River and lake water body low-concentration phage efficient enrichment method based on multi-stage collaborative technology

Abstract

The invention relates to an efficient enrichment method for low-concentration bacteriophages in river and lake water bodies, which remarkably improves the recovery rate and the concentration multiple of the bacteriophages through a multi-stage synergistic technology in combination with physical interception, chemical precipitation and biological targeted adsorption. The method specifically comprises the following steps: pretreatment: removing large particles and suspended solids through gradient filtration and low-speed centrifugation; ultrafiltration concentration: adopting a hollow fiber ultrafiltration membrane with the molecular weight cut-off of 30-100 kDa, and realizing 10-50 times concentration under the pressure of 0.1-0.3 MPa; chemical precipitation strengthening: optimizing synergistic precipitation conditions of PEG and NaCl, and reducing phage inactivation in combination with low-temperature standing; specific adsorption of host bacteria: combining the host bacteria in the logarithmic phase with bacteriophage according to the volume ratio of 1: 100, and carrying out pH regulation and control elution to obtain a high-purity enriched solution. The method has the advantages that the total concentration multiple reaches 500-1000 times, and the recovery rate is gt; the activity retention rate is gt; and the method is suitable for a complex water body environment. Through a multi-technology combination strategy and targeted adsorption design, an efficient tool is provided for environmental monitoring, phage therapy and microbiological research.

Description

Technical Field

[0001] The present invention relates to a method for efficiently enriching low-concentration phages in river and lake waters based on multi-level collaborative technology, belonging to the field of environmental microorganism separation technology. Background Art

[0002] Bacteriophages are a type of virus that specifically infects bacteria and are widely used in fields such as biological control, genetic engineering, and biomedicine. Their characteristics make bacteriophages have important application potential in combating bacterial infections, regulating microbial communities, and serving as gene carriers. Bacteriophages can specifically recognize and attack specific bacteria, which makes them an ideal biological treatment tool, especially today when drug-resistant bacteria are becoming increasingly serious. In the agricultural field, bacteriophages are used as natural biological pesticides, which helps control plant diseases, reduce the use of chemical pesticides, and thus reduce the impact on the environment. In the field of water ecological restoration, bacteriophages have important value in ecological regulation of river and lake waters, eutrophication treatment, etc.

[0003] However, despite the broad application prospects of bacteriophages in various fields, the concentration of bacteriophages in river and lake waters is usually low (<10³ PFU / mL), which has become a major obstacle in practical applications. Studies have found that the concentration of bacteriophages in river and lake waters is often insufficient to support effective applications and research, mainly due to the complex ecological environment in the water body and competition with other microorganisms. Traditional enrichment methods, such as precipitation, physical centrifugation, and filtration, have the following problems: 1. The efficiency of a single step is low, and it is difficult to cope with complex water body interference (such as suspended particles, organic matter); 2. High centrifugal force is likely to damage the structure of bacteriophages; 3. The chemical precipitation method has low purity and requires secondary purification; 4. Traditional adsorption materials (such as anion resins) have poor selectivity. These methods not only take a long time but may also cause a decrease in the activity of bacteriophages during the enrichment process, or even loss, thus affecting the effects of subsequent experiments and applications.

[0004] In response to the above problems, existing technologies have tried to improve efficiency through various methods, but have not solved the following key challenges: The existing technology (CN 114085829 B) adsorbs viruses through modified resins, relying on charge interactions, but has insufficient specificity for bacteriophages; while the plant virus enrichment method (CN 110283792 B) increases the concentration through stepwise extraction, but does not solve the problem of complex water body interference. Therefore, the present invention breaks through the limitations of single technologies by innovatively designing a multi-level collaborative technology chain to achieve efficient and highly active enrichment of low-concentration bacteriophages. Summary of the Invention

[0005] The present invention provides a method for efficiently enriching low-concentration phages in river and lake waters based on multi-stage collaborative technology. The aim is to address the problems of low efficiency, poor selectivity, and insufficient retention of activity in traditional methods, and to propose a method for efficiently enriching low-concentration phages in river and lake waters based on multi-stage collaborative technology, which is applicable to environmental monitoring, phage therapy, and microbial ecological research. Through multi-stage collaborative technology, combined with physical interception, chemical precipitation, and biological targeted adsorption, the recovery rate and concentration multiple of phages can be significantly improved.

[0006] The technical solution of the present invention: A method for efficiently enriching low-concentration phages in river and lake waters based on multi-stage collaborative technology, comprising the following steps: (1) Pretreatment: Remove large particles and suspended matter through gradient filtration and low-speed centrifugation; (2) Ultrafiltration concentration: Use a low-adsorption membrane material (whether a general definition of the low-adsorption membrane material can be made, only restricting it to polyacrylonitrile ultrafiltration membrane will result in too small a protection range) to ultrafilter and concentrate the pretreated liquid, and dynamically adjust the pressure during concentration; (3) Chemical precipitation: Add PEG and NaCl to the concentrated solution, let it stand at low temperature, and then centrifuge to collect the precipitate; (4) Host bacteria targeted adsorption: Use host bacteria to specifically bind phages, and elute through pH regulation to obtain a host bacteria-phage complex; (5) Elution and purification: Elute the host bacteria-phage complex to remove unbound components; Centrifuge to collect the phages after elution to obtain a high-purity phage solution.

[0007] In the pretreatment of step (1), the gradient filtration is to filter using three-stage filters of 100 μm, 10 μm, and 0.45 μm, the rotation speed of the low-speed centrifugation is 3000 - 5000 rpm, and the centrifugation time is 10 - 15 minutes.

[0008] In the pretreatment of step (1), it also includes adding 1 mM EDTA to chelate metal ions and reduce membrane fouling.

[0009] In the ultrafiltration concentration of step (2), the low-adsorption membrane material is a hollow fiber membrane module made of polyacrylonitrile or nitrocellulose with a pore size of 0.22 μm, the cut-off molecular weight is 30 - 100 kDa, and the ultrafiltration membrane with a cut-off molecular weight of 50 kDa is the preferred solution; The dynamic adjustment of pressure is to use a low pressure of 0.1 MPa in the initial stage to reduce membrane fouling, and gradually increase it to 0.3 MPa in the later stage to increase the flux, so as to achieve a concentration of 10 - 50 times.

[0010] In step (3) of chemical precipitation, the PEG is PEG-8000 with a concentration of 5-20 w / v%, and the NaCl concentration is 0.1-0.5 M; the low-temperature standing means standing the mixed solution at 4°C for 1-2 hours, followed by centrifugation at a rotational speed of 8000-12000 rpm for 15-20 minutes. The precipitate is collected and the supernatant is discarded.

[0011] The addition amount of PEG-8000 is 10 w / v%, and the addition amount of NaCl is 0.2 M.

[0012] In step (4) of host bacteria targeted adsorption, the host bacteria match the target phage and are cultured to the logarithmic growth phase with a concentration of 108-109 CFU / mL; the precipitated phage is mixed with the host bacteria and reacted for 1-2 hours to obtain a mixed solution to promote the binding of the phage and the host bacteria; in the pH-regulated elution, the pH value of the mixed solution is adjusted to 5.0-7.0, and the unbound phage is eluted by the pH change, and the bound host bacteria-phage complex is collected.

[0013] The host bacteria are Escherichia coli ATCC 25922 or halophilic vibrio.

[0014] In step (5) of elution and purification, in the elution, PBS buffer or glycine-HCl buffer is used, and the neutralizing solution is Tris-HCl, and it is washed 2-3 times; the rotational speed of the centrifugation is 8000-10000 rpm and the time is 10-15 minutes.

[0015] Advantages of the present invention: 1) The total concentration multiple of this method can reach 500-1000 times, the recovery rate > 85%, and the activity retention rate > 90%, meeting the detection requirements of low-concentration samples. Moreover, more than 95% of non-target impurities are removed through the host bacteria adsorption step, which is applicable to complex water environments. At the same time, it provides convenience for subsequent detection and prevents harm caused by the failure to detect due to the low content of phages in the water body.

[0016] 2) The core innovation of the present invention lies in the multi-technology combination strategy and targeted adsorption design, which solves the technical bottlenecks of low efficiency and poor selectivity of traditional methods, and provides an efficient tool for environmental monitoring, phage therapy and microbial research.

[0017] 3) The present invention does not require ultracentrifugation or chromatography equipment, is suitable for field sampling, has no toxic reagents throughout the process, and the eluent is biodegradable. Description of the Drawings

[0018] Appendix Figure 1 is the technical flow chart of the method for highly efficient enrichment of low-concentration phages in river and lake water bodies. Detailed Embodiments

[0019] An efficient enrichment method for low-concentration phages in river and lake waters, comprising the following steps: a) Pretreatment: Remove large particles and suspended solids through gradient filtration and low-speed centrifugation. The specific steps are as follows: Perform gradient filtration using 100 μm, 10 μm, and 0.45 μm three-stage filter membranes to remove large particle substances and suspended solids in the water sample.

[0020] Centrifuge the filtered liquid at a low speed, with the rotation speed set at 3000 - 5000 rpm and the centrifugation time at 10 - 15 minutes to further remove residual suspended solids and precipitates.

[0021] b) Ultrafiltration concentration: Use a polyacrylonitrile (PAN) hollow fiber ultrafiltration membrane with a molecular weight cut-off of 30 - 100 kDa, and an ultrafiltration membrane with a molecular weight cut-off of 50 kDa is the preferred option. Concentrate 10 - 50 times under a pressure of 0.1 - 0.3 MPa; Dynamically adjust the pressure. In the initial stage, use a low pressure of 0.1 MPa to reduce membrane fouling, and gradually increase it to 0.3 MPa in the later stage to increase the flux, thereby achieving 10 - 50 times concentration.

[0022] c) Chemical precipitation: Add PEG-8000 and NaCl to the concentrated solution, and centrifuge to collect the precipitate after standing at low temperature. The specific steps are as follows: Add PEG-8000 to the concentrated solution, with a concentration range of 5% - 20% (w / v), to promote the precipitation of phages.

[0023] At the same time, add NaCl, with a concentration range of 0.1 - 0.5 M, to increase the ionic strength of the solution and further promote the aggregation of phages.

[0024] Let the mixture stand at 4°C for 1 - 2 hours, then centrifuge after standing. Set the rotation speed at 8000 - 12000 rpm and the centrifugation time at 15 - 20 minutes. Collect the precipitate part and discard the supernatant.

[0025] Among them, the addition amount of PEG-8000 is 10% (w / v), and the addition amount of NaCl is 0.2 M is the preferred option.

[0026] d) Host bacteria targeted adsorption: Utilize host bacteria to specifically bind phages and elute through pH regulation. The specific steps are as follows: Select host bacteria that match the target phages, culture them to the logarithmic growth phase, and the concentration reaches 108 - 109 CFU / mL.

[0027] Mix the precipitated phages with the host bacteria, and keep them reacting at a suitable temperature (such as 37°C) for 1 - 2 hours to promote the binding of phages and host bacteria.

[0028] By adjusting the pH value of the mixed solution, for example, pH 5.0 - 7.0, the binding efficiency between phage and host bacteria is improved. Unbound phages are eluted using the pH change, and the bound host bacteria - phage complex is collected.

[0029] The host bacteria are bacteria that specifically bind to the target phage, including Escherichia coli ATCC 25922 or Vibrio parahaemolyticus.

[0030] e) Elution and purification: The host bacteria - phage complex is eluted and washed 2 - 3 times using a buffer (such as PBS buffer) to remove unbound components.

[0031] The eluted phages are collected by centrifugation at a rotational speed of 8000 - 10000 rpm for 10 - 15 minutes to obtain a high - purity phage solution.

[0032] The elution buffer is 0.1 M glycine - HCl (pH 3.0), and the neutralization solution is 1 M Tris - HCl (pH 9.0).

[0033] In the pretreatment of step a), it further includes adding 1 mM EDTA during the pretreatment stage to chelate metal ions and reduce membrane fouling.

[0034] The technical solution of the present invention will be further explained below with reference to the accompanying drawings.

[0035] Refer to the attached Figure 1 , a method for efficient enrichment of low - concentration phages in river - lake water bodies based on a multi - stage collaborative technology, which realizes efficient enrichment through a four - stage collaborative technology chain.

[0036] 1. Gradient filtration and low - speed centrifugation: Remove particles > 0.45 μm and retain phages in the supernatant; 2. Ultrafiltration concentration: Retain phages based on molecular weight differences and exclude dissolved organic matter (DOM); 3. Chemical precipitation enhancement: PEG / NaCl induces aggregation to further concentrate and remove small - molecule impurities; 4. Host - bacteria targeted adsorption: Utilize the specific binding between phage and host to achieve high - purity enrichment.

[0037] The specific steps are as follows: 1. Pretreatment stage Purpose: Eliminate particles and microorganisms > 0.45 μm in water bodies to reduce membrane fouling and interference in subsequent steps.

[0038] Operation process: Gradient filtration: sequentially pass through a 100 μm nylon mesh (to remove branches, leaves, and grit), a 10 μm glass fiber filter membrane (to retain plankton), and a 0.45 μm polyethersulfone filter membrane (to remove bacteria and eukaryotic microorganisms).

[0039] Low-speed centrifugation: Take the filtered liquid in a centrifuge tube, centrifuge at 800 - 1500×g for 10 minutes, discard the precipitate, and retain the supernatant.

[0040] Technical advantages: Gradient filtration avoids clogging of a single filter membrane and extends the lifespan of the ultrafiltration membrane; low-speed centrifugation retains phages (sedimentation coefficient < 200 S) in the supernatant, while suspended particles (> 500 S) sediment to the bottom of the tube.

[0041] 2. Ultrafiltration concentration stage Purpose: Retain phages (typical size 20 - 200 nm) based on molecular weight differences and exclude small molecule impurities (such as DOM).

[0042] Equipment and parameters: Ultrafiltration membrane: Hollow fiber membrane module (material: polyacrylonitrile), molecular weight cut-off 30 kDa (corresponding particle size about 30 nm); Operating conditions: Transmembrane pressure 0.1 - 0.3 MPa, flow rate 10 - 20 L / h, temperature 4 - 25°C; Concentration factor: Adjusted to 10 - 50 times according to the initial volume.

[0043] Key innovation: Use of low-adsorption membrane materials to reduce non-specific binding of phages on the membrane surface (verification data: adsorption loss rate < 5%); dynamic pressure regulation: initial low pressure (0.1 MPa) to avoid membrane fouling, and gradually increase the pressure later (up to 0.3 MPa) to increase the flux.

[0044] 3. Chemical precipitation enhancement stage Purpose: Induce phage aggregation and precipitation through PEG / NaCl to further concentrate and remove soluble impurities.

[0045] Reagents and steps: Precipitant preparation: 10% PEG-8000 (w / v) and 5 M NaCl mother liquor, pre-cooled to 4°C; Addition ratio: Add 10 mL of PEG mother liquor and 2 mL of NaCl mother liquor to every 100 mL of ultrafiltration concentrate (final concentration: 10% PEG + 1 M NaCl); Precipitation conditions: Stand at 4°C for 2 hours, centrifuge at 10,000×g for 20 minutes, and discard the supernatant; Resuspension solution: The precipitate was gently resuspended with SM Buffer (50 mM Tris-HCl, 100 mM NaCl, 8 mM MgSO4, pH 7.5) to 1 / 10 of the original volume.

[0046] Optimization parameters: PEG molecular weight selection: 8000 Da can balance the precipitation efficiency and mildness (comparative experiment: the recovery rate of PEG-6000 is 15% lower, and PEG-10000 causes a 20% decrease in activity); Ionic strength regulation: 1 M NaCl can neutralize the surface charge of phages and promote the hydrophobic interaction between PEG and phages.

[0047] 4. Host bacteria targeted adsorption stage Purpose: Utilize the specific binding of phages to host bacteria to achieve selective enrichment and impurity removal.

[0048] Implementation steps: Host bacteria culture: The target strain (such as Escherichia coli ATCC 25922) was inoculated into LB medium and cultured with shaking at 37°C until OD 600 = 0.6 (mid-logarithmic phase, cell concentration ≈ 1×10 8 CFU / mL); Adsorption reaction: The phage solution and the bacterial solution were mixed at a ratio of 1:100 (v / v), and incubated with shaking (150 rpm) at 37°C for 30 minutes; Centrifugal collection: Centrifuge at 5000×g for 5 minutes, discard the supernatant, and retain the precipitate of the cell-phage complex; Elution and neutralization: The precipitate was eluted with 0.1 M glycine-HCl buffer (pH 3.0) with shaking for 10 minutes, immediately neutralized to pH 7.0 by adding 1 M Tris-HCl (pH 9.0), and the supernatant was taken after centrifugation as the final product.

[0049] Technical advantages: It has specific adsorption performance, only the target phages are captured by the surface receptors of host bacteria, and impurities (such as free DNA, humic acid) are not adsorbed; it can be eluted gently, and the low pH destroys the phage-host binding, avoiding the use of strong denaturants (such as SDS).

[0050] To verify the experimental effect of the present invention, a comparative example was used for effect verification.

[0051] Example 1: A water sample from a eutrophic lake (initial concentration: 80 PFU / mL) 1. Pretreatment: The volume loss rate of 10 L of the water sample after gradient filtration was <5%; 2. Ultrafiltration concentration: The volume of the retentate was 500 mL (concentrated 20 times), and the phage recovery rate was 92%; 3. Chemical precipitation: Final volume 50 mL (concentrated 10 times), recovery rate 78%; 4. Host bacteria adsorption: Final volume 5 mL (concentrated 10 times), recovery rate 95%; Total concentration multiple: 20×10×10 = 2000 times, total recovery rate = 92%×78%×95% = 68.3%, active retention rate > 90%.

[0052] Example 2: A mixed sample of brackish water at the estuary of a certain river (initial concentration: 30 PFU / mL) 1. Pretreatment: Add EDTA (final concentration 1 mM) to chelate metal ions and prevent membrane fouling; 2. Ultrafiltration concentration: Use a 50 kDa cut-off membrane and concentrate 30 times; 3. Chemical precipitation: Optimize the PEG concentration to 9% and NaCl to 0.8 M, and the recovery rate is increased to 85%; 4. Host bacteria adsorption: Use a salt-tolerant strain (such as Vibrio parahaemolyticus) and extend the adsorption time to 45 minutes; Total concentration multiple: 30×15×12 = 5400 times, total recovery rate > 70%.

[0053] Thus, through systematic innovation, this patent provides an efficient solution for the development of phage resources in environmental water bodies.

Claims

1. A method for highly efficient enrichment of low-concentration phages in river and lake water bodies based on multi-level collaborative technology, characterized in that It includes the following steps: (1) Pretreatment: Remove large particles and suspended matters through gradient filtration and low-speed centrifugation; (2) Ultrafiltration concentration: Use low-adsorption membrane materials to ultrafilter and concentrate the pretreated liquid, and dynamically adjust the pressure during concentration; (3) Chemical precipitation: Add PEG and NaCl to the concentrated liquid to induce aggregation, and centrifuge to collect the precipitate after standing at low temperature; (4) Host bacteria targeted adsorption: Utilize the specific binding of host bacteria to phages, and elute through pH regulation to obtain host bacteria-phage complexes; (5) Elution and purification: Elute the host bacteria-phage complexes to remove unbound components; Centrifuge to collect the eluted phages to obtain a high-purity phage solution.

2. The method for highly efficiently enriching low-concentration phages in river and lake water bodies based on a multi-level collaborative technology according to claim 1, wherein the In step (1) pretreatment, the gradient filtration is to filter using three-stage filter membranes of 100 μm, 10 μm and 0.45 μm, the rotation speed of the low-speed centrifugation is 3000 - 5000 rpm, and the centrifugation time is 10 - 15 minutes.

3. The method for highly efficient enrichment of low-concentration phages in river and lake water bodies based on multi-level collaborative technology according to claim 1 or 2, characterized in that In step (1) pretreatment, adding 1 mM EDTA to chelate metal ions and reduce membrane fouling is also included.

4. A method for highly efficient enrichment of low-concentration phages in river and lake water bodies based on multi-level collaborative technology, characterized in that In step (2) ultrafiltration concentration, the low-adsorption membrane material is a hollow fiber membrane module made of polyacrylonitrile or nitrocellulose with a pore size of 0.22 microns, and the molecular weight cut-off is 30 - 100 kDa; The dynamic pressure adjustment is to use a low pressure of 0.1 MPa in the initial stage to reduce membrane fouling, and gradually increase it to 0.3 MPa in the later stage to increase the flux, so as to achieve a concentration of 10 - 50 times.

5. The method for highly efficiently enriching low-concentration phages in river and lake water bodies based on a multi-level collaborative technology according to claim 1, wherein In step (3) chemical precipitation, the PEG is PEG-8000, the concentration is 5 - 20 w / v%, and the NaCl concentration is 0.1 - 0.5 M; The low-temperature standing is to let the mixture stand at 4°C for 1 - 2 hours, then centrifuge after standing, the centrifugation speed is 8000 - 12000 rpm, the time is 15 - 20 minutes, collect the precipitate part and discard the supernatant.

6. The high-efficiency enrichment method for low-concentration phages in river and lake water bodies based on a multi-level collaborative technology according to claim 5, wherein The addition amount of PEG-8000 is 10 w / v%, and the addition amount of NaCl is 0.2 M.

7. The method for highly efficient enrichment of low-concentration phages in river and lake water bodies based on multi-level collaborative technology according to claim 1, characterized in that the In step (4) host bacteria targeted adsorption, the host bacteria match the target phage, are cultured to the logarithmic growth phase, and the concentration is 108 - 109 CFU / mL; Mix the precipitated phages with the host bacteria, and react for 1 - 2 hours to obtain a mixture to promote the binding of phages and host bacteria; In the pH-regulated elution, adjust the pH value of the mixture to 5.0 - 7.0, and use the pH change to elute the unbound phages, and collect the combined host bacteria-phage complexes.

8. A method for highly efficient enrichment of low-concentration phages in river and lake water bodies based on multi-level collaborative technology according to claim 1 or 7, characterized in that The host bacteria are Escherichia coli ATCC 25922 or Halobacterium halodurans.

9. The method for highly efficiently enriching low-concentration phages in river and lake water bodies based on a multi-level collaborative technology according to claim 1, characterized in that In step (5) elution and purification, in the elution, PBS buffer or glycine-HCl buffer is used, the neutralizing solution is Tris-HCl, and wash 2 - 3 times; The centrifugation speed is 8000 - 10000 rpm, and the time is 10 - 15 minutes.

10. The high-efficiency enrichment method for low-concentration phages in river and lake water bodies based on multi-level collaborative technology according to claim 4, characterized in that The molecular weight cut-off of the hollow fiber membrane module made of polyacrylonitrile is 50 kDa.

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