Preparation method of agar gel microspheres, prepared agar gel microspheres and application

Abstract

The invention belongs to the field of preparation of biopolymer materials, and particularly relates to a preparation method of agar gel microspheres, the agar gel microspheres prepared by the preparation method and application of the agar gel microspheres. Aiming at the problems of large particle size range, poor particle size uniformity and the like of the existing hydrogel microspheres, the invention provides the preparation method of the agar gel microspheres, which comprises the following steps: (1) mixing a bacterial suspension and an agar aqueous solution, and carrying out primary preheating to obtain a water phase; (2) an oily solvent and a surfactant are mixed and subjected to secondary preheating, an oil phase is obtained, and the surfactant is selected from at least two of a span series surfactant, a Tween series surfactant, Triton X-100 and ABIL EM 90; and (3) adding the water phase into the oil phase, stirring at 45-75 DEG C, curing, and demulsifying to obtain the agar gel microspheres. The hydrogel microspheres prepared by the method are appropriate in particle size range and good in particle size uniformity.

Description

Technical Field

[0001] This invention belongs to the field of biopolymer material preparation, specifically relating to a method for preparing agarose gel microspheres, the agarose gel microspheres prepared therefrom, and their applications. Background Technology

[0002] Environmental functional microorganisms are a group of microorganisms that actively participate in and promote the material cycle of ecosystems, assist in the degradation and transformation of environmental pollutants, and maintain the stable operation of ecosystems under environmental stress through their specific metabolic activities or physiological characteristics in the natural environment. These microorganisms play multiple key roles in ecosystems; for example, photosynthetic bacteria and heterotrophic microorganisms respectively play roles in carbon cycling and fixation. In addition to decomposing organic carbon, nitrogen-fixing bacteria, nitrifying bacteria, and denitrifying bacteria work together to complete the key steps of the nitrogen cycle. Regarding pollutant metabolism, white-rot fungi can decompose polycyclic aromatic hydrocarbons (PAHs), some bacteria and fungi can cope with heavy metal pollution through different mechanisms, and extreme-environment microorganisms such as halophilic archaea and acidophilic thiobacilli maintain their ecological functions under harsh conditions such as high salinity and acidity. Developing and identifying environmentally functional microorganisms is of great significance. From a resource utilization perspective, it may uncover microorganisms with new functions, providing new biocatalysts or biotransformation pathways for industrial production and bioenergy development, bringing new economic growth points.

[0003] Currently, the efficient isolation and cultivation of microorganisms with important environmental functions from samples has become a research focus for microbiologists worldwide. Existing technologies have disclosed methods for generating biopolymer microspheres to embed and immobilize bioactive substances such as cells and enzymes. For example, methods for preparing biopolymer microspheres (such as agar, sodium alginate, and other fibers, natural gel polymers, etc.) include emulsification solidification, jet cooling, chemical cross-linking, and microfluidic methods. Chinese patent (publication number CN105950472A) discloses a method for co-culturing microorganisms. Based on a microfluidic chip, it first disperses insoluble calcium salts in an aqueous phase containing polymers (agar), then adds a surfactant and a gel initiator to an oil phase (paraffin oil), and finally mixes the aqueous and oil phases to prepare hydrogel microspheres. However, it has the following problems: 1. It mainly utilizes the chemical cross-linking between insoluble calcium salts and gel initiators to prepare hydrogel microspheres, which is a complex and time-consuming process; 2. Its preparation process is limited by the microfluidic chip, making it difficult to achieve high-throughput cultivation and screening of microorganisms. Emulsion-curing is currently the most studied and widely used method for preparing microspheres due to its simple equipment operation, relatively simple chemical composition, and high throughput. This method involves adding the aqueous phase of heated and melted agar solution to mineral oil containing surfactants to form a water-in-oil (W / O) emulsion. After cooling, the emulsion is washed with a washing solution to obtain agar gel microspheres. Although the emulsion-curing method can solve the problem of high-throughput microsphere preparation, the hydrogel microspheres prepared by it have problems such as a large particle size range and poor particle size uniformity. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for preparing agarose gel microspheres, the agarose gel microspheres prepared therefrom, and their applications. The hydrogel microspheres prepared by the method of this invention have a suitable particle size range and good particle size uniformity.

[0005] A first aspect of the present invention provides a method for preparing agarose gel microspheres, the method comprising the following steps:

[0006] (1) Mix the bacterial suspension and agar solution, and preheat once to obtain the aqueous phase;

[0007] (2) The oily solvent and the surfactant are mixed and preheated twice to obtain the oil phase. The surfactant is selected from at least two of the following: Span series surfactants, Tween series surfactants, Triton X-100 and ABIL EM 90.

[0008] (3) Add the aqueous phase to the oil phase, stir at 45~75℃, and then solidify and demulsify to obtain the agar gel microspheres; the agar gel microspheres have a structure in which microorganisms are embedded in agar, and their particle size ranges from 4.5 to 35.0 μm, and the particle size distribution width is 0.64~1.30.

[0009] The inventors have discovered that during the microbial encapsulation and cultivation process, agar gel microspheres with a large particle size range and poor particle size uniformity, after encapsulating microorganisms, create microbial cultivation environments of different sizes due to their large particle size differences. As the microorganisms are cultivated, the differences in the cultivation environment gradually increase, and some microorganisms in the smaller particle size microspheres have already broken through the microspheres and entered the liquid environment, while some microorganisms in the larger particle size agar microspheres have not broken through, which is not conducive to high-throughput microbial cultivation and screening. To this end, the inventors creatively utilize an oily solvent containing at least two surfactants (e.g., at least two of Span series surfactants, Tween series surfactants, Triton X-100, and ABIL EM 90) as the oil phase and an agar aqueous solution carrying microorganisms as the aqueous phase. The aqueous phase is then added to the oil phase and stirred to form water-in-oil droplets. During this process, at least two surfactants work together to form precursor microspheres with suitable and uniform particle size. Then, through steps such as curing and demulsification, agar gel microspheres with suitable (particle size range of 4.5~35.0 μm) and uniform (particle size distribution width value of 0.64~1.30) particle size are obtained, which are embedded with microorganisms.

[0010] Preferably, in step (1), the concentration of agar in the aqueous phase is 0.5~2.0 wt%;

[0011] The concentration of microorganisms in the bacterial suspension is cells / ml;

[0012] The concentration of agar in the agar aqueous solution is 1.0~4.0 wt%;

[0013] The volume ratio of the agar aqueous solution to the bacterial suspension is 1:1.5 to 1.5:1;

[0014] The temperature of the first preheating is 45~75℃;

[0015] The preheating time is 15-20 minutes.

[0016] Preferably, the bacterial suspension is prepared by the following steps:

[0017] i) First, filter the environmental sample through a 20-50 μm filter membrane to remove impurities and obtain the filtrate. The environmental sample includes natural water bodies, soil, air, activated sludge, or laboratory cultured bacterial solution.

[0018] ii) Add 5 to 10 times the volume of sterile water to the filtrate and centrifuge.

[0019] iii) Repeat step ii) at least 3 times to obtain a bacterial suspension. Among them, for microorganisms in atmospheric environmental samples, a feasible sampling method is to use the "FA-5 liquid impact microbial aerosol sampler" to continuously collect microorganisms at a sampling flow rate of 7~15 L / min for 12~24h. After collecting the microorganisms in the atmosphere into water, the above-mentioned filter membrane filtration and impurity removal steps are performed.

[0020] Preferably, in step (2), the oily solvent is selected from at least one or a combination of mineral oil, vegetable oil, synthetic fats, silicone oil and animal fats;

[0021] The total concentration of surfactants in the oil phase is 1.0~4.0 vol%

[0022] The mixing is carried out under ultrasonic treatment, the power of which is 100~500 W and the time is 15~30 minutes.

[0023] The temperature of the secondary preheating is 45~75℃;

[0024] The secondary preheating time is 15-20 minutes.

[0025] More preferably, the mineral oil is selected from at least one or a combination of paraffin oil, petrolatum, white mineral oil, and kerosene;

[0026] The vegetable oil is selected from at least one or a combination of olive oil, soybean oil, and sunflower seed oil.

[0027] The synthetic oil contains at least one of fatty acid esters and polyolefin oils;

[0028] The silicone oil contains at least one of dimethyl silicone oil and cyclomethyl silicone oil;

[0029] The animal fats include at least one of lanolin and beeswax.

[0030] Preferably, in step (2), the Span series surfactants include at least one of SPAN 60 and SPAN 85;

[0031] The Tween series surfactants include at least one of TWEEN 60 and TWEEN 85.

[0032] More preferably, the surfactant is selected from: a mixture of Span series surfactants and Tween series surfactants or a mixture of Triton X-100 and ABIL EM 90.

[0033] This invention preferably uses a mixture of Span and Tween surfactants, or a mixture of Triton X-100 and ABIL EM 90, as surfactants. Specifically, Span 80 is used as an example of a Span surfactant, and Tween 80 is used as an example of a Tween surfactant. The combination of these two surfactants achieves tight interfacial co-adsorption through their similar oleic acid hydrophobic tail chains. Simultaneously, the rigid sorbitol ring of Span 80 and the flexible polyoxyethylene chain of Tween 80 form a dense and stable composite interfacial film, significantly enhancing the anti-agglomeration ability of the droplets, thereby achieving high uniformity in droplet size. The combination of ABIL EM 90 and Triton X-100 achieves rapid interfacial spreading and initial formation of ultrafine droplets through the ultra-flexible siloxane chain of ABIL EM 90. At the same time, the anchoring effect of the rigid benzene ring structure of Triton X-100 enhances the mechanical strength of the interfacial film, effectively suppressing Ostwald ripening. The two surfactants form a collaborative mechanism of "highly efficient emulsification and strong stability," jointly ensuring the uniformity and stability of the microsphere particle size.

[0034] More preferably, the volume ratio of the Span and Tween surfactants in the mixture is 1.5:1 to 1:1.5. This specific ratio range ensures that the two surfactants achieve optimal molecular arrangement density and synergistic effect at the oil-water interface. Taking SPAN 80 and TWEEN 80 as examples, within this range, the strong interfacial anchoring ability provided by SPAN 80 and the steric stabilization effect provided by TWEEN 80 achieve the best balance, forming a composite interfacial film with excellent strength and elasticity. This most effectively suppresses the aggregation of emulsion droplets, resulting in the narrowest particle size distribution and the highest uniformity of the final agargel microspheres. Beyond this ratio range, the performance of the interfacial film will decrease, leading to poorer microsphere particle size uniformity.

[0035] The volume ratio of ABIL EM 90 to Triton X-100 in the mixture of Triton X-100 and ABIL EM 90 is 45:1 to 5:1, preferably 40:1 to 10:1. ABIL EM 90, with its superspreadability and extremely low interfacial tension, acts as the primary emulsifier, efficiently forming fine initial droplets. A small amount of Triton X-100 acts as an auxiliary stabilizer; its rigid molecular structure enhances the interfacial film strength, effectively inhibiting droplet maturation and aggregation. The two work synergistically to achieve a balance between emulsification efficiency and long-term stability. The required proportion of ABIL EM 90 is significantly higher because, although its organosilicon structure provides excellent emulsification efficiency, the resulting interfacial film is too flexible, requiring Triton X-100 as an anchor point to provide rigid support. However, the latter's potential biotoxicity necessitates strict dosage control. Therefore, while ensuring stability, ABIL EM 90 still dominates to achieve a balance between efficient emulsification and biocompatibility.

[0036] Preferably, in step (3), the volume ratio of the aqueous phase to the oil phase is 1:6 to 1:14;

[0037] The stirring speed is 1000~3000 rpm;

[0038] The stirring process takes 3 to 8 minutes.

[0039] The curing is carried out under the following conditions: at a temperature of 0℃ to 4℃, stirring at a speed of 1250 to 2000 rpm for 30 seconds to 2 minutes;

[0040] Hexane is used as a solvent in the demulsification process.

[0041] More preferably, the demulsification is carried out in the following steps: adding n-hexane and centrifuging, repeating ≥5 times; wherein, the amount of n-hexane added each time is 0.5 to 2 times the total volume of the aqueous phase and the oil phase in step (3).

[0042] Preferably, the preparation method further includes washing after demulsification; the washing is performed using physiological saline; the washing is performed according to the following steps: adding physiological saline and centrifuging, repeating ≥3 times; wherein, the amount of physiological saline added each time is 0.5 to 2 times the total volume of the aqueous phase and the oil phase in step (3).

[0043] A second aspect of the present invention provides an agar gel microsphere prepared according to the above-described preparation method, wherein the agar gel microsphere has a structure in which microorganisms are embedded in agar, and its particle size ranges from 4.5 to 35.0 μm, and its particle size distribution width is 0.64 to 1.30, preferably 0.64 to 0.90.

[0044] A third aspect of the present invention provides a method for isolating microorganisms from environmental samples, the method comprising the following steps:

[0045] (1) The above agar gel microspheres were transferred to an in-situ water environment containing sterilized environmental samples and cultured under simulated environmental sample conditions;

[0046] (2) After culturing, the microorganisms are obtained by centrifugation and filtration.

[0047] In this disclosure, agarose gel microspheres of suitable and uniform particle size are placed in an in-situ water body containing sterilized environmental samples (or a liquid culture medium containing sterilized original microbial growth environment materials) for in-situ culture under simulated original environmental conditions. After a period of culture, the growth of microorganisms with in-situ environmental functions inside the agarose gel microspheres is not significantly different, and they can basically break through the agarose gel microspheres and enter the liquid environment simultaneously. Under centrifugation, non-functional microorganisms embedded in the denser agarose gel microspheres will form precipitates, achieving precise separation from functional microorganisms suspended in the liquid environment. The uniform particle size of the agarose microspheres has a consistent specific surface area, which can ensure the consistency of material transport and information transmission, create a unified microenvironment, and enable different bacteria to obtain the same growth starting point, laying the foundation for stable culture and precise screening of functional microorganisms.

[0048] Preferably, the conditions of the simulated environmental sample are as follows: temperature 4~40℃, shaker speed 50~150 rpm, and light intensity 1000~10000 lumens.

[0049] The beneficial effects of this invention are:

[0050] The method of this invention effectively solves the problems of excessively large particle size, wide distribution, complex preparation process, and long operation time of agarose gel microspheres prepared by traditional emulsification and solidification methods. Simultaneously, the method for culturing and applying microspheres in this invention enables high-throughput and efficient cultivation of environmental functional microorganisms, solving the challenge of in-situ functional verification.

[0051] The application method of this invention allows microbial single cells to be embedded in an agarose gel microsphere culture system, achieving co-culture of microspheres and in-situ samples. This co-culture mode not only facilitates the cultivation and functional verification of difficult-to-culture microorganisms but also fully leverages the utilization value of samples. It is particularly beneficial for samples that are difficult to obtain, have a small number of microorganisms, or are challenging to process, effectively improving the utilization efficiency of valuable samples. Attached Figure Description

[0052] Figure 1 This is a schematic flowchart of a method for preparing agarose gel microspheres according to an embodiment of the present invention.

[0053] Figure 2The diameter response surface plots of agarose gel microspheres prepared under the conditions of 0.8–1.5 wt% aqueous phase concentration, 45–65 °C emulsification stirring temperature, and SPAN 80-TWEEN 80 mixture as oil phase are shown.

[0054] Figure 3 The diagram shows the diameter response surface analysis of agarose gel microspheres under the conditions of 0.8–1.5 wt% aqueous phase concentration, 45–65 °C emulsification stirring temperature, and ABIL EM 90-Triton X-100 oil phase.

[0055] Figure 4 This is a microscopic image of the agarose gel microspheres prepared in Example 1.

[0056] Figure 5 The image shows the size distribution of the agar gel microspheres prepared in Example 1.

[0057] Figure 6 This is a microscopic image of the agarose gel microspheres prepared in Example 2.

[0058] Figure 7 This is a statistical diagram showing the size distribution of the agar gel microspheres prepared in Example 2.

[0059] Figure 8 This is a microscopic image of the agarose gel microspheres prepared in Example 3.

[0060] Figure 9 This is a statistical diagram showing the size distribution of the agar gel microspheres prepared in Example 3.

[0061] Figure 10 This is a microscopic image of the agarose gel microspheres prepared in Example 4.

[0062] Figure 11 This is a statistical diagram showing the size distribution of the agar gel microspheres prepared in Example 4.

[0063] Figure 12 This is a microscopic observation of the agarose gel microspheres of Example 11 before in situ culture under 20 mg / L tetracycline pressure.

[0064] Figure 13 This is a microscopic observation of the agar gel microspheres of Example 11 after in situ culture under 20 mg / L tetracycline pressure.

[0065] Figure 14 The heavy water-labeled Raman spectrum of the river water functional drug-resistant microorganisms isolated after culture in Example 11 under a pressure of 20 mg / L tetracycline.

[0066] Figure 15This is a statistical graph showing the labeling rate of functional drug-resistant microorganisms from river water isolated after culture in Example 11 at a pressure of 20 mg / L tetracycline.

[0067] Figure 16 This is a statistical diagram showing the size distribution of the agarose gel microspheres prepared in Example 5.

[0068] Figure 17 This is a microscopic image of the agarose gel microspheres prepared in Example 5.

[0069] Figure 18 This is a statistical diagram showing the size distribution of the agarose gel microspheres prepared in Example 6.

[0070] Figure 19 This is a microscopic image of the agarose gel microspheres prepared in Example 6.

[0071] Figure 20 The image shows the size distribution of the agarose gel microspheres prepared in Example 7.

[0072] Figure 21 This is a microscopic image of the agarose gel microspheres prepared in Example 7.

[0073] Figure 22 The image shows the size distribution of the agarose gel microspheres prepared in Example 8.

[0074] Figure 23 This is a microscopic image of the agarose gel microspheres prepared in Example 8.

[0075] Figure 24 This is a microscopic image of the agarose gel microspheres prepared in Comparative Example 4.

[0076] Figure 25 The size distribution of the agarose gel microspheres prepared for Comparative Example 2 is shown in the statistical diagram.

[0077] Figure 26 This is a microscopic image of the agarose gel microspheres prepared in Comparative Example 2.

[0078] Figure 27 The size distribution of the agarose gel microspheres prepared for Comparative Example 3 is shown in the statistical diagram.

[0079] Figure 28 This is a microscopic image of the agarose gel microspheres prepared in Comparative Example 3. Detailed Implementation

[0080] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0081] The method for preparing agarose gel microspheres disclosed herein solves the problems of large sphere size, uneven particle size, and cumbersome technical operations in the preparation process of emulsification and solidification methods. Figure 1 The diagram illustrates an exemplary method for preparing agarose gel microspheres according to the present invention.

[0082] In one embodiment of the present invention, the bacterial suspension is prepared as follows: Microorganisms in an environmental sample are separated, washed, and resuspended in sterile water to obtain a bacterial suspension. The sources of the environmental samples include, but are not limited to, natural water bodies such as rivers and seawater, soil, atmospheric samples, activated sludge, or laboratory-cultured bacterial solutions. For the separation and washing method of the environmental samples, a 20-50 μm filter membrane can be used to filter the environmental samples to remove large particulate impurities. Sterile water with a volume of 5-10 times the filtrate volume is added to the filtrate, and the sample is centrifuged at 2000-5000 rpm for 3-10 minutes. The supernatant is removed, and the steps of adding sterile water and centrifuging are repeated at least three times. Finally, the centrifuged bacterial precipitate is resuspended in sterile water to obtain the bacterial suspension. The concentration of microorganisms in the bacterial suspension is controlled at 10... 5 ~10 7 The cell / mL concentration is favorable for subsequent microbial encapsulation. Sterile water is obtained by conventionally autoclaving deionized water, using a steam sterilizer at 103.4 kPa and 121.3°C for 15–30 minutes. For atmospheric samples containing microorganisms, a feasible sampling method is to use an "FA-5 liquid impact microbial aerosol sampler" to continuously collect samples at a flow rate of 7–15 L / min for 12–24 hours. After collecting atmospheric microorganisms into the water, the samples are then filtered to remove impurities as described above.

[0083] In one embodiment of the present invention, the agar aqueous solution is prepared as follows: Agar and sterile water are mixed and heated to melt, yielding an agar aqueous solution with a concentration of 1.0~4.0 wt%. As an example, 0.5~2 g of agar is taken and melted in 50 mL of sterile water to obtain an agar aqueous solution. The melting temperature is 80~100℃, and the melting time is 5~10 minutes. The agar of the present invention is a natural high-molecular-weight polysaccharide extracted from seaweed. Its chemical composition mainly includes neutral agarose composed of D-galactose and 3,6-lacto-L-galactose, and acidic sulfur agar with negatively charged groups such as sulfate and carboxyl groups. This natural polymer possesses unique physicochemical properties, exhibiting not only good coagulation and stability but also the ability to form stable complexes with various substances. As a carrier material for bacterial microbial immobilization, agar has significant advantages. First, the hydrophilic groups in its molecular structure endow it with excellent hydration capabilities, enabling the construction of a suitable microenvironment for microbial survival. Furthermore, the natural polysaccharide backbone possesses excellent biocompatibility, minimizing its impact on bacterial activity. Second, the pore structure of the agar gel network is tunable, allowing the formation of microsphere carriers with varying pore sizes, providing ample space for bacterial colonization and metabolism. In addition, the negatively charged groups on the agar molecular chains enhance the binding force with bacteria through electrostatic interactions, while the increased mechanical strength after chemical cross-linking prevents structural damage during immobilization, ensuring bacterial stability and reusability in application. This invention utilizes agar as a carrier material for immobilizing bacterial microorganisms, combining biocompatibility, structural controllability, and mechanical stability.

[0084] In one embodiment of the present invention, the aqueous phase is prepared as follows: An agar aqueous solution and a bacterial suspension are mixed at a volume ratio of 1:1.5 to 1.5:1, and preheated once at a temperature of 45 to 75°C to obtain the aqueous phase. The preheating time is 15 to 20 minutes. The volume ratio of the agar aqueous solution to the bacterial suspension is set to 1:1.5 to 1.5:1 to ensure that the concentration of agar in the diluted aqueous phase is 0.5 to 2.0 wt%. This preheating can be performed using a constant temperature method, such as a water bath, and the constant temperature of the prepared aqueous phase is 45 to 75°C.

[0085] In one embodiment of the present invention, the oil phase is prepared as follows: Two or more surfactants are added to an oily solvent, followed by mixing under ultrasonic treatment (e.g., ultrasonic treatment frequency of 20-100 kHz, power of 100-500 W, time of 15-30 minutes), and then preheated a second time at a temperature of 45-75°C for 15-20 minutes to obtain the oil phase. The oily solvent is preferably selected from at least one of mineral oils (such as paraffin oil, petrolatum, white mineral oil, kerosene), vegetable oils (olive oil, soybean oil, sunflower oil), synthetic oils (such as fatty acid esters, polyolefin oils), silicone oils (such as dimethyl silicone oil, cyclomethyl silicone oil), and animal fats (such as lanolin, beeswax), preferably mineral oil, and more preferably paraffin oil. The total concentration of surfactants in the oil phase is preferably controlled to be 1.0% to 4.0% (volume concentration). For example, 300-600 μL of the ultrasonically mixed oil phase is dispensed into centrifuge tubes for a second preheating. The surfactant is selected from at least two of the Span series (e.g., SPAN 60, SPAN 85), Tween series (e.g., TWEEN 60, TWEEN 85), Triton X-100, and ABIL EM 90. Preferably, the surfactant used is a mixture of Span series surfactants and Tween series surfactants, or a mixture of Triton X-100 and ABIL EM 90. More preferably, the surfactant used is a mixture of two or more of SPAN 80, TWEEN 80, Triton X-100, and ABIL EM 90. Most preferably, a composite surfactant is used: ① a mixture of Span series surfactants and Tween series surfactants (e.g., SPAN 80 and TWEEN 80) at a volume ratio of 1.5:1 to 1:1.5; ② a mixture of ABIL EM 90 and Triton X-100 at a volume ratio of 45:1 to 5:1, preferably 40:1 to 1:1.

[0086] In one embodiment of the present invention, the microspheres are formed as follows: A preheated aqueous phase is added to a preheated oil phase (e.g., a centrifuge tube containing 300-600 μL of oil phase), and stirred for a certain time in a bath at 45-75°C to form water-in-oil (W / O) emulsion droplets. The droplets are then placed in a 0-4°C environment (e.g., a 0°C ice-water mixture) and stirred for 30 seconds to 2 minutes to cool and solidify, forming agarose gel microspheres. The aqueous phase and oil phase are mixed at a volume ratio of 1:6 to 1:14. During the stirring process to form the W / O emulsion droplets, magnetic stirring can be used at a speed of 1000-3000 rpm for 3-8 minutes. During solidification, the stirring speed is 1250-2000 rpm.

[0087] In one embodiment of the present invention, the agarose gel microspheres are demulsified as follows: Hexane is added to the mixed solution containing the agarose gel microspheres for demulsification. The mixture is stirred at 500-2000 rpm for 50-150 s, then centrifuged at 500-2000×g for 30-60 seconds. The upper emulsion is discarded by pipetting from top to bottom, retaining the lower microsphere solid precipitate. The volume of hexane added is 0.5-2 times the total volume of the aqueous and oil phases. This demulsification step is repeated at least 5 times. In another embodiment of the present invention, the agarose gel microspheres are washed as follows. Add a certain amount of physiological saline to the precipitate of demulsified agar gel microspheres, stir at 500-2000 rpm for 50-150 seconds, then centrifuge at 500-2000×g for 5-10 minutes. The liquid will separate into layers. Use a pipette to remove the upper organic layer and the lower water layer from top to bottom, retaining the bottom precipitate. The amount of physiological saline added should be 0.5-2 times the total volume of the aqueous and oil phases. Repeat the above washing steps at least 3 times until the supernatant is clear. For example, add 0.5-2 mL of physiological saline to a centrifuge tube containing the agar microsphere precipitate, open the tube and ventilate in a sterile environment for 5-15 minutes until the hexane in the centrifuge tube has completely evaporated. Repeat the above washing process at least 3 times until there is no obvious organic odor. The physiological saline is a 0.85% NaCl aqueous solution. The hexane washing should be repeated at least 3 times. If the supernatant is still turbid after centrifugation, increase the number of physiological saline washings until the supernatant is clear.

[0088] In one embodiment of the present invention, agarose gel microspheres are used to isolate and culture environmental functional microorganisms. Specifically, the method for isolating and culturing environmental functional microorganisms using agarose gel microspheres includes in-situ culture and centrifugation analysis.

[0089] In one embodiment of the present invention, in situ culture includes: adding 5-50 mL of sterilized water initially isolated from the environmental sample to a sterile culture tube to provide the basic nutrients required for in situ growth of microorganisms; furthermore, functional substances can be selectively added according to the type of target functional microorganisms. Agar gel microspheres generated at the bottom are transferred to a freshly prepared in situ culture environment using a pipette, and cultured under certain environmental conditions to allow the functional microorganisms inside the microspheres to proliferate, overflow from the microcapsules, and accumulate and grow in a free liquid environment. Preferably, the sterilization method for the water from which the environmental sample was isolated is filtration sterilization, i.e., using a 0.22 μm filter membrane to filter the liquid in the environmental sample to remove all bacteria from the liquid. The purpose of adding the functional substances is to achieve targeted screening of functional microorganisms. By controlling the selective pressure of the culture environment, microbial communities with specific environmental functions can be precisely enriched, including but not limited to the following methods: for screening resistant functional microorganisms: adding antibiotics (such as tetracycline 10-50 mg / L) or heavy metal ions (such as Cd). 2+ (5~100 mg / L)

[0090] Screening for microorganisms with pollutant degradation capabilities: The target pollutant (e.g., phenol 50-500 mg / L) was added as the sole carbon source; all added substances were sterilized using a 0.22 μm filter membrane, and the concentration was determined through preliminary experiments. The specific environmental conditions were identical to those of the environmental samples, such as suitable culture conditions for the river water microorganisms: temperature 4-20℃, shaker speed 50-150 rpm, and light intensity within the range of 1,000-10,000 lumens.

[0091] In one embodiment of the present invention, centrifugation includes: centrifuging the in-situ culture environment after cultivation under low-speed gravity centrifugation conditions; using a pipette to aspirate the supernatant bacterial solution and filtering it through a 20-50 μm filter membrane to remove broken microsphere residues and other microspheres containing non-functional microorganisms, while retaining the filtered bacterial solution containing the target functional microorganisms. The low-speed gravity centrifugation conditions are 500-2000 rpm for 3-5 minutes.

[0092] Performance testing:

[0093] The particle size distribution range / cumulative particle size distribution of agarose gel microspheres was tested using optical microscopy image analysis and statistical methods.

[0094] The microstructure of agarose gel microspheres was observed and photographed using an optical microscope.

[0095] The particle size distribution width of agar gel microspheres is calculated using the following formula:

[0096] Particle size distribution width value = (D 90 -D 10 ) / D 50 ,

[0097] in:

[0098] -D 10 This represents the particle size (μm) corresponding to 10% of the particles in the cumulative particle size distribution.

[0099] -D 50 This represents the particle size (μm) corresponding to 50% of the particles in the cumulative particle size distribution.

[0100] -D 90 This represents the particle size (μm) corresponding to 90% of the particles in the cumulative particle size distribution.

[0101] Example 1:

[0102] Prepare agar gel microspheres containing river water microorganisms at an agar concentration of 0.8 wt% using the following steps:

[0103] (1) Preparation of the aqueous phase:

[0104] River water samples were collected from the Yitong River (43.8819°W, 125.3518°N) in Changchun City, Jilin Province in October 2024. After obtaining the river water samples using a sampler, they were immediately placed in sterile sealed bags and transported and stored in the dark at 4°C.

[0105] Take 10 mL of river water and filter it using a 50 μm filter membrane. Add 50 mL of sterile water to the filtrate and centrifuge at 4000 rpm for 5 minutes. Remove the supernatant. Repeat the above steps of adding sterile water and centrifuging three times. Finally, resuspend the centrifuged bacterial pellet in sterile water to obtain a bacterial suspension. Dilute the sample according to the counting results to obtain a concentration of 10. 7 Cells / mL bacterial suspension;

[0106] Take 0.8 g of agar powder into an Erlenmeyer flask, add 50 mL of sterile water, seal the flask with sealing film, and heat it in an electric furnace until the agar is completely melted to form an agar aqueous solution. Mix 1 mL of bacterial suspension with 1 mL of agar aqueous solution at a volume ratio of 1:1, and place it in a 65℃ water bath for 15 minutes to prepare an aqueous phase containing microorganisms with an agar concentration of 0.8 wt%.

[0107] (2) Preparation of the oil phase:

[0108] Add 1.0 mL of SPAN 80 and 1.0 mL of TWEEN 80 to paraffin oil (CAS No.: 8020-83-5), then bring the volume to 50 mL with mineral oil. Sonicate the mixture at 50 kHz and 200 W for 30 minutes to obtain the oil phase. Aliquot 450 μL of the mixture into 2 mL centrifuge tubes and preheat the tubes in a 65°C water bath for 15 minutes to obtain the oil phase.

[0109] (3) Formation of agarose gel microspheres:

[0110] Add 50 μL of the aqueous phase to a centrifuge tube containing the oil phase, and add a 4×8 mm type A magnetic stir bar. Stir at 1500 rpm for 5 minutes in a 65°C water bath. Then place the centrifuge tube in an ice-water mixture and stir at 1500 rpm for 1 minute to cool and solidify.

[0111] (4) Demulsification of agarose gel microspheres:

[0112] Add 500 μL of n-hexane to the centrifuge tube after step (3), stir at 1000 rpm for 150 seconds, then centrifuge at 1000×g for 30 seconds. Use a pipette to remove the upper emulsion from top to bottom, keeping the lower microsphere solid precipitate. Repeat this step 5 times.

[0113] (5) Washing of agarose gel microspheres:

[0114] Add 500 μL of physiological saline to the microsphere precipitate that has been demulsified with hexane, stir at 1000 rpm for 150 seconds, and then centrifuge at 500×g for 10 minutes. The liquid will separate into layers. Use a pipette to aspirate from top to bottom, discard the upper organic layer and the lower water layer, and keep the bottom precipitate. Repeat this step 3 times. Figure 4 Meso-microstructure diagram and Figure 5 The particle size distribution curves show that the agar gel microspheres prepared in Example 1 have a particle size range of 4.58 to 11.01 μm, and the calculated particle size distribution width is 0.87.

[0115] Example 2:

[0116] River microorganism-encapsulated agar gel microspheres with an agar concentration of 0.8 wt% were prepared according to Example 1, with the only difference being the preparation of the oil phase in step (2): 1.0 mL of ABIL EM 90 and 25 μL of Triton X-100 were added to mineral oil, and then the volume was adjusted to 50 mL with mineral oil. The mixture was sonicated at 50 kHz and 200 W for 30 minutes to obtain a mixture. 450 μL of the mixture was dispensed into 2 mL centrifuge tubes, and the centrifuge tubes were preheated in a 65℃ water bath for 15 minutes to obtain the oil phase.

[0117] Figure 6 Meso-microstructure diagram and Figure 7 The particle size distribution curves show that the agar gel microspheres prepared in Example 2 have a particle size range of 10.02 to 23.31 μm, and the calculated particle size distribution width is 0.84.

[0118] Example 3:

[0119] The preparation of river water microorganism-encapsulated agar gel microspheres with an agar concentration of 1.5 wt% was carried out in accordance with Example 1. The only difference was in the preparation of the aqueous phase in step (1): 1.5 g of agar powder was placed in an Erlenmeyer flask, 50 mL of sterile water was added and the flask was sealed with a sealing film, and the agar was heated in an electric furnace to completely melt the agar to form an agar aqueous solution. The above bacterial suspension (1 mL) and the agar aqueous solution (1 mL) were mixed at a volume ratio of 1:1 and placed in a 65°C water bath for constant temperature preheating for 15 minutes to prepare an aqueous phase containing microorganisms with an agar concentration of 1.5 wt%.

[0120] Figure 8 Meso-microstructure diagram and Figure 9 The particle size distribution curves show that the agar gel microspheres prepared in Example 3 have a particle size range of 8.77 to 23.72 μm, and the calculated particle size distribution width is 1.08.

[0121] Example 4:

[0122] River water microorganism-encapsulated agar gel microspheres with an agar concentration of 0.8 wt% were prepared according to Example 1, with the only difference being:

[0123] (1) Preparation of the aqueous phase:

[0124] Take 0.8 g of agar powder into an Erlenmeyer flask, add 50 mL of sterile water and seal with sealing film. Heat in an electric furnace to completely melt the agar to form an agar aqueous solution. Mix 1 mL of bacterial suspension with 1 mL of agar aqueous solution at a volume ratio of 1:1 and place it in a 45℃ water bath for 15 minutes to prepare an aqueous phase containing microorganisms with an agar concentration of 0.8 wt%.

[0125] (2) Preparation of the oil phase:

[0126] Add 1.0 mL of SPAN 80 and 1.0 mL of TWEEN 80 to the mineral oil, then bring the volume to 50 mL with mineral oil. Sonicate the mixture at 50 kHz and 200 W for 30 minutes to obtain the oil phase. Aliquot 450 μL of the mixture into 2 mL centrifuge tubes and preheat the tubes in a 45°C water bath for 15 minutes to obtain the oil phase.

[0127] (3) Formation of agarose gel microspheres:

[0128] Add 50 μL of the aqueous phase to a centrifuge tube containing the oil phase, and add a 4×8 mm type A magnetic stir bar. Stir at 1500 rpm for 5 minutes in a 45°C water bath. Then place the centrifuge tube in an ice-water mixture and stir at 1500 rpm for 1 minute to cool and solidify.

[0129] Figure 10 Meso-microstructure diagram and Figure 11 The particle size distribution curves show that the agar gel microspheres prepared in Example 4 have a particle size range of 6.05 to 11.72 μm and a particle size distribution width of 0.65.

[0130] Example 5:

[0131] River water microorganism-encapsulated agar gel microspheres with an agar concentration of 0.8 wt% were prepared according to Example 1, with the only difference being:

[0132] (1) Preparation of the aqueous phase:

[0133] Take 0.8 g of agar powder into an Erlenmeyer flask, add 50 mL of sterile water, seal the flask with sealing film, and heat it in an electric furnace until the agar is completely melted to form an agar aqueous solution. Mix 1 mL of bacterial suspension with 1 mL of agar aqueous solution at a volume ratio of 1:1, and place it in a 60℃ water bath for 15 minutes to prepare an aqueous phase containing microorganisms with an agar concentration of 0.8 wt%.

[0134] (2) Preparation of the oil phase:

[0135] Add 1.0 mL of ABIL EM 90 and 25 μL of Triton X-100 to mineral oil, then bring the volume to 50 mL with mineral oil. Sonicate at 50 kHz and 200 W for 30 minutes to prepare a mixture. Aliquot 450 μL of the mixture into 2 mL centrifuge tubes and preheat the tubes in a 60°C water bath for 15 minutes to obtain the oil phase.

[0136] (3) Formation of agarose gel microspheres:

[0137] Add 50 μL of the aqueous phase to a centrifuge tube containing the oil phase, and simultaneously add a 4×8 mm type A magnetic stir bar. Stir at 1500 rpm for 5 minutes in a 60℃ water bath. Then place the centrifuge tube in an ice-water mixture and stir at 1500 rpm for 1 minute to cool and solidify. The size distribution of the resulting agarose gel microspheres is shown in the figure below. Figure 16 As shown in the image. Microscopic observation of agarose gel microspheres, as shown. Figure 17 As shown.

[0138] Example 6:

[0139] River water microorganism-encapsulated agar gel microspheres with an agar concentration of 0.8 wt% were prepared according to Example 2, with the only difference being:

[0140] (1) Preparation of the aqueous phase:

[0141] Take 0.8 g of agar powder into an Erlenmeyer flask, add 50 mL of sterile water, seal the flask with sealing film, and heat it in an electric furnace until the agar is completely melted to form an agar aqueous solution. Mix 1 mL of bacterial suspension with 1 mL of agar aqueous solution at a volume ratio of 1:1, and place the mixture in a 45°C water bath for 15 minutes to prepare an aqueous phase containing microorganisms with an agar concentration of 0.8 wt%.

[0142] (2) Preparation of the oil phase:

[0143] Add 1.0 mL of ABIL EM 90 and 25 μL of Triton X-100 to mineral oil, then bring the volume to 50 mL with mineral oil. Sonicate the mixture at 50 kHz and 200 W for 30 minutes to obtain the oil phase. Aliquot 450 μL of the mixture into 2 mL centrifuge tubes and preheat the tubes in a 45°C water bath for 15 minutes to obtain the oil phase.

[0144] (3) Formation of agarose gel microspheres:

[0145] Add 50 μL of the aqueous phase to a centrifuge tube containing the oil phase, and simultaneously add a 4×8 mm type A magnetic stir bar. Stir at 1500 rpm for 5 minutes in a 45°C water bath. Then place the centrifuge tube in an ice-water mixture and stir at 1500 rpm for 1 minute to cool and solidify. The size distribution of the resulting agarose gel microspheres is shown in the figure below. Figure 18 As shown in the image. Microscopic observation of agarose gel microspheres, as shown. Figure 19 As shown.

[0146] Example 7:

[0147] River microorganism-embedded agar gel microspheres with an agar concentration of 0.8 wt% were prepared according to Example 2, with the only difference being: the preparation of the oil phase in step (2): 1.0 mL of ABIL EM 90 and 50 μL of Triton X-100 were added to mineral oil, and then the volume was adjusted to 50 mL with mineral oil. The mixture was sonicated at 50 kHz and 200 W for 30 minutes to obtain a mixture. 450 μL of the mixture was dispensed into 2 mL centrifuge tubes, and the centrifuge tubes were placed in a 65℃ water bath for 15 minutes to obtain the oil phase. The size distribution of the obtained agar gel microspheres is shown in the figure below. Figure 20 As shown in the image. A microscopic observation of the obtained agarose gel microspheres is shown below. Figure 21 As shown.

[0148] Example 8:

[0149] River microorganism-embedded agar gel microspheres with an agar concentration of 0.8 wt% were prepared according to Example 2, with the only difference being: the preparation of the oil phase in step (2): 1.0 mL of ABIL EM 90 and 100 μL of Triton X-100 were added to mineral oil, and then the volume was adjusted to 50 mL with mineral oil. The mixture was sonicated at 50 kHz and 200 W for 30 minutes to obtain a mixture. 450 μL of the mixture was dispensed into 2 mL centrifuge tubes, and the centrifuge tubes were placed in a 65℃ water bath for 15 minutes to obtain the oil phase. The size distribution of the obtained agar gel microspheres is shown in the following figure. Figure 22 As shown in the image. A microscopic observation of the obtained agarose gel microspheres is shown below. Figure 23 As shown.

[0150] Comparative Example 1

[0151] River water microorganism-encapsulated agar gel microspheres with an agar concentration of 0.8 wt% were prepared according to Example 2, with the only difference being:

[0152] (1) Preparation of the aqueous phase:

[0153] Take 0.8 g of agar powder into an Erlenmeyer flask, add 50 mL of sterile water, seal the flask with sealing film, and heat it in an electric furnace until the agar is completely melted to form an agar aqueous solution. Mix 1 mL of bacterial suspension with 1 mL of agar aqueous solution at a volume ratio of 1:1, and place the mixture in a 30°C water bath for 15 minutes to prepare an aqueous phase containing microorganisms with an agar concentration of 0.8 wt%.

[0154] (2) Preparation of the oil phase:

[0155] Add 1.0 mL of ABIL EM 90 and 25 μL of Triton X-100 to mineral oil, then bring the volume to 50 mL with mineral oil. Sonicate the mixture at 50 kHz and 200 W for 30 minutes to obtain the oil phase. Aliquot 450 μL of the mixture into 2 mL centrifuge tubes and preheat the tubes in a 30°C water bath for 15 minutes to obtain the oil phase.

[0156] (3) Formation of agarose gel microspheres:

[0157] Add 50 μL of the aqueous phase to a centrifuge tube containing the oil phase, and add a 4×8 mm type A magnetic stir bar. Stir at 1500 rpm for 5 minutes in a 30°C water bath. Then place the centrifuge tube in an ice-water mixture and stir at 1500 rpm for 1 minute to cool and solidify.

[0158] Comparative Example 2

[0159] River microorganism-embedded agar gel microspheres with an agar concentration of 0.8 wt% were prepared according to Example 2, with the only difference being: the preparation of the oil phase in step (2): 1.025 mL of Triton X-100 was added to mineral oil, and then the volume was adjusted to 50 mL with mineral oil. The mixture was sonicated at 50 kHz and 200 W for 30 minutes to prepare a mixture. 450 μL of the mixture was dispensed into 2 mL centrifuge tubes, and the centrifuge tubes were placed in a 65℃ water bath for 15 minutes to obtain the oil phase. The size distribution of the obtained agar gel microspheres is shown in the figure below. Figure 25 As shown in the image. A microscopic observation of the obtained agarose gel microspheres is shown below. Figure 26 As shown.

[0160] Comparative Example 3

[0161] River microorganism-encapsulated agar gel microspheres with an agar concentration of 0.8 wt% were prepared according to Example 2, with the only difference being the preparation of the oil phase in step (2): 1.025 mL of ABIL EM 90 was added to mineral oil, and then the volume was adjusted to 50 mL with mineral oil. The mixture was sonicated at 50 kHz and 200 W for 30 minutes to obtain a mixture. 450 μL of the mixture was dispensed into 2 mL centrifuge tubes, and the centrifuge tubes were placed in a 65℃ water bath for 15 minutes to obtain the oil phase. The size distribution of the obtained agar gel microspheres is shown in the following figure. Figure 27 As shown in the image. A microscopic observation of the obtained agarose gel microspheres is shown below. Figure 28 As shown.

[0162] Comparative Example 4:

[0163] River microorganism-encapsulated agar gel microspheres with an agar concentration of 0.8 wt% were prepared according to Example 2, with the only difference being: the preparation of the oil phase in step (2): 1.0 mL of ABIL EM 90 and 500 μL of Triton X-100 were added to mineral oil, and then the volume was adjusted to 50 mL with mineral oil. The mixture was sonicated at 50 kHz and 200 W for 30 minutes to obtain a mixture. 450 μL of the mixture was dispensed into 2 mL centrifuge tubes, and the centrifuge tubes were placed in a 65℃ water bath for 15 minutes to obtain the oil phase. The resulting agar gel microspheres are shown in the microscopic observation image. Figure 24 As shown.

[0164] Table 1 shows the preparation process and performance parameters of the agar gel microspheres obtained in Examples 1-8 and Comparative Examples 1-4:

[0165]

[0166] Comparing Comparative Example 1, Example 6, Example 5, and Example 1, it can be seen that as the water bath temperature gradually increases between 45 and 65°C, the particle size of the resulting agar gel microspheres increases sequentially, and the particle size distribution width gradually widens. However, in Comparative Example 1, due to its excessively low water bath temperature, agar solidification occurred during the aqueous phase preparation step, making it impossible to obtain regularly shaped agar microspheres. Considering that water bath temperatures exceeding 65°C begin to affect microbial activity, and exceeding 75°C can even lead to mass microbial death, this invention controls the water bath temperature between 45 and 75°C, preferably between 45 and 65°C.

[0167] Comparing Examples 2, 7, 8, and 4, it is evident that as the volume ratio of ABIL EM 90 to Triton X-10 in the surfactant changes, the particle size of the resulting agar gel microspheres gradually decreases, while the particle size distribution width gradually increases. Example 2 is the most suitable for microbial culture. (See also...) Figure 24In Comparative Example 4, the particle size was too small, and the observation accuracy through an optical microscope was no longer sufficient for detection.

[0168] Comparing Comparative Example 2, Comparative Example 3 and Example 2, it can be seen that Example 2, which adds two surfactants, can form a composite interfacial film with better performance through the synergistic effect between the components (the former optimizes the interfacial molecular arrangement, balances anchoring and steric hindrance, and the latter performs efficient emulsification and enhances film strength). This effectively inhibits emulsion droplet aggregation and Ostwald curing, and improves the uniformity and stability of microsphere particle size.

[0169] This invention first performs Gaussian curve fitting based on the distribution diagram under each condition to obtain the highest value of the normal distribution. Then, using the highest value of the Gaussian distribution under each condition as the average diameter under that condition, surface fitting is performed. The optimal range for the average diameter under two surfactants, agar concentration, and melting temperature is examined. Figures 2-3 The figures shown are fitted with surfactants SPAN 80 and TWEEN 80 respectively. Figure 2 ) and ABIL EM 90-Triton X-100 ( Figure 3 The response surface methodology for preparing microspheres using the oil phase under the conditions of 0.8–1.5 wt% aqueous agar concentration and 45–65 °C emulsification stirring temperature in this technical step showed that the optimal average diameter of microspheres prepared by the oil phase containing SPAN 80 (1% by volume) and TWEEN 80 (1% by volume) surfactants was 6.84–15.28 μm, which is basically consistent with Example 1 of this invention. The optimal average diameter of microspheres prepared by the oil phase containing Triton X-100 (0.05% by volume) and ABIL EM 90 (1% by volume) surfactants was 16.05–24.85 μm, which is basically consistent with Example 2 of this invention.

[0170] Example 11:

[0171] The agar gel microspheres prepared in Example 2 were used to perform in situ culture and isolation of tetracycline-resistant microorganisms from river water microorganisms, including the following steps:

[0172] (1) In situ culture:

[0173] A 40 mL river water sample was filtered through a 0.22 μm filter membrane and added to a sterile culture tube as an in situ culture environment. Tetracycline was added at a final concentration of 20 mg / L. The agarose gel microspheres prepared in Example 2 were transferred to the freshly prepared in situ culture environment using a pipette and cultured for 5 days at 15°C, 100 rpm, and 5000 lumens of light.

[0174] (2) Centrifugal separation:

[0175] After in situ culture, the culture environment was centrifuged at 2000 rpm for 3 minutes. The upper bacterial culture was aspirated with a pipette and filtered through a 20 μm filter membrane. The filtered bacterial culture contained tetracycline-resistant functional microorganisms.

[0176] Figure 12 and Figure 13 The changes in morphology and microbial growth of agar gel microspheres before and after culture are shown under a conventional optical microscope. Figure 12 This technology demonstrates its ability to independently and completely encapsulate and immobilize river microorganisms within agarose gel microspheres. Figure 13 The results show that after culturing the microspheres for 5 days, microorganisms with tetracycline resistance were broken up and grew into the liquid environment (right), while non-tetracycline resistant microorganisms remained in the microspheres (left). Gravity centrifugation can achieve precise separation of functional microorganisms in samples from complex environments.

[0177] To verify that all bacteria in the retained filtrate of Example 11 possessed tetracycline resistance, a heavy water-Raman spectroscopy resistance verification experiment was conducted. The filtrate from Example 11 was added to an in-situ environment containing 20 mg / L tetracycline and 30 vol% heavy water for in-situ culture and labeling for 12 hours. The Raman spectra of the labeled bacteria were measured using a Raman spectrometer. The results showed that almost all bacteria obtained in Example 11 exhibited heavy water labeling peaks (2040~2300 cm⁻¹) at 20 mg / L tetracycline concentration. -1 This proves that these bacteria possess tetracycline resistance. Figure 14 Raman spectroscopy identification of 1831 cultured microorganisms showed a heavy water labeling rate greater than 96% (1776 cells were labeled). Figure 15 This study verified that the method can achieve high-throughput culture and accurate isolation of tetracycline-resistant microorganisms in river water.

Claims

1. A method for preparing agarose gel microspheres, characterized in that, The preparation method includes the following steps: (1) Mix the bacterial suspension and agar solution, and preheat once to obtain the aqueous phase; (2) The oily solvent and the surfactant are mixed and preheated twice to obtain the oil phase. The surfactant is selected from at least two of the following: Span series surfactants, Tween series surfactants, Triton X-100 and ABIL EM 90. (3) Add the aqueous phase to the oil phase, stir at 45~75℃, and then solidify and demulsify to obtain the agar gel microspheres; the agar gel microspheres have a structure in which microorganisms are embedded in agar, and their particle size ranges from 4.5 to 35.0 μm, and the particle size distribution width is 0.64~1.

30.

2. The preparation method according to claim 1, characterized in that, In step (1), The concentration of agar in the aqueous phase is 0.5~2.0 wt%; The concentration of microorganisms in the bacterial suspension is cells / ml; The concentration of agar in the agar aqueous solution is 1.0~4.0 wt%; The volume ratio of the agar aqueous solution to the bacterial suspension is 1:1.5 to 1.5:1; The temperature of the first preheating is 45~75℃; The preheating time is 15-20 minutes.

3. The preparation method according to claim 1, characterized in that, The bacterial suspension is prepared by the following steps: i) First, filter the environmental sample through a 20-50 μm filter membrane to remove impurities and obtain the filtrate. The environmental sample includes natural water bodies, soil, air, activated sludge, or laboratory cultured bacterial solution. ii) Add 5 to 10 times the volume of sterile water to the filtrate and centrifuge. iii) Repeat step ii) at least 3 times to obtain a bacterial suspension.

4. The preparation method according to claim 1, characterized in that, In step (2), The oily solvent is selected from at least one or a combination of mineral oil, vegetable oil, synthetic fats, silicone oil and animal fats; The total concentration of surfactants in the oil phase is 1.0~4.0 vol% The mixing is carried out under ultrasonic treatment, the power of which is 100~500 W and the time is 15~30 minutes. The temperature of the secondary preheating is 45~75℃; The secondary preheating time is 15-20 minutes.

5. The preparation method according to claim 4, characterized in that, The mineral oil is selected from at least one or a combination of paraffin oil, petrolatum, white mineral oil, and kerosene. The vegetable oil is selected from at least one or a combination of olive oil, soybean oil, and sunflower seed oil. The synthetic oil contains at least one of fatty acid esters and polyolefin oils; The silicone oil contains at least one of dimethyl silicone oil and cyclomethyl silicone oil; The animal fats include at least one of lanolin and beeswax.

6. The preparation method according to claim 1, characterized in that, In step (2), the Span series surfactants include at least one of SPAN 60 and SPAN 85; The Tween series surfactants include at least one of TWEEN 60 and TWEEN 85.

7. The preparation method according to claim 6, characterized in that, The surfactant is selected from: a mixture of Span series surfactants and Tween series surfactants or a mixture of Triton X-100 and ABIL EM 90.

8. The preparation method according to claim 7, characterized in that, The volume ratio of Span surfactant to Tween surfactant in the mixture is 1.5:1 to 1:1.

5. The volume ratio of ABIL EM 90 to Triton X-100 in the mixture of Triton X-100 and ABIL EM 90 is 40:1 to 1:

1.

9. The preparation method according to claim 1, characterized in that, In step (3), The volume ratio of the aqueous phase to the oil phase is 1:6 to 1:14; The stirring speed is 1000~3000 rpm; The stirring process takes 3 to 8 minutes. The curing is carried out under the following conditions: at a temperature of 0℃ to 4℃, stirring at a speed of 1250 to 2000 rpm for 30 seconds to 2 minutes; Hexane is used as a solvent in the demulsification process.

10. The preparation method according to claim 9, characterized in that, The demulsification is carried out in the following steps: adding n-hexane and centrifuging, repeating ≥5 times; wherein, the amount of n-hexane added each time is 0.5 to 2 times the total volume of the aqueous phase and the oil phase in step (3).

11. The preparation method according to claim 1, characterized in that, The preparation method further includes washing after demulsification; the washing is performed using physiological saline; the washing is performed according to the following steps: adding physiological saline and centrifuging, repeating ≥3 times; wherein, the amount of physiological saline added each time is 0.5 to 2 times the total volume of the aqueous phase and the oil phase in step (3).

12. The agar gel microspheres prepared by the preparation method according to any one of claims 1-11, characterized in that, The agar gel microspheres have a structure in which microorganisms are embedded in agar, and their particle size ranges from 4.5 to 35.0 μm, with a particle size distribution width of 0.64 to 1.

30.

13. A method for isolating microorganisms from environmental samples, characterized in that, The method includes the following steps: (1) The agar gel microspheres of claim 12 are transferred to an in-situ water culture environment containing sterilized environmental samples and cultured under simulated environmental sample conditions; (2) After culturing, the microorganisms are obtained by centrifugation and filtration.

14. The method according to claim 13, characterized in that, The conditions for the simulated environmental sample were as follows: temperature 4~40℃, shaker speed 50~150 rpm, and light intensity 1000~10000 lumens.

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