A method for preparing and applying an anisotropic Pickering emulsion stabilized by a hydrophobic H-SiO2@PNIPAM hybrid microgel.
By preparing hydrophobic H-SiO2@PNIPAM hybrid microgels as emulsifiers, temperature-sensitive Pickering emulsions were prepared, solving the stability and operational complexity problems of traditional emulsion culture methods, and achieving efficient anaerobic culture and rapid isolation of Bifidobacteria.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2024-09-09
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies struggle to prepare emulsions that are both stable and unstable for microbial culture, especially for the efficient anaerobic culture of Bifidobacteria, and traditional methods are costly and complex to operate.
By preparing hydrophobic H-SiO2@PNIPAM hybrid microgels as emulsifiers, thermosensitive Pickering emulsions were prepared. The phase inversion of the emulsion was achieved by utilizing temperature changes, enabling efficient cultivation and rapid separation of microorganisms.
It enables low-cost and high-efficiency microbial culture, reduces the risk of contamination, simplifies the operation process, and improves the efficiency of microbial culture.
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Figure CN119351246B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of nanomaterial preparation and bioculture technology, specifically relating to a method for preparing a hydrophobic H-SiO2@PNIPAM hybrid microgel-stabilized opposite-transformation Pickering emulsion and its application. Background Technology
[0002] Bifidobacteria are strict anaerobic bacteria isolated from the infant gut, and they have wide applications in food fermentation, human health, and cosmetics. Furthermore, after cultivation, inactivation, and decomposition, Bifidobacteria can yield a metabolite—Bifida ferment lysate—a valuable active ingredient in cosmetics, possessing whitening, sun protection, antioxidant, and anti-aging effects. Therefore, the efficient cultivation of Bifidobacteria has attracted widespread attention from researchers. However, as an obligate anaerobic, or strict anaerobic bacterium, Bifidobacteria require demanding and complex cultivation conditions. Traditional cultivation methods include the most basic deoxygenation method, air removal method, and chemical reducing agent method, commonly used anaerobic tank method, anaerobic bag method, and the advanced anaerobic glove box method. However, these methods suffer from drawbacks such as high requirements, lack of universality, and excessive cost. Therefore, there is an urgent need in this field for an effective, convenient, and low-cost method for the anaerobic cultivation of microorganisms.
[0003] In recent years, researchers have used each droplet of emulsion as a miniature culture vessel for microbial cultivation. The advantages of using emulsion droplets as a compartmentalized space for microbial culture are: microorganisms are randomly confined to tiny liquid compartments; the oil in the outer phase isolates oxygen, creating an anaerobic environment suitable for anaerobic bacteria growth; it also increases microbial dispersibility and nutrient transport, and reduces product inhibition, allowing metabolic products and microbial numbers to accumulate more rapidly than in macroscopic systems (e.g., shake flasks), while also reducing the risk of culture contamination. However, culturing bacteria with emulsions requires high emulsion stability, while releasing bacteria from the inner phase after cultivation necessitates timely emulsion demulsification. This presents a paradoxical requirement that the emulsion be both stable and unstable, an effect that existing emulsions struggle to achieve.
[0004] As particle-stabilized emulsions, the properties of Pickering emulsions largely depend on the properties of the particles. Microgels, as soft particles that are responsive without modification, are excellent candidate particles for preparing stimulus-responsive Pickering emulsions. However, most microgels are very hydrophilic, making it difficult to stabilize W / O emulsions and encapsulate bacteria in the inner phase. Furthermore, the responsiveness of microgels can only lead to incomplete demulsification, making it difficult to release and recover all bacteria from the inner phase.
[0005] Therefore, it is essential to prepare a hydrophobic responsive microgel particle to stabilize the W / O Pickering emulsion and further utilize this emulsion system for efficient cultivation of Bifidobacteria. Summary of the Invention
[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.
[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.
[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a hydrophobic H-SiO2@PNIPAM hybrid microgel-stabilized opposite-transformation Pickering emulsion.
[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing a hydrophobic H-SiO2@PNIPAM hybrid microgel-stabilized opposite-transformation Pickering emulsion, comprising,
[0010] Oil dispersion of H-SiO2@PNIPAM hybrid microgel was prepared;
[0011] Preparation of Pickering emulsion: The oil dispersion of H-SiO2@PNIPAM hybrid microgel was mixed with the aqueous phase and emulsified by vortexing to obtain the Pickering emulsion;
[0012] By changing the volume of the oil phase or the emulsification temperature, the emulsion can be switched between oil-in-water and water-in-oil.
[0013] As a preferred embodiment of the preparation method described in this invention, the oil dispersion of the H-SiO2@PNIPAM hybrid microgel is prepared by a method comprising:
[0014] NIPAM, BIS and 570 dye was dissolved in deionized water and deoxygenated by passing N2 through it in a 45°C water bath for 40 minutes.
[0015] Then, 0.02 g / mL KPS solution was added to the solution, and the temperature was gradually increased to 70 °C under a nitrogen atmosphere to wait for the reaction to start.
[0016] One hour after the reaction was initiated, silane coupling agent KH-570 was added to the solution, and polymerization continued at 70°C for 5 hours.
[0017] After the reaction was completed, the PNIPAM microgel was centrifuged and washed at least four times with deionized water. After washing, the PNIPAM microgel was dispersed in deionized water in the form of particles to obtain a PNIPAM microgel dispersion.
[0018] Ethanol and ammonia were added to the PNIPAM microgel dispersion and mixed evenly. The ethanol solution of TEOS was then added dropwise using a syringe pump while stirring. Two hours after the injection was completed, the silane coupling agent diethoxydimethylsilane was added, and the reaction continued for another 3 hours.
[0019] After the reaction was completed, the particles were washed at least five times with ethanol and toluene, and the resulting particles were dispersed in the oil phase to obtain the oil dispersion.
[0020] As a preferred embodiment of the preparation method described in this invention, wherein: The concentration of dye 570 is 1 mg / mL, and the solvent is 10% vol ethanol.
[0021] As a preferred embodiment of the preparation method described in this invention, wherein: the NIPAM, BIS, The ratio of 570 dye, deionized water, KPS solution, and silane coupling agent KH-570 is 2g:0.06g:500μL:150mL:5mL:240μL.
[0022] As a preferred embodiment of the preparation method described in this invention, the concentration of the PNIPAM microgel dispersion is 1 wt%; the ratio of the PNIPAM microgel dispersion, ethanol, ammonia, ethanol solution of TEOS and silane coupling agent diethoxydimethylsilane is 30 mL: 120 mL: 6 mL: 15 mL: 600 μL.
[0023] As a preferred embodiment of the preparation method described in this invention, the oil phase includes any one of toluene, cyclopentamethoxysiloxane, dodecane, and n-hexane;
[0024] Emulsification temperatures include 10℃, 20℃, 25℃, 30℃, 35℃, 40℃, and 50℃.
[0025] The oil phase volumes included 55 vol%, 58 vol%, 60 vol%, 62 vol%, 63.5 vol%, 65 vol%, 66.6 vol%, 68 vol%, and 69 vol%.
[0026] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of reverse-transformation Pickering emulsion in the efficient culture and rapid separation of anaerobic bacteria in microcompartments.
[0027] As a preferred embodiment of the application described in this invention, it includes: using an oil dispersion of H-SiO2@PNIPAM hybrid microgel as the oil phase and an anaerobic bacterial suspension as the aqueous phase, vortex emulsification is performed at 37°C to form a temperature-sensitive water-in-oil Pickering emulsion.
[0028] Temperature-sensitive water-in-oil Pickering emulsion was used as a micro-bioreactor system for the cultivation of anaerobic bacteria.
[0029] After the culture is completed, the emulsion is re-emulsified by changing the temperature, causing an inversion and transforming it into an oil-in-water Pickering emulsion. This completely releases the anaerobic bacteria after the internal fermentation, allowing for the separation of the bacterial broth and microbial metabolites after fermentation.
[0030] In a preferred embodiment of the application described in this invention, the anaerobic bacteria are any one of Bifidobacterium, Lactobacillus plantarum, Lactobacillus helveticus, Gram-positive spore-forming bacteria, Streptococcus mutans, yeast, Clostridium butyricum, and Oxalic acid bacteria.
[0031] As a preferred embodiment of the application described in this invention, the culture time is 0h, 3h, 6h, 12h, 24h, or 48h.
[0032] Beneficial effects of this invention:
[0033] (1) The present invention can prepare hydrophobic hybrid microgel nanoparticles by simple precipitation polymerization and sol-gel method. The resulting hybrid microgels have a uniform particle size distribution and a size of about 500 nm in the dry state. The hybrid microgel nanoparticles prepared by this method retain the temperature responsiveness of the PNIPAM microgel core, which can swell and shrink in water with temperature changes.
[0034] (2) The present invention uses the prepared hybrid microgel as an emulsifier to prepare a stable water-in-oil Pickering emulsion, and can make the emulsion temperature responsive. The emulsion can be changed from water-in-oil to oil-in-water by simple temperature adjustment or change of oil phase volume. The operation is simple and the cost is low.
[0035] (3) The present invention uses a suspension of Bifidobacterium as the aqueous phase, which can encapsulate Bifidobacterium inside the droplets of water-in-oil Pickering emulsion. Under normal culture conditions, it can achieve efficient culture of Bifidobacterium. Compared with conventional culture methods such as anaerobic bags and anaerobic glove boxes, it is cheaper, easier to operate, reduces the risk of bacterial contamination, and has universality. Attached Figure Description
[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:
[0037] Figure 1 This is a scanning electron microscope (SEM) image of the PNIPAM microgel in an embodiment of the present invention.
[0038] Figure 2 This is a scanning electron microscope (SEM) image of the hydrophobic H-SiO2@PNIPAM hybrid microgel in an embodiment of the present invention.
[0039] Figure 3 The figures show the air-water contact angle measurements of PNIPAM microgel (Figure a) and H-SiO2@PNIPAM hybrid microgel (Figure b) in the embodiments of the present invention.
[0040] Figure 4 This is a density gradient (DLS) graph showing the particle size variation of PNIPAM microgels and H-SiO2@PNIPAM hybrid microgels in water as a function of temperature in embodiments of the present invention.
[0041] Figure 5 The images show the appearance (left) and optical microscope image (right) of the water-in-oil Pickering emulsion stabilized by H-SiO2@PNIPAM hybrid microgel as an emulsifier in this embodiment of the invention.
[0042] Figure 6 The images show the appearance of different types of Pickering emulsions stabilized by H-SiO2@PNIPAM hybrid microgels as emulsifiers under different oil phase volumes in embodiments of the present invention.
[0043] Figure 7 The images show the appearance of different types of Pickering emulsions stabilized by H-SiO2@PNIPAM hybrid microgels as emulsifiers at different temperatures in embodiments of the present invention.
[0044] Figure 8 The images show the appearance (left) and laser confocal microscope image (right) of the water-in-oil Pickering emulsion when the oil phase is cyclopentamethoxysiloxane and the aqueous phase is MRS culture medium, as described in this embodiment of the invention.
[0045] Figure 9 The images show optical microscope images of Bifidobacteria encapsulated inside droplets of a water-in-oil Pickering emulsion (left) and the complete release of Bifidobacteria after inversion (right) in an embodiment of the present invention.
[0046] Figure 10 The bar chart shows the concentration of Bifidobacteria at different culture times for three culture methods (conventional MRS culture, MRS oil-coated culture, and Pickering emulsion culture) in the embodiments of the present invention. Detailed Implementation
[0047] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.
[0048] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.
[0049] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.
[0050] In this embodiment of the invention, NIPAM was purchased from TCI reagents, and the product batch was KIAM-JX;
[0051] BIS was purchased from Aladdin Reagents, product batch number J2309050;
[0052] Dye 570 was purchased from Polysciences;
[0053] KPS was purchased from Sinopharm Reagent, product batch number 20210413;
[0054] The silane coupling agent KH-570 was purchased from Sinopharm Reagent, batch number 20210205;
[0055] TEOS was purchased from SigmaAldrich, product batch number S8250158228;
[0056] Diethoxydimethylsilane was purchased from TCI Reagents, batch number 6Z7LE-PI.
[0057] Example 1
[0058] Preparation of hydrophobic H-SiO2@PNIPAM hybrid microgel nanoparticles:
[0059] (1) Preparation of PNIPAM microgels
[0060] 2g NIPAM, 0.06g BIS and 500μL 570 dye (1 mg / mL, 10% vol ethanol) was dissolved in 150 mL of deionized water and deoxygenated by passing N2 through it in a 45 °C water bath for 40 min. Then, 5 mL of KPS solution (containing 0.1 g) was added to the solution, and the temperature was gradually increased to 70 °C under a nitrogen atmosphere to wait for the reaction to start.
[0061] One hour after the reaction was initiated, 240 μL of silane coupling agent KH-570 was added to the solution, and polymerization was continued at 70 °C for 5 hours.
[0062] After the reaction was completed, the PNIPAM microgel was washed at least four times by centrifugation with deionized water at 10,000 rpm for 10 min each time. After washing, the PNIPAM microgel was dispersed in deionized water in the form of particles and stored in a refrigerator at 4°C.
[0063] The prepared PNIPAM microgels were characterized by electron microscopy, and the results are as follows: Figure 1 As shown, the prepared PNIPAM microgels have a uniform particle size distribution, with a particle size of approximately 500 nm.
[0064] (2) Preparation of hydrophobic H-SiO2@PNIPAM hybrid microgels
[0065] Add 30 mL of PNIPAM microgel dispersion with a solid content of 1 wt% to a 250 mL round bottom flask, then add 120 mL of ethanol and 6 mL of ammonia water and mix well. While stirring, add 15 mL of TEOS ethanol solution (TEOS to anhydrous ethanol volume ratio of 1:14) dropwise into the flask using a syringe pump.
[0066] Two hours after the injection was completed, 600 μL of the silane coupling agent diethoxydimethylsilane was added, and the reaction continued for another 3 hours.
[0067] After the reaction is complete, the particles are washed at least five times with ethanol and toluene, and are finally dispersed directly in the oil phase in the form of particles, or the particles are dispersed in deionized water and freeze-dried into powder for subsequent characterization.
[0068] The prepared hydrophobic H-SiO2@PNIPAM hybrid microgel was characterized by electron microscopy, and the results are as follows: Figure 2 As shown, a layer of silica nanoparticles has been grown in situ on the surface of the PNIPAM microgel, and the hybrid microgel has a uniform particle size distribution with a particle size of about 500 nm.
[0069] The air-water contact angles of the prepared hydrophobic H-SiO2@PNIPAM hybrid microgels and pure PNIPAM microgels were measured, and the results are as follows: Figure 3As shown, the contact angle of the hydrophobically modified H-SiO2@PNIPAM hybrid microgel is as high as 111.4°, proving that it has been successfully hydrophobically modified.
[0070] The aqueous dispersions of the prepared hydrophobic H-SiO2@PNIPAM hybrid microgel and pure PNIPAM microgel were subjected to temperature-dependent particle size distribution (DLS) tests, and the results are as follows: Figure 4 As shown, the H-SiO2@PNIPAM hybrid microgel retains its temperature-sensitive properties after hydrophobic modification, and can swell and shrink with temperature changes.
[0071] Example 2
[0072] Pickering emulsion stabilized by hydrophobic H-SiO2@PNIPAM hybrid microgel:
[0073] The H-SiO2@PNIPAM hybrid microgel prepared in Example 1(2) was prepared as a toluene dispersion with a particle concentration of 2wt%. 2.66 mL of the dispersion was mixed with 1.33 mL of deionized water and vortexed at a certain temperature for 30 s to obtain a W / O type Pickering emulsion.
[0074] Figure 5 The image shows the appearance of the W / O type Pickering emulsion (left) and an optical microscope image (right).
[0075] Example 3
[0076] W / O type Pickering emulsion droplets as a means of efficient culture of anaerobic bacteria in microcompartments and isolation of endophase anaerobic bacteria:
[0077] The H-SiO2@PNIPAM hybrid microgel prepared in Example 1(2) was prepared as a cyclopentamethoxysiloxane dispersion with a particle concentration of 2wt%. 2mL of the dispersion was mixed with 1mL of MRS culture medium suspension containing Bifidobacterium, and vortexed at 37°C for 30s to obtain a W / O type Pickering emulsion.
[0078] The emulsion was then placed in a 37°C incubator for incubation.
[0079] After culturing for a period of time, the emulsion is cooled to room temperature. After cooling, 1 mL is added and the mixture is vortexed again for 30 seconds to emulsify. This will cause an inversion and give an O / W type emulsion, which will release all the Bifidobacteria in the original inner phase droplets.
[0080] Add 2 ml of LMR (Low MnS) medium. Due to gravity, the bacterial culture will sink, making it easier to separate.
[0081] pass Figure 9The optical microscope images show the encapsulation (left) and isolation of Bifidobacteria (right).
[0082] Comparative Example 1
[0083] The oil phase volume of the W / O type Pickering emulsion obtained in Example 2 was changed to 55 vol%, 58 vol%, 60 vol%, 62 vol%, 63.5 vol%, 65 vol%, 66.6 vol%, 68 vol%, and 69 vol%, and emulsified by vortexing for 30 seconds to obtain Pickering emulsions of different emulsion types.
[0084] Figure 6 The appearance diagram of the emulsion shows the change in emulsion type. As the volume of the oil phase increases, the emulsion changes from an oil-in-water type to a water-in-oil type.
[0085] Comparative Example 2
[0086] By changing the oil phase volume of the W / O type Pickering emulsion obtained in Example 2 to 60 vol%, and then vortexing it for 30 seconds at different temperatures (10°C, 20°C, 25°C, 30°C, 35°C, 40°C, and 50°C), different types of Pickering emulsions can also be obtained.
[0087] Figure 7 The appearance diagram of the emulsion shows the transformation of the emulsion type. As the temperature increases, the emulsion can change from an oil-in-water type to a water-in-oil type.
[0088] Comparative Example 3
[0089] The oil phase of the W / O Pickering emulsion in Example 2 was changed to cyclopentamethoxysiloxane with a volume of 66.6 vol%, and the aqueous phase was changed to MRS culture medium with a volume of 33.3 vol%. The emulsification was carried out by vortexing at a constant temperature of 37°C for 30 seconds to obtain the W / O Pickering emulsion.
[0090] Depend on Figure 8 As can be observed from the emulsion appearance diagram and laser confocal microscope image, stable W / O type Pickering emulsions can still be prepared using MRS as the aqueous phase.
[0091] Comparative Example 4
[0092] Comparison of culture rates of anaerobic bacteria:
[0093] (1) After centrifuging and collecting the Bifidobacterium in the MRS bacterial suspension, the Bifidobacterium was redispersed and diluted with MRS medium to an absorbance of 0.88 and placed in a constant temperature incubator at 37℃. This was recorded as the control group.
[0094] (2) 2 mL of cyclopentamethoxane was spread on the top layer of the Bifidobacterium suspension of the same concentration, and the suspension was placed in a constant temperature incubator at 37℃ and incubated. This was recorded as experimental group 1.
[0095] (3) The H-SiO2@PNIPAM hybrid microgel prepared in Example 1(2) was prepared into a cyclopentamethoxysiloxane dispersion with a particle concentration of 2wt%. 2mL of the dispersion was mixed with 1mL of MRS culture medium suspension containing Bifidobacterium (concentration same as the control group). After vortexing at 37℃ for 30s, a W / O type Pickering emulsion was obtained. The emulsion was then placed in a 37℃ constant temperature incubator for culture and was recorded as experimental group 2.
[0096] (4) After culturing for 3h, 6h, 12h, 24h, and 48h, the OD of the bacterial suspension in the three groups of experiments was measured respectively. 600 value.
[0097] Depend on Figure 10 It can be seen that the W / O type Pickering emulsion droplets, as microchambers, are far more efficient at culturing anaerobic bacteria than conventional culture.
[0098] In summary, this invention provides a method for efficient microcompartmental culture and rapid separation of anaerobic bacteria based on thermosensitive microgels. First, an emulsifier for the emulsion culture system, namely H-SiO2@PNIPAM hybrid microgel, was prepared. This hybrid microgel exhibits significantly increased hydrophobicity compared to pure PNIPAM microgels, thus stabilizing W / O type Pickering emulsions. Simultaneously, this hybrid microgel is thermosensitive, undergoing a volume phase inversion according to temperature changes, switching between hydrophobic and hydrophilic states. Therefore, the phase inversion of the stable Pickering emulsion can be controlled by adjusting the temperature and water-to-oil ratio. Subsequently, using this emulsion system for efficient culture and separation experiments of anaerobic bacteria, the culture efficiency was more than twice that of conventional cultures.
[0099] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.
Claims
1. A method for preparing a hydrophobic H-SiO2@PNIPAM hybrid microgel-stabilized opposite-transformation Pickering emulsion, characterized in that: include, Oil dispersion of H-SiO2@PNIPAM hybrid microgel was prepared; Preparation of Pickering emulsion: The oil dispersion of H-SiO2@PNIPAM hybrid microgel was mixed with the aqueous phase and emulsified by vortexing to obtain the Pickering emulsion; In this process, changing the volume of the oil phase or altering the emulsification temperature allows the emulsion to switch between oil-in-water and water-in-oil states. The preparation method of the oil dispersion of the H-SiO2@PNIPAM hybrid microgel includes: Dissolve NIPAM, BIS and PolyFluor® 570 dye solutions in deionized water and deoxygenate them by passing N2 through them in a 45°C water bath for 40 min. Then, 0.02 g / mL KPS solution was added to the solution, and the temperature was gradually increased to 70 °C under a nitrogen atmosphere to wait for the reaction to start. After 1 hour of reaction initiation, silane coupling agent KH-570 was added to the solution, and polymerization continued at 70°C for 5 hours. After the reaction was completed, the PNIPAM microgel was centrifuged and washed at least four times with deionized water. After washing, the PNIPAM microgel was dispersed in deionized water in the form of particles to obtain a PNIPAM microgel dispersion. Ethanol and ammonia were added to the PNIPAM microgel dispersion and mixed evenly. The ethanol solution of TEOS was then added dropwise using a syringe pump while stirring. Two hours after the injection was completed, the silane coupling agent diethoxydimethylsilane was added, and the reaction continued for another 3 hours. After the reaction was completed, the particles were washed with ethanol and toluene at least five times, and the resulting particles were dispersed in the oil phase to obtain the oil dispersion. The oil phase is any one of toluene, cyclopentadimethylsiloxane, dodecane, and n-hexane; The emulsification temperature is 10℃, 20℃, 25℃, 30℃, 35℃, 40℃ or 50℃; The oil phase volumes were 55 vol%, 58 vol%, 60 vol%, 62 vol%, 63.5 vol%, 65 vol%, 66.6 vol%, 68 vol%, or 69 vol%. The aqueous phase is an anaerobic bacterial suspension.
2. The preparation method according to claim 1, characterized in that: The concentration of the PolyFluor® 570 dye solution is 1 mg / mL, and the solvent is 10% vol ethanol.
3. The preparation method according to claim 1 or 2, characterized in that: The concentration of the PNIPAM microgel dispersion is 1 wt%.
4. The preparation method according to claim 2, characterized in that: The ratio of NIPAM, BIS, PolyFluor® 570 dye solution, deionized water, KPS solution, and silane coupling agent KH-570 is 2g:0.06g:500μL:150mL:5mL:240μL.
5. The preparation method according to claim 3, characterized in that: The ratio of the PNIPAM microgel dispersion, ethanol, ammonia, ethanol solution of TEOS, and silane coupling agent diethoxydimethylsilane is 30 mL: 120 mL: 6 mL: 15 mL: 600 μL.
6. The application of the opposite-transformation Pickering emulsion prepared by any of the preparation methods described in claims 1 to 5 in the efficient microcompartment culture and rapid separation of anaerobic bacteria.
7. The application as described in claim 6, characterized in that: include, A temperature-sensitive water-in-oil Pickering emulsion was formed by vortex emulsification of an oil dispersion of H-SiO2@PNIPAM hybrid microgel as the oil phase and an anaerobic bacterial suspension as the aqueous phase at 37°C. Temperature-sensitive water-in-oil Pickering emulsion was used as a micro-bioreactor system for the cultivation of anaerobic bacteria. After the culture is completed, the temperature is changed and the emulsion is re-emulsified to cause an inversion, transforming it into an oil-in-water Pickering emulsion. This completely releases the anaerobic bacteria after the internal fermentation and separates the bacterial broth and microbial metabolites after fermentation.
8. The application as described in claim 7, characterized in that: The anaerobic bacteria are any one of Bifidobacterium, Lactobacillus plantarum, Lactobacillus helveticus, Streptococcus mutans, yeast, Clostridium butyricum, and Oxalic acid bacteria.
9. The application as described in claim 7, characterized in that: The incubation time is 3h, 6h, 12h, 24h or 48h.