Air microorganism green capture membrane material and preparation method and application thereof

Abstract

The present invention discloses a green capture membrane material for air microorganisms and its preparation method and application, belonging to the technical field of filtration membrane materials. The technical scheme is as follows: the green capture membrane material has a honeycomb porous cross-linked interconnected network structure, and its particle retention rate is 99.3%. The capture amount and survival rate of air microorganisms can reach 3.2 times and 1.5 times of commercial gel filtration membranes, respectively, and have lower pressure drop, higher capture efficiency and microbial survival rate than commercial gel filtration membranes. The green capture membrane material is prepared by phase separation of solute and solvent through gel confinement, and its non-covalent cross-linked structure can automatically disintegrate after encountering water, which can greatly simplify the elution and extraction process after bioaerosol sampling, and realize the maximum sample transfer, collection and recovery ability of captured microorganisms. The green capture membrane material for air microorganisms disclosed by the present invention has good biocompatibility, simple and green preparation process, and by combining with ATP bioluminescence method, it realizes the rapid detection of microorganisms after capture, and has important application prospects in bioaerosol pollution monitoring.

Description

Technical Field

[0001] The invention belongs to the technical field of filtration membrane materials, and in particular relates to a green air microorganism capture membrane material and a preparation method and application thereof. Background Art

[0002] Bioaerosol pollution seriously affects air quality. Inhalation of aerosols containing infectious pathogens can lead to a variety of diseases and complications, posing a continuous threat to public health and social stability. The organisms hidden in bioaerosols can remain suspended in the air for a long time and spread over long distances with air currents, which poses a severe challenge to the prediction and prevention of infectious diseases. The collection and rapid detection of airborne pathogens can not only timely assess the risk of transmission and infection, but also provide a scientific basis for the formulation of infectious disease prevention policies and early diagnosis. Due to the extremely low concentration of airborne microorganisms in the atmosphere, the capture and enrichment of airborne microorganisms has become a key prerequisite for their accurate detection.

[0003] Compared with the passive gravity sedimentation sampling method, a variety of active bioaerosol sampling techniques have been developed to accelerate the capture and collection of airborne microorganisms, including impact sampling, cyclone sampling, filtration sampling, electrostatic adsorption sampling, condensation and other sampling methods. Among them, the filtration sampling method relying on porous filter media has the advantages of high collection efficiency, low cost, high durability and convenient operation, which has attracted extensive attention and research. However, the raw materials for the preparation of porous air filter media currently mainly rely on non-degradable synthetic polymers and inorganic materials, etc., which usually produce toxic and harmful chemicals and serious carbon emissions during the entire manufacturing process, are environmentally unfriendly and have poor biocompatibility. Importantly, during the bioaerosol sampling and subsequent elution of the captured material, unfriendly raw materials and processing techniques can cause potential biotoxicity and sterilization effects on the captured airborne microorganisms, resulting in unnecessary inactivation and sample loss. In the subsequent quantitative analysis of the collected microorganisms, this additional sample loss and inactivation caused by the filter membrane can lead to a serious underestimation of the microbial population in the bioaerosol, thereby greatly reducing the accuracy of the detection. In this case, we need to develop more green and friendly air microorganism green capture membrane materials to achieve dual guarantees of the quantity and quality of microorganism collection based on the green capture of air microorganisms. Summary of the invention

[0004] The present invention provides a green capture membrane material for air microorganisms and a preparation method and application thereof. The green capture membrane material is made of all biomass and has the characteristics of high collection efficiency, good biocompatibility and high microbial survival rate for bioaerosols, and can meet the dual needs of efficient capture and rapid detection of air microorganisms.

[0005] The technical solution of the present invention is:

[0006] In the first aspect, a green air microorganism capture membrane material is disclosed, which has a honeycomb-shaped cross-linked network structure. The particle retention rate of the green capture membrane material is 99.3%, and the capture amount and survival rate of air microorganisms can be 3.2 times and 1.5 times that of commercial gel filtration membranes, respectively. The pore size is 0.1-40μm and the thickness is 0.10-0.40mm.

[0007] Preferably, the green capture membrane material has the characteristics of high collection efficiency, good biocompatibility, and high microbial survival rate, and can meet the dual needs of efficient capture and rapid detection of airborne microorganisms.

[0008] Preferably, the green capture membrane material has the function of automatically disintegrating when in contact with water, and the disintegration time is 1-2 minutes, which simplifies the elution and extraction process after bioaerosol sampling and realizes the maximum transfer, collection and recovery capabilities of captured microorganisms.

[0009] Preferably, the green capture membrane material is prepared by phase separation of solute and solvent through gel confinement; wherein the solute is at least two of starch, cellulose, gum, gelatin, pectin, fructan, dextran, soybean polysaccharide and pullulan, and the solvent includes at least ethanol and ethyl acetate.

[0010] Preferably, when the solute is soybean polysaccharide and gelatin, the mass ratio of soybean polysaccharide to gelatin is (3-17):8.

[0011] In a second aspect, a method for preparing the air microorganism green capture membrane material is disclosed, comprising the following steps:

[0012] 1) Preparation of the initial membrane solution: dissolve the solute in water, heat and stir until completely dissolved to form a transparent aqueous solution, then add ethanol and continue stirring to obtain the initial membrane solution;

[0013] 2) Gel formation: The initial membrane solution is transferred into a container and cooled to form a gel; this process requires gradient cooling, which means that the initial membrane solution is first slowly cooled to room temperature and then placed in a refrigerator to avoid a sudden drop in temperature, which would prevent the formation of an ordered membrane structure.

[0014] 3) Gel-confined phase separation: adding ethanol to the gel in step 2) to perform phase separation and induce gel dehydration and precipitation;

[0015] 4) Membrane formation: Add solvent to continue inducing gel dehydration and precipitation until the filter membrane is fully formed to obtain a white solid;

[0016] 5) Drying of the membrane: Drying the formed white solid to obtain a stable green air microorganism capture membrane material.

[0017] Preferably, in step 1), the volume ratio of water to ethanol in the initial membrane solution is 1:(0.2-0.9); the preparation temperature of the initial membrane solution is 50-90° C., and the stirring time is 30-90 min.

[0018] Preferably, the ratio of the transfer volume of the initial membrane solution to the inner surface area of ​​the vessel in step 2) is 0.05-0.50 mL / cm 2 , the cooling time is 5-30min.

[0019] Preferably, in step 3), the volume ratio of ethanol to membrane solution is 1-5:1-5, the phase separation temperature is 10-50°C, and the time is 5-12h; in step 4), the solvent is at least two of methyl acetate, methanol, ethyl acetate, ethanol, acetone, dimethyl sulfoxide, dichloromethane and petroleum ether, the volume ratio of the solvent to the membrane solution is 1-4:1-4, the molding temperature is 10-60°C, and the time is 1-8h.

[0020] The third aspect discloses the application of the green air microorganism capture membrane material in gas filtration, air microorganism capture and bioaerosol detection. The green capture membrane material is combined with the ATP bioluminescence method after sampling to perform rapid air microorganism detection.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The green air microorganism capture membrane material prepared by the present invention not only has a honeycomb-shaped cross-linked interconnected network structure, good air permeability and high structural stability, but also has higher capture efficiency and microorganism survival rate in terms of air microorganism capture than commercial gel filtration membranes. The capture amount and survival rate of air microorganisms can be 3.2 times and 1.5 times that of commercial gel filtration membranes, respectively, and the particle retention rate is 99.3%.

[0023] (2) The present invention uses all-biomass materials as raw materials with good biocompatibility. Not only is the preparation method simple and effective, environmentally friendly, green and sustainable, and the product is degradable, but the rich functional groups inside the natural product are also conducive to the effective assembly and regulation of the porous structure inside the capture membrane material.

[0024] (3) The green air microorganism capture membrane material prepared by the present invention has the function of automatically disintegrating when exposed to water, which can greatly simplify the elution and extraction process after bioaerosol sampling. The disintegration time on the culture dish is more than 2 times shorter than that of commercial gel filtration membrane, and can achieve the maximum sample transfer, collection and recovery capabilities of captured microorganisms.

[0025] (4) The air microorganism green capture membrane material prepared by the present invention can be combined with the ATP bioluminescence detection method after filtration sampling to perform rapid quantitative detection of the captured microorganisms. The detection time is shortened by at least 288 times compared with the traditional colony culture counting method, realizing the effective combination of filtration sampling and ATP detection, and has broad application prospects in bioaerosol pollution monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 are scanning electron microscope images of the front side (a), the back side (b) and the cross section (c) of the air microorganism green capture membrane material prepared according to Example 1;

[0027] Figure 2 : are SEM comparison images of air microorganism green capture membrane materials prepared under different volumes of anhydrous ethanol, wherein (a) and (b) are the front and back sides of the membrane material prepared in Comparative Example 1, (c) and (d) are the front and back sides of the membrane material prepared in Example 2, and (e) and (f) are the front and back sides of the membrane material prepared in Comparative Example 2, respectively;

[0028] Figure 3 This is a SEM picture of the green air microorganism capture membrane material automatically disintegrating when it comes into contact with water;

[0029] Figure 4 are membrane materials prepared under different comparative examples, wherein (a) is a photograph of the membrane material prepared in comparative example 5, (b) is a photograph of the membrane material prepared in comparative example 6, and (c) is a SEM image of the membrane material prepared in comparative example 14;

[0030] Figure 5 It is a comparison chart of the growth curve of E. coli in the presence and absence of air microbial green capture membrane material (SGFM);

[0031] Figure 6 This is a comparison chart of microbial activity after three different microorganisms were co-incubated with different concentrations of air microbial green capture membrane materials;

[0032] Figure 7 This is a SEM comparison of the air microorganism green capture membrane material before and after bioaerosol sampling;

[0033] Figure 8 It is a photo of agar plates after colony culture after sampling of air microorganisms green capture membrane materials, commercial gel filtration membrane, impact sampler and natural sedimentation at different aerosol concentrations;

[0034] Fig. 9The figure is a comparison of the number of colonies captured by the air microbial green capture membrane material (SGFM) and the commercial gel filtration membrane (C-FM), where (a) is a photo of an agar plate in which the filter membrane is dissolved after sampling and a portion of the membrane solution is taken out for culture counting to avoid colony overload, and (b) is a comparison of the total number of colonies captured by different membrane materials at different aerosol concentrations;

[0035] Fig.10 It is a comparison of the pressure drop (a) and the captured microorganism survival rate (b) between the air microorganism green capture membrane material (SGFM) and the commercial gel filtration membrane (C-FM). In the figure, SGFM-P is the pressure drop of the membrane material prepared in Comparative Example 1.

[0036] Fig.11 It is the test result between the luminescence intensity (RLU) and the colony count (CFU) of three different microorganisms by ATP bioluminescence method;

[0037] Fig.12 It is the test result between the luminescence intensity (RLU) and the colony count (CFU) of three different microorganisms by ATP bioluminescence method with the participation of air microbial green capture membrane material (SGFM);

[0038] Fig.13 This is a comparison chart between the test results of the air microorganism green capture membrane material using the culture method and the ATP bioluminescence method after bioaerosol sampling. DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0040] Example 1

[0041] The air microorganism green capture membrane material is prepared by the following method:

[0042] 1) Preparation of the initial membrane solution: Dissolve soybean polysaccharide (0.2 g) and gelatin (0.4 g) in 10.0 mL of deionized water, place the mixture in a constant temperature water bath at 60°C to fully dissolve and form a transparent aqueous solution, then drop 4.0 mL of anhydrous ethanol under continuous stirring, and continue stirring for 30 min to obtain a stable and clear initial membrane solution;

[0043] 2) Gel formation: 0.8 mL of the initial membrane solution was transferred into the dish. The ratio of the transferred volume of the initial membrane solution to the inner surface area of ​​the dish was 0.10 mL / cm 2 After the initial membrane solution is cooled naturally to room temperature, the membrane solution is placed in a refrigerator at 4°C to continue cooling and refrigerated for 15 minutes to form a gel;

[0044] 3) Gel-confined phase separation: Take out 0.8 mL of the gel in step 2), add 1.0 mL of anhydrous ethanol for phase separation, the phase separation temperature is 20° C., the time is 5 h, and the gel is induced to dehydrate and precipitate;

[0045] 4) Membrane formation: add 2.0 mL of a mixed solvent of ethyl acetate and ethanol in a volume ratio of 1:1, and continue to induce gel dehydration and precipitation until the filter membrane is completely formed. The forming temperature is 20°C and the time is 5 hours to obtain a white solid;

[0046] 5) Drying of the membrane: The formed white solid was freeze-dried for 5 hours to obtain a green air microorganism capture membrane material with a pore size of 3 μm and a thickness of 0.28 mm.

[0047] like Figure 1 The SEM images of the front, back and cross-section of the air microorganism green capture membrane material prepared in Example 1 show that its surface is a honeycomb-like micro-nano porous structure, and its interior contains a multi-layer interpenetrating network structure. Among them, the macroporous skeleton structure can provide rigid support, allowing air to pass quickly and at high flux, and the smaller pore network can ensure the size selectivity of the capture membrane material and the effective retention of microbial pollutants. This special morphology gives the green capture membrane material a high gas flow rate, high filtration efficiency and high sample enrichment ability.

[0048] Example 2

[0049] The air microorganism green capture membrane material is prepared by the following method:

[0050] 1) Preparation of the initial membrane solution: Dissolve soybean polysaccharide (0.2 g) and gelatin (0.4 g) in 10.0 mL of deionized water, place the mixture in a constant temperature water bath at 60°C to fully dissolve and form a transparent aqueous solution, then drop 5.0 mL of anhydrous ethanol under continuous stirring, and continue stirring for 30 min to obtain a stable and clear initial membrane solution;

[0051] 2) Gel formation: 1.0 mL of the initial membrane solution was transferred into the dish. The ratio of the transferred volume of the initial membrane solution to the inner surface area of ​​the dish was 0.14 mL / cm 2 After the initial membrane solution is cooled naturally to room temperature, the membrane solution is placed in a refrigerator at 4°C to continue cooling and refrigerated for 15 minutes to form a gel;

[0052] 3) Gel-confined phase separation: Take out 1.0 mL of the gel in step 2), add 1.0 mL of anhydrous ethanol for phase separation, the phase separation temperature is 20° C., the time is 5 h, and the gel is induced to dehydrate and precipitate;

[0053] 4) Membrane formation: add 2.0 mL of a mixed solvent of ethyl acetate and ethanol in a volume ratio of 1:1, and continue to induce gel dehydration and precipitation until the filter membrane is completely formed. The forming temperature is 20°C and the time is 5 hours to obtain a white solid;

[0054] 5) Drying of the membrane: The formed white solid was freeze-dried for 5 hours to obtain a green air microorganism capture membrane material with a pore size of 3 μm and a thickness of 0.30 mm.

[0055] The SEM images of the front and back sides of the prepared green capture membrane material are shown in Figure 2 As shown in c and d, c is the front side and d is the back side. Water is added to the prepared green capture membrane material to study the performance of membrane disintegration and release. Specifically, the dried green capture membrane material is cut into long strips, and 10.0 μL of deionized water is dripped into the edge area near one side. The SEM image of the green capture membrane material after adding water is shown in FIG. Figure 3 As shown in the figure, it can be seen that the green capture membrane material has the property of automatically disintegrating when it comes into contact with water. Since its preparation and structure regulation process mainly rely on non-covalent bond forces, the green capture membrane material obtained will quickly disintegrate after coming into contact with water and transform back into a membrane solution state. After the membrane material is completely disintegrated, the air microorganisms captured on the membrane material can be fully released into the water. Therefore, the addition of water can easily achieve the re-release of the sample captured by the green capture membrane material.

[0056] Comparative Example 1

[0057] Different from Example 2, the structure of this comparative example is controlled by changing the volume ratio of anhydrous ethanol in the initial membrane solution. Specifically, the amount of anhydrous ethanol added in step 1) is 0 mL, and the rest is the same as Example 2. The SEM images of the front and back of the prepared membrane material are as follows: Figure 2 As shown in a and b, a is the front side and b is the back side.

[0058] Comparative Example 2

[0059] Different from Example 2, this comparative example controls the structure by changing the volume ratio of anhydrous ethanol in the initial membrane solution. Specifically, the amount of anhydrous ethanol added in step 1) is 10.0 mL, and an excess of ethanol is added to the initial membrane solution. The rest is the same as Example 2. The SEM images of the front and back of the prepared membrane material are as follows: Figure 2 As shown in e and f, e is the front side and f is the back side.

[0060] from Figure 2 It can be seen that with the increase of ethanol volume in the initial membrane solution, the microstructure of the obtained green capture membrane material also changed significantly. Among them, the stacking particle size of the biomacromolecules increased significantly with the increase of ethanol amount, the surface structure became rougher, and the pore size and porosity increased significantly.

[0061] Comparative Example 3

[0062] Different from Example 2, in step 1), soybean polysaccharide is replaced by sodium alginate, and the rest is the same as Example 2. At this time, the viscosity of the membrane solution will be greatly increased, and the biomacromolecules cannot be precipitated in the subsequent phase separation process, and the filter membrane cannot be formed.

[0063] Comparative Example 4

[0064] Different from Example 2, gelatin was replaced with agar in step 1) of this comparative example, and the rest was the same as Example 2. At this time, the membrane solution could not form a gel, the biomacromolecules could not be precipitated in the subsequent phase separation process, and the filter membrane could not be formed.

[0065] Comparative Example 5

[0066] The difference from Example 2 is that the amount of raw materials in step 1) of this comparative example is changed to soybean polysaccharide (0.1 g) and gelatin (0.4 g), and the rest is the same as Example 2. Although the membrane solution at this time can form a gel, the biomacromolecules precipitated after the subsequent phase separation treatment cannot be effectively adhered together, and the filter membrane after drying is very thin. Figure 4 As shown in a.

[0067] Comparative Example 6

[0068] Different from Example 2, this comparative example does not include step 2), and anhydrous ethanol is directly added to carry out the phase separation process. Figure 4 As shown in b, the structure of the filter membrane after drying is broken and the thickness is relatively thin, and it is impossible to obtain a complete filter membrane with a certain mechanical strength.

[0069] Comparative Example 7

[0070] Different from Example 2, in step 2) of this comparative example, 0.5 mL of the initial membrane solution was transferred into the dish, and the ratio of the transferred volume of the initial membrane solution to the inner surface area of ​​the dish was 0.7 mL / cm 2 , and the rest is the same as Example 2. Although the membrane solution can still form a gel after low-temperature refrigeration, after the gel confined phase separation process, very little white solid is precipitated, and an effective filter membrane structure cannot be formed.

[0071] Comparative Example 8

[0072] Different from Example 2, in step 3) of this comparative example, the phase separation time is adjusted to 1 hour, and the rest is the same as Example 2. At this time, the biomacromolecules in the membrane solution have just begun to precipitate, and the white solid formed is very small, and an effective filter membrane structure cannot be formed.

[0073] Comparative Example 9

[0074] The difference from Example 2 is that in step 3) of this comparative example, anhydrous ethanol is replaced by methanol, and the rest is the same as Example 2. At this time, the biomacromolecules in the membrane solution cannot be precipitated, and the next step of molding cannot be performed.

[0075] Comparative Example 10

[0076] The difference from Example 2 is that this comparative example does not include step 4), and the rest is the same as Example 2. After the membrane solution in the vessel completes the phase separation of the gel confinement, the obtained white solid is still in a viscous state, and the filter membrane cannot be formed and cannot be dried in the next step.

[0077] Comparative Example 11

[0078] Different from Example 2, the solvent (ethyl acetate and ethanol) was replaced with ethyl acetate in step 4) of this comparative example, and the rest was the same as Example 2. The membrane solution remained viscous after immersion for 5 hours, and the filter membrane could not be formed, nor could the next drying process be carried out.

[0079] Comparative Example 12

[0080] The difference from Example 2 is that in step 4) of this comparative example, the solvent (ethyl acetate and ethanol) is replaced by methanol, and the rest is the same as Example 2. The membrane solution remains viscous after immersion for 5 hours, and the filter membrane cannot be formed.

[0081] Comparative Example 13

[0082] Different from Example 2, the membrane forming time in step 4) of this comparative example is 0.5h, and the rest is the same as Example 2. At this time, the filter membrane forming time is too short, and the precipitate still remains viscous and cannot be dried.

[0083] Comparative Example 14

[0084] The difference from Example 2 is that freeze drying is replaced by oven drying in step 5) of this comparative example, and the rest is the same as Example 2. The white filter membrane becomes curled and transparent after drying, and its microstructure is as follows Figure 4 As shown in c, the original cross-connected porous network structure completely disappears, so freeze-drying is the best way to maintain the porous network structure of the capture membrane material.

[0085] Example 3 Biocompatibility test of airborne microorganism green capture membrane material

[0086] The biocompatibility test of the air microorganism green capture membrane material (hereinafter referred to as SGFM) prepared by the present invention is specifically as follows: 60 mg of the air microorganism green capture membrane material prepared in Example 2 was dissolved in LB culture medium (2 mL, 30 mg / mL), and then Escherichia coli (E. coli) was added for co-incubation. The culture conditions were 37°C and the oscillation speed was set to 200 rpm. The OD value of the mixed culture solution was tested by an ELISA instrument at regular intervals, and the possible inhibitory effect of the membrane solution on bacterial growth was detected by drawing the growth curve under co-culture. Figure 5 As shown, the addition of air microorganism green capture membrane material not only does not inhibit the proliferation of bacteria, it can be seen that the air microorganism green capture membrane material has no antibacterial effect.

[0087] The biocompatibility of air microorganism green capture membrane materials is mainly evaluated by traditional plate culture and colony counting methods. The air microorganism green capture membrane material is dissolved in the culture medium, and culture solutions of different concentrations are obtained by gradient dilution. Then, they are mixed with Escherichia coli, Staphylococcus aureus (S.aureus) and Candida albicans (C.albicans) at different concentrations. The mixed bacterial suspension is evenly spread on the agar plate, and the colonies are counted after 48 hours of culture. The biocompatibility of the air microorganism green capture membrane material is calculated by counting the number of colonies at different concentrations. Among them, Escherichia coli and Staphylococcus aureus are cultured in LB medium at 37°C; Candida albicans is cultured in YPD medium at 30°C. Figure 6 As shown, as the membrane concentration increased from 0 to 30 mg / mL, the three microorganisms were always able to maintain high activity, indicating that the prepared air microorganism green capture membrane material has excellent biocompatibility for different microorganisms.

[0088] Example 4 Testing of the Air Microorganism Capture Performance of the Air Microorganism Green Capture Membrane Material

[0089] The test of air microorganism capture performance was mainly evaluated by a bioaerosol simulation experiment in a closed atomization chamber (800mm×600mm×800mm). Different bioaerosol environments were created by changing the concentration and type of bacterial suspension in the aerosol generator. Among them, the air flow rate of the aerosol generator was set to 10L / min, the generation time was 2min, and the aerosol concentration, particle size, temperature and humidity inside the atomization chamber were monitored in real time by a particle size analyzer (AQ Guard, Palas, Germany). The air microorganism green capture membrane material prepared in Example 2 was uniformly cut into a circle with a diameter of 25mm and installed on an air sampler (Sartorius MD8, Germany) for filtration sampling. At the same time, the commercial gel filtration membrane (Sartorius, Germany, hereinafter referred to as C-FM) was used as a benchmark product in the industry for benchmarking research. It was developed by Sartorius, Germany, and is currently the only water-soluble air filtration membrane on the market. During use, it was also cut into a circle with a diameter of 25mm and installed on an air sampler for microbial capture. In addition, impactor sampling and natural precipitation sampling were also compared under the same aerosol conditions.

[0090] like Figure 7 As shown, by comparing the microstructural changes on the surface of the air microorganism green capture membrane material of the present invention before and after sampling, it can be found that a large amount of particulate matter is attached to its surface after aerosol sampling, but its network structure is still stable without collapse and cracks. It can be seen that the air microorganism green capture membrane material prepared by the present invention has a stable porous structure and effective particulate matter retention performance.

[0091] After sampling, the green capture membrane material after filtration sampling is directly attached to an agar plate, and the amount of captured microorganisms is detected by culture method. Since the air microorganism green capture membrane material prepared by the present invention and the commercial gel filtration membrane both have the function of disintegrating when exposed to water, it is convenient to coat it on the surface of the plate for colony culture. Compared with the disintegration time of 5 minutes of the commercial gel filtration membrane, the air microorganism green capture membrane material can be completely disintegrated within 1.5 minutes, with a faster disintegration speed.

[0092] Through the air microorganism capture test under different bioaerosol concentrations, it can be found that compared with commercial gel filtration membrane, impactor sampling and natural sedimentation sampling, the air microorganism green capture membrane material prepared by the present invention has better aerosol collection ability. Under the condition of collecting the same volume of air, more air microorganisms can be captured. The results are as follows: Figure 8 After sampling at high concentrations of bioaerosol, the membrane material is dissolved and a portion of the membrane solution is taken out for culture, which can effectively avoid the overload of the colonies on the culture dish ( Fig. 9a), and the number of colonies captured by the filter membrane can be calculated more accurately. 7 When bioaerosol is generated by 1000 CFU / mL, the number of colonies captured by the membrane material prepared by the present invention can reach 3.2 times that of commercial gel filtration membrane ( Fig. 9 b).

[0093] The pressure gauge test showed that the air microorganism green capture membrane material had a lower pressure drop value than the commercial gel filtration membrane, reflecting its better air permeability ( Fig.10 a). The number of particles in the air before and after the filtration membrane was tested by a particle size analyzer, and the filtration efficiency of the air microorganism green capture membrane material and the commercial gel filtration membrane was calculated to be 99.3% and 98.9%, respectively, further proving its excellent air microorganism interception ability. In addition, by testing the survival rate of captured microorganisms on the membrane material, it was found that the air microorganism green capture membrane material of the present invention is more beneficial to maintaining the activity of microorganisms ( Fig.10 b), the survival rate of microorganisms captured by the membrane material prepared by the present invention can still be maintained at more than 80% after 1 hour. On the contrary, the survival rate of microorganisms in the commercial gel filtration membrane has dropped to 60%. Further extending the time to 2 hours, a significant difference in the survival rate between the two membrane materials can still be observed. At this time, the survival rate of microorganisms captured by the membrane material prepared by the present invention is 1.5 times that of the commercial gel filtration membrane. It can be seen that the air microorganism green capture membrane material prepared by the present invention has higher sampling efficiency and microorganism survival rate in air microorganism capture.

[0094] Example 5: Combination of airborne microorganism green capture membrane material and ATP bioluminescence method

[0095] ATP bioluminescence method can realize rapid quantitative detection of microorganisms. By directly dripping bacterial suspension on the membrane material, the air microorganism green capture membrane material prepared in Example 2 is combined with the microorganism. Then PBS buffer is added to fully dissolve the membrane, centrifuged at 8000rpm for 3min, and the enriched microorganisms are obtained after removing the supernatant. After the lysate fully lyses the microorganisms, luciferin / luciferase is added for bioluminescence detection, and the linear relationship between the luminescence value (RLU) and the number of microorganisms (CFU) on the membrane material is established to measure the effectiveness of the combination with the ATP bioluminescence method.

[0096] Fig.11 and Fig.12 The linear relationship between bioluminescence value and microbial quantity in the presence or absence of membrane material, Fig.11 There are only three kinds of microorganisms. Fig.12It is an air microorganism green capture membrane material and three kinds of microorganisms. It can be seen that the air microorganism green capture membrane material prepared by the present invention can be effectively combined with the ATP bioluminescence method without affecting the detection accuracy of the ATP bioluminescence method, and the content of microorganisms can be effectively estimated through the corresponding linear relationship.

[0097] Example 6: Rapid detection of bioaerosols captured by airborne microorganism green capture membrane materials

[0098] After the sampling of the air microorganism green capture membrane material prepared in Example 2 is completed, the membrane material is removed and transferred to a sterile centrifuge tube, PBS buffer is added for full dissolution, and then centrifuged at 8000 rpm for 3 minutes, and the supernatant is removed to obtain the enriched microorganisms. After the microorganisms are fully lysed by the lysate, luciferin / luciferase is added for bioluminescence, and the number of microorganisms captured on the membrane material is calculated by the established linear relationship between the luminescence value (RLU) and the number of microorganisms (CFU), thereby indirectly calculating the concentration of air microorganisms in the aerosol.

[0099] The detection time required by the culture method usually takes more than 48 hours. Compared with the traditional colony culture counting method, the ATP detection method only takes 10 minutes to complete. Fig.13 The results of the green air microorganism capture membrane material prepared by the present invention after sampling at different aerosol concentrations are compared by the culture method and the ATP method. Compared with the traditional colony culture counting method, the ATP detection method not only has a shorter test time, but also has more accurate test results. This is mainly because most of the microorganisms in the air are not culturable, and the ATP detection method can identify the number of live bacteria without difference, and can better reflect the actual aerosol concentration.

[0100] Although the present invention has been described in detail by combining the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. Air microorganism green capture membrane material, characterized in that: The green capture membrane material has a honeycomb-shaped cross-linked interconnected network structure, the particle retention rate of the green capture membrane material is 99.3%, the pore size is 0.1-40µm, and the thickness is 0.10-0.40mm; The green capture membrane material is prepared by phase separation of solute and solvent through gel confinement; wherein the solute is at least two of starch, cellulose, gum, gelatin, pectin, fructan, dextran, soybean polysaccharide and pullulan, and the solvent includes at least ethanol and ethyl acetate; When the solute is soybean polysaccharide and gelatin, the mass ratio of soybean polysaccharide to gelatin is (3-17):8; The method for preparing the air microorganism green capture membrane material comprises the following steps: 1) Preparation of the initial membrane solution: dissolve the solute in water, heat and stir until completely dissolved to form a transparent aqueous solution, then add ethanol and continue stirring to obtain the initial membrane solution; 2) Gel formation: transfer the initial membrane solution into a container and cool it to form a gel; 3) Gel-confined phase separation: Add ethanol to the gel in step 2) to perform phase separation and induce gel dehydration and precipitation; 4) Membrane formation: Add solvent to continue inducing gel dehydration and precipitation until the filter membrane is fully formed to obtain a white solid; 5) Drying of the membrane: Drying the formed white solid to obtain a stable green air microorganism capture membrane material; The ratio of the initial membrane solution transfer volume to the inner surface area of ​​the vessel in step 2) is 0.05-0.50 mL / cm 2 , cooling time is 5-30min; In step 3), the volume ratio of ethanol to membrane solution is 1-5:1-5, the phase separation temperature is 10-50°C, and the time is 5-12h; in step 4), the solvent is at least two of methyl acetate, methanol, ethyl acetate, ethanol, acetone, dimethyl sulfoxide, dichloromethane and petroleum ether, the volume ratio of the solvent to the membrane solution is 1-4:1-4, the molding temperature is 10-60°C, and the time is 1-8h.

2. The air microorganism green capture membrane material according to claim 1, characterized in that: The green capture membrane material has the function of automatically disintegrating when in contact with water, and the disintegration time is 1-2 minutes.

3. The air microorganism green capture membrane material according to claim 1, characterized in that: In step 1), the volume ratio of water to ethanol in the initial membrane solution is 1:(0.2-0.9); the preparation temperature of the initial membrane solution is 50-90°C, and the stirring time is 30-90min.

4. An application of the air microorganism green capture membrane material according to any one of claims 1 to 3 in gas filtration, air microorganism capture and bioaerosol detection, characterized in that: The green capture membrane material is combined with the ATP bioluminescence method after sampling for rapid detection of air microorganisms.

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