CN114752083A - Method for in-situ culture of bacterial cellulose hydrogel microspheres by using super-hydrophobic interface - Google Patents
Method for in-situ culture of bacterial cellulose hydrogel microspheres by using super-hydrophobic interface Download PDFInfo
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- 239000005016 bacterial cellulose Substances 0.000 title claims abstract description 100
- 239000004005 microsphere Substances 0.000 title claims abstract description 75
- 230000003075 superhydrophobic effect Effects 0.000 title claims abstract description 72
- 239000000017 hydrogel Substances 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000011065 in-situ storage Methods 0.000 title claims abstract description 33
- 235000002837 Acetobacter xylinum Nutrition 0.000 claims abstract description 32
- 239000007788 liquid Substances 0.000 claims abstract description 20
- 244000005700 microbiome Species 0.000 claims abstract description 12
- 241001136169 Komagataeibacter xylinus Species 0.000 claims abstract 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 24
- 238000005507 spraying Methods 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 230000007935 neutral effect Effects 0.000 claims description 6
- 239000002105 nanoparticle Substances 0.000 claims description 3
- 239000000377 silicon dioxide Substances 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- 241000589180 Rhizobium Species 0.000 claims description 2
- 241000192023 Sarcina Species 0.000 claims description 2
- 238000009629 microbiological culture Methods 0.000 claims description 2
- 230000035755 proliferation Effects 0.000 claims description 2
- 241000589155 Agrobacterium tumefaciens Species 0.000 claims 1
- 239000001963 growth medium Substances 0.000 abstract description 32
- 238000012258 culturing Methods 0.000 abstract description 11
- 238000002360 preparation method Methods 0.000 abstract description 6
- 244000235858 Acetobacter xylinum Species 0.000 description 29
- 230000008569 process Effects 0.000 description 9
- 239000000499 gel Substances 0.000 description 8
- 238000010411 cooking Methods 0.000 description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
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- 241000589158 Agrobacterium Species 0.000 description 1
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- YASYEJJMZJALEJ-UHFFFAOYSA-N Citric acid monohydrate Chemical compound O.OC(=O)CC(O)(C(O)=O)CC(O)=O YASYEJJMZJALEJ-UHFFFAOYSA-N 0.000 description 1
- 208000005189 Embolism Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
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- DGLRDKLJZLEJCY-UHFFFAOYSA-L disodium hydrogenphosphate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].OP([O-])([O-])=O DGLRDKLJZLEJCY-UHFFFAOYSA-L 0.000 description 1
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Abstract
The invention relates to a method for in-situ culture of bacterial cellulose hydrogel microspheres by using a super-hydrophobic interface, belonging to the technical field of preparation of bacterial cellulose hydrogel microspheres. The culture medium containing the microorganisms capable of producing the bacterial cellulose is sprayed on the super-hydrophobic interface, a large number of tiny acetobacter xylinum culture medium liquid drops are formed on the surface of the super-hydrophobic interface, acetobacter xylinum in the tiny culture medium liquid drops can proliferate and secrete the bacterial cellulose, and the bacterial cellulose hydrogel microspheres are formed. The method for in-situ culturing the bacterial cellulose hydrogel microspheres by using the super-hydrophobic interface has the advantages of simple operation, good balling quality and easy control and classification of the microsphere size.
Description
Technical Field
The invention relates to the technical field of preparation of bacterial cellulose hydrogel microspheres, in particular to a method for in-situ culture of bacterial cellulose hydrogel microspheres by using a super-hydrophobic interface.
Background
The bacterial cellulose hydrogel is a natural polymer material, has good biocompatibility and degradability, and is widely applied to the fields of biomedical devices, cosmetics, tissue engineering scaffolds, wound dressings and the like. The bacterial fiber hydrogel is prepared into microspheres, and can be applied to the fields of chromatographic analysis, solid carrier synthesis, protein fixation, drug sustained release, embolism treatment and the like. The existing bacterial cellulose hydrogel microspheres mostly utilize regenerated bacterial cellulose to prepare gel microspheres, the natural structure of the bacterial cellulose cannot be reserved, the in-situ culture bacterial cellulose hydrogel can keep the natural structure of the bacterial cellulose, but the preparation process is difficult, the method is less, and the patent number is as follows: the invention of CN201610363780.5 discloses a method for preparing bacterial cellulose hydrogel by dynamic culture, but has the defects of poor balling quality, low yield, difficult separation and the like. How to prepare the bacterial cellulose hydrogel more efficiently by the in-situ culture method is worth thinking.
Disclosure of Invention
The invention provides a method for in-situ culture of bacterial cellulose hydrogel microspheres by using a super-hydrophobic interface, aiming at solving the problem that the prior art cannot effectively prepare the bacterial cellulose hydrogel. The super-hydrophobic material has extremely strong hydrophobic capability, and the tiny liquid drops can keep a good spherical state on a super-hydrophobic interface. According to the invention, a culture medium containing microorganisms (preferably acetobacter xylinum) capable of producing bacterial cellulose is sprayed on the super-hydrophobic interface, a large number of tiny acetobacter xylinum culture medium liquid drops are formed on the surface of the super-hydrophobic interface, and acetobacter xylinum in the tiny culture medium liquid drops can proliferate and secrete the bacterial cellulose to form the bacterial cellulose hydrogel microspheres. The method for in-situ culturing the bacterial cellulose hydrogel microspheres by using the super-hydrophobic interface has the advantages of simple operation, good balling quality and easy control and classification of the microsphere size.
According to the purpose of the invention, the method for in-situ culture of the bacterial cellulose hydrogel microspheres by using the super-hydrophobic interface comprises the following steps:
(1) spraying a microbial culture solution capable of producing bacterial cellulose onto the sterilized superhydrophobic interface to form a culture solution droplet;
(2) The microorganism capable of producing the bacterial cellulose secretes the bacterial cellulose, and when the bacterial cellulose is filled in the whole liquid drop, the bacterial cellulose hydrogel microspheres are obtained.
Preferably, the surface of the superhydrophobic interface is coated with silica nanoparticles.
Preferably, the microorganism is acetobacter xylinum, agrobacterium, rhizobium or sarcina.
Preferably, the microorganisms in the broth are in the exponential proliferation phase.
Preferably, the diameter of the bacterial cellulose hydrogel microspheres is 200um-300 um.
Preferably, after the step (2), the bacterial cellulose hydrogel microspheres are washed away from the super-hydrophobic interface by using a NaOH solution and collected, and are repeatedly steamed and boiled until the bacterial cellulose hydrogel microspheres become white or transparent, and finally are treated to be neutral by using ultrapure water.
Generally, compared with the prior art, the technical scheme conceived by the invention mainly has the following technical advantages:
the invention provides a method for in-situ culture of bacterial cellulose hydrogel microspheres by using a super-hydrophobic interface. The behavior of secreting the bacterial cellulose by the microorganism (preferably acetobacter xylinum) capable of producing the bacterial cellulose has oxygen taxis, the super-hydrophobic material has extremely strong hydrophobic capacity, and the micro liquid drops can keep a good spherical state on a super-hydrophobic interface. After the super-hydrophobic interface is subjected to aseptic treatment, a culture medium containing microorganisms capable of producing bacterial cellulose is sprayed on the super-hydrophobic interface, a large number of tiny culture medium droplets are formed on the surface of the super-hydrophobic interface and are placed in a proper environment for culture, in the culture process, microorganisms capable of producing bacterial cellulose in the tiny culture medium droplets can proliferate and secrete the bacterial cellulose, after sufficient culture time, the bacterial cellulose secreted by the microorganisms capable of producing the bacterial cellulose can fill the whole droplets to form gel, and the culture medium and other impurities are removed by cooking with NaOH solution to obtain the prepared bacterial cellulose hydrogel microspheres. The method for in-situ culturing the bacterial cellulose hydrogel microspheres by using the super-hydrophobic interface has the advantages of simple operation, good balling quality and easy control and grading of the microsphere size.
Drawings
FIG. 1 is a schematic diagram of in situ culture of bacterial cellulose hydrogel microspheres using a superhydrophobic interface according to the present invention.
FIG. 2 is a specific step of in situ culturing bacterial cellulose hydrogel microspheres by using a super-hydrophobic interface according to the present invention.
FIG. 3 is a photo-mirror and electron microscope photograph of bacterial cellulose hydrogel microspheres cultured in situ using a superhydrophobic interface.
FIG. 4 is a photograph of in situ culturing bacterial cellulose hydrogel microspheres using a superhydrophobic interface.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention. In addition, the technical features involved in the respective embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The invention provides a method for in-situ culture of bacterial cellulose hydrogel microspheres by using a super-hydrophobic interface. The behavior of the acetobacter xylinum 2 for secreting bacterial cellulose has oxygen taxis, the super-hydrophobic material has extremely strong hydrophobic capacity, and the micro liquid drops can keep a good spherical state on a super-hydrophobic interface. After the super-hydrophobic interface 1 is subjected to aseptic treatment, the stable contact angle of the surface of the super-hydrophobic interface is more than 150 degrees, and the rolling contact angle is less than 10 degrees. Spraying a culture medium containing acetobacter xylinum on the super-hydrophobic interface 1, forming a large number of tiny acetobacter xylinum culture medium liquid drops 3 on the surface of the super-hydrophobic interface 1, placing the tiny acetobacter xylinum culture medium liquid drops in a proper environment for culture, wherein the acetobacter xylinum 2 in the tiny culture medium liquid drops can proliferate and secrete bacterial cellulose 4 in the culture process, after sufficient culture time, the bacterial cellulose 4 secreted by the acetobacter xylinum 2 can be filled in the whole liquid drops to form gel, and removing the culture medium and other impurities by cooking with NaOH solution to obtain the prepared bacterial cellulose hydrogel microspheres 5. The method for in-situ culturing the bacterial cellulose hydrogel microspheres by using the super-hydrophobic interface has the advantages of simple operation, good balling quality and easy control and grading of the microsphere size.
According to the invention, the acetobacter xylinum 2 secreting bacterial cellulose has aerotaxis, and can secrete bacterial cellulose 4 at a liquid-gas junction with sufficient oxygen.
According to the invention, the super-hydrophobic interface 1 is made by coating silica nanoparticles on a paper surface, has super-hydrophobic performance, and can keep a spherical shape on the surface of the super-hydrophobic interface by micro liquid drops.
According to the invention, the method for in-situ culture of the bacterial cellulose hydrogel microspheres by using the super-hydrophobic interface comprises the following specific steps:
step 1: preparing an ultra-hydrophobic interface 1, sterilizing the ultra-hydrophobic interface at high temperature and high pressure, and preparing a certain amount of exponentially-multiplied acetobacter xylinum culture medium and a sterile sprayer.
And 2, step: a sterile environment is prepared.
And step 3: and (3) filling the exponentially propagated acetobacter xylinum culture medium into a sterile spray can, spraying above the super-hydrophobic interface, and naturally dropping fog drops on the super-hydrophobic interface to form micro culture medium drops 3.
And 4, step 4: culturing in sterile environment at suitable temperature for 3 days while maintaining humidity above 80%.
And 5: and (3) flushing the grown gel microspheres containing the culture medium from the super-hydrophobic interface 1 by using a NaOH solution, collecting, repeatedly cooking until the bacterial cellulose hydrogel microspheres become white or transparent, and then treating the gel microspheres to be neutral by using pure water to obtain the prepared bacterial cellulose hydrogel microspheres 5.
According to the invention, the superhydrophobic interface 1 required in step 1 has superhydrophobic property, the contact angle is more than 150 degrees, and the tiny culture medium liquid drop 3 can keep a spherical shape on the surface.
According to the present invention, the exponentially-enriched acetobacter xylinum 2 involved in the step 1 can proliferate and secrete the bacterial cellulose 4 at the fastest rate.
According to the invention, the culture medium for the acetobacter xylinum required in the step 1 is prepared from 6.8g/L disodium hydrogen phosphate dodecahydrate, 5g/L yeast extract, 5g/L peptone, 1.5g/L citric acid monohydrate and 20g/L glucose. The acetobacter xylinum culture medium can provide enough nutrition for the growth of acetobacter xylinum 2 and the secretion of bacterial cellulose 4.
According to the present invention, the sterile environment prepared in step 2 is a clean bench environment in the laboratory.
According to the invention, the spraying process referred to in step 3 is carried out in a sterile environment, in a laboratory using an ultraclean bench. The aseptic environment avoids the pollution of environmental bacteria, and ensures that the acetobacter xylinum 2 in the tiny culture medium liquid drop 3 can normally grow and secrete the bacterial cellulose 4 after spraying.
According to the present invention, the optimum culture temperature of Acetobacter xylinum 2 is 30 ℃ and the culture environment temperature in step 4 is generally 30 ℃.
According to the present invention, the continuous culture for 3 days in step 4 can be adjusted according to the specific growth conditions of Acetobacter xylinum 2 and the secretion conditions of bacterial cellulose 4.
According to the present invention, the humidity of 80% in step 4 can be adjusted between 60% and 100% according to the microsphere formation conditions, in order to prevent evaporation of water from the micro-droplets 3 of the medium from affecting the growth of Acetobacter xylinum 2 and secretion of bacterial cellulose 4.
According to the invention, the purpose of repeatedly cooking by using NaOH solution in the step 5 is to kill the acetobacter xylinum 2 existing in the gel microspheres and simultaneously remove the residual culture medium and endotoxin generated in the growth process of the acetobacter xylinum, and the NaOH used for cooking is generally 1 mol/L. The ultrapure water is treated to be neutral so as to treat the bacterial cellulose hydrogel microspheres to be neutral for subsequent use.
According to the invention, the culture process of the bacterial cellulose hydrogel microspheres 5 is completed in situ on the surface of the superhydrophobic interface 1.
According to the method, the operation is convenient, the process is simple, and the large-scale production can be realized by enlarging the surface area of the super-hydrophobic interface.
According to the invention, the bacterial cellulose hydrogel microspheres prepared by the method for in-situ culturing the bacterial cellulose hydrogel microspheres by utilizing the super-hydrophobic interface can control the size of the micro-culture medium liquid drop 3 through a spraying process, so that the control of the size of the microspheres is realized.
According to the invention, the fiber density of the bacterial cellulose hydrogel microspheres prepared by the method for in-situ culturing the bacterial cellulose hydrogel microspheres by utilizing the super-hydrophobic interface is related to the culture time, and the bacterial cellulose hydrogel microspheres 5 with different fiber densities can be adaptively produced by changing the culture time within a certain time range.
Example 1
In order to explain the principle of the method for in situ culturing bacterial cellulose hydrogel microspheres by using a super-hydrophobic interface in the present invention, the embodiment will be described with reference to fig. 1. As shown in FIG. 1 (a), in this example, a culture medium of Acetobacter xylinum which proliferates exponentially was sprayed on the superhydrophobic interface 1 to form minute culture medium droplets 3. As shown in fig. 1 (b), during the subsequent cultivation process, the tiny culture medium liquid drop 3 will be exposed to the air, the spherical shape can allow oxygen to enter the liquid drop from each surface of the liquid drop uniformly, and provide necessary material conditions for the acetobacter xylinum 2 to secrete the bacterial cellulose 4, and after a period of cultivation, as shown in fig. 1 (c), the bacterial cellulose 4 secreted by the acetobacter xylinum 2 can directly form the spherical bacterial cellulose hydrogel microspheres 5.
Example 2
In order to describe the process of the method for in-situ culture of bacterial cellulose hydrogel microspheres by using a superhydrophobic interface described in embodiment 1, a specific preparation process of in-situ culture of bacterial cellulose hydrogel microspheres by using a superhydrophobic interface according to the present invention will be described with reference to fig. 2.
Step 1: preparing a super-hydrophobic interface 1, sterilizing the super-hydrophobic interface at high temperature and high pressure, and preparing a certain amount of exponentially-multiplied acetobacter xylinum culture medium and a sterile sprayer.
And 2, step: a sterile environment is prepared.
And step 3: and (3) filling the exponentially propagated acetobacter xylinum culture medium into a sterile spray can, spraying above the super-hydrophobic interface, and naturally dropping fog drops on the super-hydrophobic interface to form micro culture medium drops 3.
And 4, step 4: culturing in sterile environment at suitable temperature for 3 days while maintaining humidity above 80%.
And (3) flushing the grown gel microspheres containing the culture medium from the super-hydrophobic interface 1 by using a NaOH solution, collecting, repeatedly cooking until the bacterial cellulose hydrogel microspheres become white or transparent, and then treating the gel microspheres to be neutral by using pure water to obtain the prepared bacterial cellulose hydrogel microspheres 5.
Example 3
In order to describe the bacterial cellulose hydrogel microspheres cultured in situ by using the super-hydrophobic interface in example 1, this embodiment will be described with reference to fig. 3. Fig. 3 (a) is a photo-mirror image of the bacterial cellulose hydrogel microsphere 5 in this embodiment, and the prepared bacterial cellulose hydrogel microsphere 5 is in a semi-transparent spherical shape and has a better roundness. Fig. 3 (b) is a field emission scanning electron microscope picture of the bacterial cellulose hydrogel microsphere 5 in this embodiment, in which a shape-retaining agent is filled inside the microsphere, and the prepared bacterial cellulose hydrogel microsphere 5 has a diameter of about 225um, a good spherical shape, and a smooth surface. Fig. 3 (c) is a cross-sectional image of a field emission scanning electron microscope of the bacterial cellulose hydrogel microsphere 5, which is not treated with a shape-retaining agent, and has a porous structure on the surface and inside.
Example 4
The method for in-situ culture of the bacterial cellulose hydrogel microspheres by using the super-hydrophobic interface, disclosed by the invention, is simple to operate, easy to expand production and capable of improving preparation efficiency. As shown in fig. 4 (a), the minute medium droplet 3 in the present embodiment appears approximately spherical on the superhydrophobic interface 1. In this embodiment, the method of the present invention is used for scale-up culture under laboratory conditions, as shown in fig. 4 (b), one culture dish is a superhydrophobic interface 1, and the culture of the bacterial cellulose hydrogel microspheres 5 is performed on a plurality of superhydrophobic interfaces, so as to improve the preparation efficiency.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. A method for in-situ culture of bacterial cellulose hydrogel microspheres by using a super-hydrophobic interface is characterized by comprising the following steps:
(1) spraying a microbial culture solution capable of producing bacterial cellulose onto the sterilized superhydrophobic interface to form a culture solution droplet;
(2) The microorganism capable of producing the bacterial cellulose secretes the bacterial cellulose, and when the bacterial cellulose is filled in the whole liquid drop, the bacterial cellulose hydrogel microspheres are obtained.
2. The method for in-situ culture of bacterial cellulose hydrogel microspheres by using the superhydrophobic interface as claimed in claim 1, wherein the surface of the superhydrophobic interface is coated with silica nanoparticles.
3. The method for in situ culture of bacterial cellulose hydrogel microspheres using a superhydrophobic interface according to claim 1 or 2, wherein the microorganism is acetobacter xylinum, agrobacterium tumefaciens, rhizobium or sarcina.
4. The method for in situ culture of bacterial cellulose hydrogel microspheres using a superhydrophobic interface of claim 1, wherein the microorganisms in the culture solution are in an exponential proliferation phase.
5. The method for in situ culture of bacterial cellulose hydrogel microspheres by using the superhydrophobic interface as claimed in claim 1, wherein the diameter of the bacterial cellulose hydrogel microspheres is 200um-300 um.
6. The method for in-situ culture of bacterial cellulose hydrogel microspheres by using a superhydrophobic interface as claimed in claim 1, wherein after the step (2), the bacterial cellulose hydrogel microspheres are washed away from the superhydrophobic interface by using NaOH solution and collected, repeatedly steamed until the bacterial cellulose hydrogel microspheres become white or transparent, and finally treated with ultrapure water to be neutral.
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| CN103695499A (en) * | 2013-12-09 | 2014-04-02 | 深圳先进技术研究院 | Method for preparing bacterial cellulose microsphere |
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