then performing electrostatic spinning to prepare the composite nanofiber membrane.

2. The method of claim 1, wherein the flocculation conditions are as follows: stirring at 50-500r/min and 45-85 deg.c for 1-30min and regulating pH to 6-11; the addition amount of the flocculant chitosan is 0.01-1%.

3. The method of claim 1, wherein the cell disruption rate in the lysing solution is 30% to 55%.

4. The method of claim 1, wherein the splitting conditions are as follows: cracking at 20-70 deg.c for 5-30min; the lyase is at least one of lysozyme, helicase, cellulase or lywallzyme; the chemical lysing agent comprises NaOH, KOH, SDS, or Triton X-100.

5. The method of claim 4, wherein the collected bacterial cells are resuspended and then added with 0.25-1M NaOH and 0.25% -0.5% SDS to obtain a final concentration as a lysing agent.

6. The method of claim 1, wherein the PEO solution is at a concentration of 1% to 10%; preparing starch into starch solution with mass fraction of 5-30%, gelatinizing at 60-90 deg.C for 10-60min; the blending ratio of the PEO solution and the starch solution is 4:1-1:4.

7. The method for preparing a composite nanofiber membrane as claimed in claim 1, wherein the blending ratio of the thallus lysate to the starch/PEO mixed solution is 9:1-1:9.

8. The method for preparing a composite nanofiber membrane as claimed in claim 1, wherein 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 ℃.

9. A composite nanofiber membrane prepared by the method of any one of claims 1 to 8.

10. The use of the composite nanofiber membrane as claimed in claim 9, in the fields of preparing non-woven fabrics, sanitary products, filter media and the like.