Separation and purification method of phage extracted by saccharification based on green solvent

Abstract

The invention relates to the technical field of biological separation and purification, and particularly discloses a method for efficiently separating and purifying bacteriophages from crude lysates of multi-drug-resistant bacteria by using a sugar precipitation extraction technology of a green solvent, and the method is suitable for separating and purifying bacteriophages of multi-drug-resistant bacteria such as klebsiella pneumoniae, escherichia coli, stenotrophomonas maltophilia and the like. According to the method, the green solvent and the saccharides are mixed to establish a sugar precipitation extraction system, and the high-efficiency separation and purification of the bacteriophage are realized by optimizing the ratio of the saccharides to the green solvent. The method is easy to operate, centrifugation is not needed, extraction can be completed within 40 minutes, the phage recovery rate can reach 82.42%, the impurity removal rate is larger than or equal to 75%, the solvent can be recycled, and the method is suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and specifically discloses a method for the separation and purification of bacteriophages based on glycosylation extraction using green solvents, which is applicable to the separation and purification of bacteriophages from multidrug-resistant bacteria. Background Technology

[0002] Phage therapy, as an effective antibiotic alternative against multidrug-resistant bacterial infections, has shown great clinical application potential due to its high specificity and safety. However, the large-scale application of this technology is severely limited by the bottlenecks of existing purification processes. Traditional methods such as cesium chloride (CsCl) density gradient ultracentrifugation and polyethylene glycol precipitation suffer from drawbacks such as high equipment investment, complex operation, limited processing capacity, and low product recovery rates, making it difficult to meet the dual requirements of economic efficiency and cost-effectiveness for commercial production.

[0003] Existing extraction technologies, such as traditional organic solvent extraction and salting-out extraction, generally suffer from drawbacks such as the volatility, flammability, and explosiveness of organic solvents, equipment corrosion caused by high concentrations of inorganic salts, and cumbersome and costly subsequent salt recovery and wastewater treatment processes. In contrast, while organic solvent-based sugar extraction technology has shown some improvement, the organic solvents used, such as acetonitrile, isopropanol, and ethyl acetate, still possess biotoxicity and volatility, limiting their application in the purification of biological products.

[0004] Unlike common organic solvents, some newly developed green solvents, such as triethyl citrate, tributyl citrate, tricresyl phosphate, triethyl phosphate, and isopropyl myristate, possess characteristics such as high boiling points, low volatility, biodegradability, low toxicity, and environmental friendliness, and are widely used in pharmaceuticals, polymer materials, and even cosmetics. If green solvents can be used to replace traditional organic solvents in the development of novel saccharification extraction technologies and applied to phage isolation, it will undoubtedly overcome the drawbacks of traditional saccharification extraction. Moreover, there are no reported applications of saccharification extraction technology in phage isolation and purification; this new technology will possess significant technological foresight and innovation.

[0005] This patent application will develop a glycolysis extraction technology based on green solvents and apply it to the efficient separation and purification of bacteriophages. The invention aims to develop a novel glycolysis extraction technology based on green solvents, promoting the environmentally friendly and sustainable development of phage preparation processes, and providing key technical support for the industrialization of large-scale, low-cost phage therapy. Summary of the Invention

[0006] The purpose of this invention is to provide a sugar precipitation extraction method based on green solvents, which can achieve high recovery rate and high purity separation of bacteriophages by optimizing the combination of sugar and green solvents and process parameters, while reducing production costs and environmental pollution.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: S1: Dissolve sugars in crude lysate containing bacteriophages to form a sugar phase mixture; S2: Add green solvent to the sugar phase mixture, mix and let stand to separate into layers, forming a three-phase system containing an upper phase, an intermediate phase and a lower phase; S3: Separate the intermediate phase to obtain purified bacteriophage; The green solvent mentioned in step S2 is one of triethyl citrate (TEC), tributyl citrate (TBC), tricresyl phosphate (TCP), triethyl phosphate (TEP), or isopropyl myristate (IPM).

[0008] Furthermore, in step S1, the sugar substance is one of glucose, sucrose, or maltose.

[0009] Furthermore, the mass concentration of the sugar is 18.0-32.0%, and the mass concentration of the green solvent is 10.0-50.0%.

[0010] Preferably, the sugar is glucose, and the green solvent is triethyl citrate.

[0011] Preferably, the glucose concentration is 28.0-30.0%.

[0012] Preferably, the mass concentration of the triethyl citrate is 20.0-40.0%.

[0013] Furthermore, the settling time for stratification described in step S2 is 40 minutes or more.

[0014] Furthermore, the green solvent can be recycled and reused three times.

[0015] Compared with the prior art, the beneficial effects of this invention are as follows: Highly efficient separation and purification: Phages are directly extracted and separated from phage lysate, with a phage recovery rate of up to 82.42% and an impurity removal rate of ≥75%. While achieving a high yield in one-step separation, proteins, pathogenic cells, and endotoxins are removed simultaneously, resulting in preliminary purification of the phages. Green and environmentally friendly: It uses non-toxic, biodegradable green solvents such as triethyl citrate, and replaces salt with sugar to avoid environmental pollution caused by volatile organic solvents and high salt content; The method is simple to operate and low in cost: no centrifugation is required, extraction can be completed within 40 minutes, the green solvent and sugar are inexpensive and recyclable, making it suitable for large-scale production; High versatility: Applicable to the isolation and purification of various bacteriophages, and has universality for bacteriophage isolation. Attached Figure Description

[0016] Figure 1 Flowchart of sugar precipitation extraction operation Figure 2 Stability of bacteriophages in different sugar solutions Figure 3 Stability of bacteriophages in different green solvents Figure 4 Effect of sugar concentration on phage recovery rate in saccharification extraction system Figure 5 Distribution behavior of bacteriophages, proteins, cells and endotoxins in glycosylation extraction systems Among them, T, M, and B refer to the upper phase, middle phase, and lower phase, respectively. Figure 6 The conductivity variation trend of the glycosylation extraction system (a) and the phase separation process (b) Detailed Implementation

[0017] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified in the embodiments, the experimental methods used are conventional methods, and the reagents or instruments used are all commercially available conventional products.

[0018] 1. Materials and methods used in the embodiments 1.1 Chemicals and Reagents Detailed information on the chemicals and reagents used is shown in Table 1:

[0019] 1.2 Bacteriological strains and bacteriophages The strains and bacteriophage information used are shown in Table 2.

[0020] ; 1.3 Main Equipment The main equipment used is shown in Table 3.

[0021] ; 1.4 Analytical and Detection Methods The analytical methods for phage titer, protein concentration, cell biomass, endotoxin and conductivity are shown in Table 4.

[0022] ; 1.4.1 Phage titer determination Phage titer refers to the number of phages per milliliter of liquid (PFU / mL). First, 0.5 mL of host bacterial suspension (OD200) is prepared. 600nm≈0.6) is spread on LB agar plates, and then the plates are dried for 30 min; then 5 μL of serially diluted phage sample is spotted on the bacterial cover layer, and then dried for 10 min; after incubation at 37℃ for 3 h, the phage plaques are observed, and the phage titer (PFU / mL) is calculated according to formula (1). The number of plaques should be in the range of 1-10 to avoid overlap and influence.

[0023] (1) 1.4.2 Protein determination Protein concentration was determined using the BCA protein assay kit, with optical density measured at 562 nm. Bovine serum albumin (BSA) was used as the standard protein, and the protein concentration in the sample was calculated based on the standard curve.

[0024] 1.4.3 Biomass Measurement The optical density (OD) value at 600 nm was measured using a UV-Vis spectrophotometer. 600 nm The study aimed to assess the biomass of host cells and other suspended solids (e.g., cell debris) in the pretreated phage lysate and glycolysis extraction system.

[0025] 1.4.4 Endotoxin quantification Endotoxin levels were measured using an endotoxin assay kit with endpoint colorimetric assay (LAL assay). The colorimetric reaction was detected at 545 nm to quantify the endotoxin concentration. Phage samples were appropriately diluted with endotoxin-free water prior to assay.

[0026] 1.5 Preparation of phage lysate 1.5.1 Host Bacterial Culture LB liquid medium, composed of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl, was used. The medium was incubated at 37°C and 200 rpm for 8 h. OD 600 ≈0.5, cell density approximately 10 9 CFU / mL.

[0027] 1.5.2 Preparation of phage lysate Add the phage stock solution with an infection multiplicity (MOI) of 0.1 to 10 mL of logarithmic phase host bacterial culture, mix well, and transfer to 50 mL of fresh LB medium. Incubate at 37°C and 200 rpm for 4-6 h to obtain phage lysate, which contains phage, released proteins, endotoxins, other components of the host bacteria, whole cells, and cell debris.

[0028] The lysate was filtered through a 0.22 μm filter membrane to remove bacterial cell fragments, yielding the phage treatment solution.

[0029] Phage lysate or processing solution can be stored in SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl, pH 7.5) at 4°C.

[0030] 1.6 Experimental Methods 1.6.1 Stability of bacteriophages in sugars and green solvents Phage lysis buffer was added to a solution of a certain concentration of sugar (glucose / sucrose / maltose) or green solvent (triethyl citrate (TEC), tributyl citrate (TBC), tricresyl phosphate (TCP), triethyl phosphate (TEP), isopropyl myristate (IPM)) and samples were taken over time to detect phage survival. Simultaneously, phage lysis buffer was added to SM buffer as a control group.

[0031] The survival percentage of bacteriophages in the sugar solution is calculated according to formula (2): (2) in, C 1 and C 0 The figures show the phage titers of the mixture after sugar addition and the control group, respectively.

[0032] When only green solvent is added, the bacteriophages are mainly in the lower phase, and the survival percentage of the bacteriophages is calculated according to formula (3): (3) in, It is the phage titer of the lower phase; and These represent the volumes of the lower phase with added green solvent and the control group, respectively.

[0033] 1.6.2 Sugar precipitation extraction Sugar was dissolved in the phage lysis buffer, green solvent was added, and the mixture was vortexed for 1 min. The mixture was allowed to stand until phase separation occurred, forming an upper phase, an intermediate phase, and a lower phase. Each phase was collected, and the phage titer, protein concentration, endotoxin, and cell mass were measured. The phage recovery rate, impurity removal rate, partition coefficient, and phase volume ratio were calculated according to formulas (4)-(7). (4) (5) (6) (7) in, (PFU / mL) and (mL) represents the phage titer and volume of the intermediate phase, respectively. (PFU / mL) and (mL) represent the phage titer and volume of the pretreated phage lysate, respectively; (mg / mL or CFU / mL) and (mg / mL or CFU / mL) are the concentrations of impurities (i.e., proteins, bacterial cells, or endotoxins) in the intermediate phase and pretreated phage lysates, respectively. (mg / mL or CFU / mL) and (mg / mL or CFU / mL) represent the concentrations of the upper and lower phases, respectively. (mL) and (mL) represents the volume of the upper phase and the lower phase, respectively.

[0035] The concentration factor (CF) of a phage is the volume of the phage lysate or treatment solution divided by the volume of the concentrated intermediate phase of the phage.

[0036] The phage separation factor (SF) for total protein and cells refers to the partition coefficient of phage in the intermediate phase and the protein or cell-enriched phase divided by the partition coefficient of protein or cells in the corresponding two phases. Protein is usually enriched in the lower phase, and cells are enriched in the upper phase.

[0037] The effect of different extraction methods on phage extraction efficiency This comparative example cites existing technical literature to detail the effects of aqueous two-phase systems and salting-out extraction systems on phage extraction.

[0038] Comparative Example 1: Literature ( Journal of Biotechnology In [2002, 93(1): 1-14], a two-phase aqueous system consisting of polyethylene glycol 400 (PEG400) and dipotassium hydrogen phosphate (K2HPO4) was used to extract and separate the crude lysate of M13 bacteriophage. The bacteriophage was partitioned into the upper phase, and the recovery rate was greater than 83%.

[0039] Comparative Example 2: Literature ( Separation and Purification Technology In this study (2019, 211: 322-329), a two-phase aqueous system consisting of polyethylene glycol 8000 (PEG8000) and potassium phosphate was used to extract phage lysates of φSan23 that had been pre-centrifuged and filtered. The phages partitioned into the lower phase, and the recovery rate was 95%.

[0040] Comparative Example 3: Literature ( Journal of Chromatography B In 2007, 854(1-2): 13-19, a two-phase aqueous system composed of PEG8000 and potassium phosphate was used to extract a small volume (300 μL) of crude T4 phage lysate. The recovery rate of phage in the upper phase was only 38%.

[0041] Comparative Example 4: Literature ( Separation and Purification Technology (2020, 242:116784) A two-step salting-out extraction method was used to separate Klebsiella pneumoniae phage phiKpS2. The first step used a salting-out extraction system consisting of 10 wt% sodium citrate and 30 wt% ethyl acetate to enrich the target phage in the lower phase, achieving a recovery rate of 99%. The second step involved adding n-propanol to the salt-rich lower phase for extraction, transferring the phage to the intermediate phase, with a recovery rate of 77%.

[0042] Comparative Example 5: Literature ( Journal of Chromatography A (2022, 1679: 463407) adopts 18% ( w / w ) ammonium citrate and 40% ( w / w A salt-out extraction system composed of ethyl acetate was used to extract Acinetobacter baumannii phage phiAB9 after centrifugation and filtration pretreatment. The phage was enriched in the intermediate phase, with a recovery rate of 91%.

[0043] In summary, existing extraction technologies have the following significant drawbacks: In a polyethylene glycol (PEG)-potassium phosphate aqueous two-phase system, bacteriophages are distributed in either the upper or lower phase, requiring further separation from the PEG in the upper phase or the salt in the lower phase, which complicates the process. PEG is expensive, limiting its large-scale industrial application; additionally, the use of salt presents challenges in recycling and wastewater treatment, and can corrode industrial equipment.

[0044] Bacteriophages are ultimately allocated to the intermediate phase in the salting-out extraction system, but ethyl acetate is a volatile compound, and there are also challenges in recovering organic salts.

[0045] Example 1: Stability screening of bacteriophages in sugars and green solvents To determine the feasibility of the sugar precipitation extraction system, the stability of bacteriophages in different sugars and green solvents was first investigated. Sugars included glucose, sucrose, and maltose, while green solvents included triethyl citrate, tributyl citrate, tricresyl phosphate, triethyl phosphate, and isopropyl myristate. Klebsiella pneumoniae bacteriophage lysate was added to 30% ( w / w Add phage titer of 1 × 10⁻⁶ to sugar or green solvent solution. 8 The survival rate of bacteriophages was investigated at PFU / mL and a temperature of 20±5 °C for 0–24 h. The stability of bacteriophages in different sugar solutions was also assessed. Figure 2 As shown, the phage maintained a high titer (>95%) in glucose, sucrose, and maltose for 24 hours, and the titer decreased by less than 1% in all samples within 6 hours, meaning that extraction within 6 hours had almost no impact. The stability results of the phage in different green solvents are as follows: Figure 3 As shown, phages maintained high titers after 24 h of exposure in green solvents, such as 92% in triethyl citrate. Retention rates for other green solvents were: tributyl citrate (91%), isopropyl myristate (87%), triethyl phosphate (77%), and tricresyl phosphate (59%). The results indicate that glucose, sucrose, maltose, and most green solvents (triethyl citrate, tributyl citrate, isopropyl myristate, and triethyl phosphate) exhibit good stability to phages and can be used for the construction of subsequent saccharification extraction systems.

[0046] Example 2: Effect of sugar on phage extraction In 30% w / w Under specific conditions, the added phage titer of triethyl citrate was 1 × 10⁻⁶. 8 The effects of glucose, sucrose, and maltose concentrations (PFU / mL) on phage recovery and impurity removal were investigated at a temperature of 20±5 °C within the sugar concentration range of 18%–32%. The results of the effect of sugar concentration on phage recovery in the glycolysis extraction system are shown below. Figure 4 As shown, when the sugar mass fraction was 24%-32%, the recovery rate of intermediate-phase bacteriophages in both the glucose and sucrose systems was higher than 40%, while the highest recovery rate of the maltose system was consistently lower than 43%. The glucose system performed best, with intermediate-phase bacteriophage recovery rates exceeding 70% under 28%-30% mass fraction conditions, reaching a peak of 82.42% at 28%. Regarding impurity removal, the comprehensive separation performance comparison of different sugars under optimal systems is shown in Table 5: the intermediate-phase protein removal rate of the glucose system with a mass fraction higher than 24% remained consistently above 70%, significantly outperforming the sucrose and maltose systems. In conclusion, the glucose system exhibits the best performance in terms of bacteriophage recovery rate, protein removal rate, and economic efficiency, with its optimal mass fraction range being 28%-30%.

[0047] ; Example 3: Effect of green solvents on phage isolation In this embodiment, the sugar content was fixed at 28% ( w / w Under glucose conditions, the effects of five green solvents—triethyl citrate, tributyl citrate, tricresyl phosphate, triethyl phosphate, and isopropyl myristate—on phage recovery and impurity removal were systematically compared. First, the effects of triethyl citrate at concentrations of 10%-50% were investigated. w / w The impact within the range, the results are as follows Figure 5As shown, when the concentration of triethyl citrate is between 20% and 40%, the recovery rate of the mesophase phage is higher than 75%, and the protein and cell removal rates are maintained above 80%. When the concentration is below 20% or above 40%, the recovery rate of the mesophase phage decreases significantly. Therefore, the applicable concentration range for triethyl citrate is 10.0%–50.0%, preferably 20.0%–40.0%.

[0048] At a fixed glucose concentration of 28% ( w / w Under the condition of a green solvent concentration of 30% (w / w), the effects of different green solvents on phage separation were further compared. The results are shown in Table 6: most glucose was distributed in the lower phase with a recovery rate greater than 92%, while more than 83% of the solvent was partitioned into the upper phase. Among them, the triethyl citrate system performed best, with a phage recovery rate of 82.42%, a protein removal rate of 89.76%, a cell removal rate of 88.49%, and an endotoxin removal rate of 81.09% in the intermediate phase. In contrast, the recovery rates of solvents such as tricresyl phosphate and triethyl phosphate were relatively low. Considering separation efficiency, phase forming ability, and biosafety, triethyl citrate is the most suitable green solvent for phage separation due to its non-toxicity, easy degradation, low volatility, and excellent phase separation characteristics.

[0049] ; Example 4 Optimization of the saccharification extraction system Based on the preliminary determination of the effects of sugar and green solvent on glycolytic extraction, the system composition and extraction conditions were further optimized. First, the mass fraction of sugar was fixed at 28% or 30%, and the mass fraction of green solvent was 30%. The effects of different combinations of sugar and green solvent on phage recovery were investigated. The results are shown in Table 7: the glycolytic extraction system composed of 28% glucose and 30% triethyl citrate showed the highest phage recovery rate, which was superior to other glycolytic extraction systems.

[0050] ; Secondly, at a glucose concentration of 28% (w / w), the effects of triethyl citrate on concentrations of 10%-50% were investigated. w / wThe effects of the triethyl citrate concentration on phage recovery, protein, cell, and endotoxin removal rates within the specified range are shown in Table 8. As the mass fraction of triethyl citrate increases, the recovery rate of the mesophase phage in the glycolytic extraction system initially increases and then decreases. The maximum recovery rate (82.42%) is achieved when the triethyl citrate concentration is 30%, and the removal rates of protein, cells, and endotoxins are also higher than under other extraction conditions, at 89.76%, 88.49%, and 81.09%, respectively. The separation factors of the phage relative to the aforementioned impurities are 149.72, 109.64, and 5.42, respectively, achieving a 46.67-fold concentration effect. Under these optimized conditions, this process achieves high phage recovery, high-purity enrichment, and efficient removal of multiple impurities, making it suitable for large-scale production.

[0051] ; Example 5: Optimization of Extraction Time Preparation of 28% ( w / w ) glucose, 30% ( w / w A glycolytic extraction system composed of triethyl citrate and crude phage lysate was used. After vortexing and settling, the conductivity of the system was monitored using a conductivity meter to determine the phase separation. If no significant change in conductivity was observed, the system was allowed to stand for another 18 hours (without centrifugation). The control group was centrifuged immediately after vortexing. The migration behavior of phages, proteins, and cells during phase separation was observed, and the removal rates of phages, proteins, and bacterial cells at different time points were measured.

[0052] Figure 6 The results show that the system initially exists as a single phase after vortex mixing (0 min). Droplets begin to form in the upper phase at 4 min. Between 6 and 16 min, these droplets aggregate, break up, and gradually decrease in number. Between 20 and 30 min, a small number of droplets float in the intermediate phase. By 40 min, the system has completely separated into three phases, the droplets have completely disappeared, and the continuous phase is clear. Conductivity monitoring shows that the conductivity of the lower phase stabilizes at 3.96 mS / cm after 18 min, while the conductivity of the upper phase continues to change until it stabilizes at 0.083 mS / cm at 40 min, indicating that the system reaches phase equilibrium within 40 min.

[0053] Further analysis of the extraction kinetics revealed that the phage recovery rate in the mesophase continuously increased with prolonged settling time, while the recovery rate in the lower phase decreased accordingly, indicating that the phage gradually migrated from the lower phase to the mesophase. After 40 min, the phage recovery rate in the mesophase stabilized, and protein and cell partitioning were completed within 40 min and 30 min, respectively. These results demonstrate that this glycosylation extraction system can achieve efficient phage separation within 40 min, without the need for centrifugation, making it suitable for large-scale production.

[0054] Example 6: Verification of the universality of the glycosylation extraction system for different bacteriophages This embodiment verifies that 28% ( w / w ) Glucose and 30% ( w / w The glycosylation extraction system based on triethyl citrate is applicable to the separation of various bacteriophages. *Escherichia coli* λ phage, *Stenotrophomonas maltophilia* phages phiSM29 and phiSM30 were selected as representative phages for extraction and separation using this system. The results are shown in Table 9: all three phages were effectively enriched in the intermediate phase, with recoveries exceeding 74%, while the removal rates of protein and host cells reached over 89% and 90%, respectively. These results demonstrate that the glucose / triethyl citrate glycosylation extraction system has good separation and purification effects on different types of bacteriophages, exhibiting good versatility and application potential.

[0055] ; Example 7: Recycling of Triethyl Citrate In 28% ( w / w Glucose / 30% w / w Solvent recovery and reuse experiments were conducted using a triethyl citrate saccharification extraction system to evaluate its impact on separation efficiency. After each extraction, the supernatant (rich in triethyl citrate) was collected, and the recovered solvent was directly used in the next round of saccharification extraction experiments. The system was recycled five times, and the results are shown in Table 10.

[0056] ; Triethyl citrate maintained a phage recovery rate of over 51% and a protein, cell, and endotoxin removal rate of over 66% within three cycles, still meeting the requirements of the separation and purification process. By the fourth use, all indicators showed a significant decrease, thus confirming its ability to be recycled three times. This example demonstrates that this green solvent possesses excellent recyclability, which is beneficial for reducing production costs and environmental pollution.

[0057] In summary, this invention establishes an efficient, green, and low-cost method for phage isolation and purification, which has good prospects for industrial application.

[0058] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A method for isolating and purifying bacteriophages using glycosylation extraction based on green solvents, characterized in that, Includes the following steps: S1: Dissolve sugars in the crude lysate of pathogens containing bacteriophages to form a sugar phase mixture; S2: Add green solvent to the sugar phase mixture, mix and let stand to separate into layers, forming a three-phase system containing an upper phase, an intermediate phase and a lower phase; S3: Separate the intermediate phase to obtain purified bacteriophage; The green solvent mentioned in step S2 is one of triethyl citrate (TEC), tributyl citrate (TBC), tricresyl phosphate (TCP), triethyl phosphate (TEP), or isopropyl myristate (IPM).

2. The method for isolating and purifying bacteriophages based on glycosylation extraction using a green solvent according to claim 1, characterized in that, The sugar substance mentioned in step S1 is one of glucose, sucrose or maltose.

3. The method for isolating and purifying bacteriophages based on glycosylation extraction using a green solvent according to claim 2, characterized in that, The mass concentration of the sugar is 18.0-32.0%, and the mass concentration of the green solvent is 10.0-50.0%.

4. The method for isolating and purifying bacteriophages based on glycosylation extraction using a green solvent according to claim 2, characterized in that, The sugar is glucose, and the green solvent is triethyl citrate.

5. The method for isolating and purifying bacteriophages based on glycosylation extraction using a green solvent according to claim 4, characterized in that, The glucose concentration is 28.0-30.0%.

6. The method for isolating and purifying bacteriophages based on glycosylation extraction using a green solvent according to claim 4, characterized in that, The mass concentration of the triethyl citrate is 20.0-40.0%.

7. The method for isolating and purifying bacteriophages based on glycosylation extraction using a green solvent according to claim 1, characterized in that, The settling time for stratification mentioned in step S2 is more than 40 minutes.

8. The method for isolating and purifying bacteriophages based on glycosylation extraction using a green solvent according to claim 1, characterized in that, The green solvent can be recycled and reused three times.

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