A bacteriophage inhibitor and its application

Abstract

The present invention discloses a bacteriophage inhibitor and its application. A bacteriophage inhibitor comprises, by weight, 30-60 parts of carboxymethylated seabuckthorn polysaccharide, 1.8-2.2 parts of citric acid, 2.3-2.7 parts of sodium citrate, 0.8-1.2 parts of D-sodium gluconate, 4-6 parts of dihydroxyethylglycine, 1.8-2.2 parts of sodium hexametaphosphate, and 0.8-1.2 parts of oxalic acid, and the amount of sodium hydroxide is added according to the pH value when the solution is prepared. The product is designed as a mixed prefabricated product; or it can be configured into liquids of various concentrations and diluted as needed when used. This product is used in a prokaryotic expression system, can effectively inhibit the contamination of cells by phages, significantly improve the efficiency and quality of scientific research and production, and provide researchers with a more relaxed fermentation environment. At the same time, the synergistic effect of the carboxymethylated seabuckthorn polysaccharide, citric acid, sodium citrate, and oxalic acid contained in the phage inhibitor can significantly improve its inhibitory effect on bacteriophages.

Description

Technical Field

[0001] The present invention relates to the technical field of bacteriophage and prokaryotic expression, in particular to a bacteriophage inhibitor and application thereof. Background Art

[0002] Prokaryotic expression systems, as a widely used gene expression technology, play a key role in scientific research fields such as protein and enzyme production, vaccine development, and genetically engineered drug development. However, prokaryotes are often threatened by bacteriophage contamination, which not only leads to abnormal cell growth and death, but also severely reduces production efficiency, affects product quality, and significantly increases R&D and production costs and time.

[0003] Currently, common methods for managing phage contamination, such as building cleanroom labs and using disinfectants, are costly, inefficient, incomplete, and can delay research and production. The phage inhibitors developed in this project precisely target the impact of phage contamination on scientific research. Simply adding a trace amount of the inhibitor during cell growth can effectively inhibit phage contamination, significantly improving the efficiency and quality of research and production.

[0004] Given the lack of products on the market that can effectively treat phage contamination without affecting the normal growth process of cells, I have made unremitting efforts and repeated experiments, and finally successfully developed a stable and reliable phage inhibitor. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a bacteriophage inhibitor and its application to solve the problems raised in the above technical background.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions:

[0007] In a first aspect, the present invention provides a phage inhibitor comprising carboxymethylated seabuckthorn polysaccharide, citric acid, sodium citrate, sodium D-gluconate, dihydroxyethylglycine, sodium hexametaphosphate, oxalic acid, and sodium hydroxide.

[0008] Preferably, a phage inhibitor comprises, by weight, 30-60 parts of carboxymethylated seabuckthorn polysaccharide, 1.8-2.2 parts of citric acid, 2.3-2.7 parts of sodium citrate, 0.8-1.2 parts of sodium D-gluconate, 4-6 parts of dihydroxyethylglycine, 1.8-2.2 parts of sodium hexametaphosphate, and 0.8-1.2 parts of oxalic acid, wherein the amount of sodium hydroxide is added according to the pH value when preparing the solution.

[0009] Preferably, the phage inhibitor product is designed as a mixed preformulation; or configured as a liquid of various concentrations, which is diluted as needed during use. The mixed preformulation is a solid mixture of each component in the formula prepared in proportion.

[0010] Preferably, when the phage inhibitor is configured into a solution with the lowest concentration, the phage inhibitor comprises 3-6 mg / L carboxymethylated seabuckthorn polysaccharide, 0.18-0.22 mg / L citric acid, 0.23-0.27 mg / L sodium citrate, 0.08-0.12 mg / L sodium gluconate, 0.4-0.6 mg / L dihydroxyethylglycine, 0.18-0.22 mg / L sodium hexametaphosphate, and 0.08-0.12 mg / L oxalic acid; and the pH is adjusted to 7.2-7.4 using sodium hydroxide.

[0011] Preferably, the phage inhibitor comprises 4 mg / L carboxymethylated seabuckthorn polysaccharide, 0.2 mg / L citric acid, 0.25 mg / L sodium citrate, 0.1 mg / L sodium D-gluconate, 0.5 mg / L dihydroxyethylglycine, 0.2 mg / L sodium hexametaphosphate, and 0.1 mg / L oxalic acid; and the pH is adjusted to 7.4 using sodium hydroxide.

[0012] Preferably, the solvent is water or culture medium.

[0013] Preferably, the preparation method of the carboxymethylated seabuckthorn polysaccharide is as follows: 400 mg of seabuckthorn polysaccharide is weighed and dissolved in 80 mL of 4 mol / L NaOH solution, then 100 mL of monochloroacetic acid with a concentration of 2 mol / L is slowly added, the reaction temperature is fixed at 60° C., and the reaction is heated in a water bath for 3 hours. After the reaction is completed, the reaction solution is cooled to room temperature, the pH of the reaction solution is adjusted to 7.0 with glacial acetic acid, and the filtrate is collected after filtration and placed in a dialysis bag (8-14 kDa) and dialyzed with distilled water for 96 hours. The solution in the dialysis bag is vacuum concentrated and freeze-dried to obtain the carboxymethylated seabuckthorn polysaccharide.

[0014] Preferably, the preparation method of sea buckthorn polysaccharide is as follows: 100g of dried sea buckthorn fruit is crushed, passed through a 40-mesh sieve, and 10 times the volume of petroleum ether is added and refluxed and extracted twice, each time for 1 hour, to remove the fat-soluble substances therein, filter, and dry the filter residue to constant weight for use; weigh the pretreated sea buckthorn filter residue, add distilled water at a material-liquid ratio of 1:10 (g / mL), and extract with the assistance of ultrasound at 70°C (ultrasonic power 180W) for 1 hour, extract twice, combine the extracts and concentrate to 100ml, add 4 times the volume of anhydrous ethanol, and stand at 4°C for 12 hours to produce precipitation, centrifuge to obtain the precipitate, redissolve it in water, and then vacuum freeze-dry to obtain crude sea buckthorn polysaccharide; use Sevag solution to remove protein from the crude sea buckthorn polysaccharide to obtain sea buckthorn polysaccharide.

[0015] Further preferably, the specific method for removing protein from crude seabuckthorn polysaccharides using Sevag solution to obtain seabuckthorn polysaccharides is as follows: weigh crude seabuckthorn polysaccharides and dissolve them in distilled water to obtain a 0.1 g / ml crude polysaccharide solution, then mix the crude seabuckthorn polysaccharide with the Sevag solution at a volume ratio of 1:3, stir thoroughly for 30 minutes, and then centrifuge at 4000 rpm for 10 minutes, collect the supernatant, and repeat the above operation 5 times; combine the supernatants, evaporate and concentrate to remove organic reagents, and then vacuum freeze-dry to obtain seabuckthorn polysaccharides. The Sevag solution is prepared by mixing chloroform and n-butanol in a ratio of 4:1 and stored in the dark.

[0016] In a second aspect, the present invention provides the use of the above-mentioned phage inhibitor in a prokaryotic expression system.

[0017] Preferably, the phage inhibitor is used to inhibit Escherichia coli phage.

[0018] Preferably, the phage inhibitor is used to inhibit Bacillus subtilis phage.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. This product significantly inhibits the adverse effects of lysogenic phages on the fermentation process and is available in two forms. The first is a high-concentration liquid additive, offering the advantages of low production costs and convenient transportation. The second is a pre-mixed product, which can be sold in combination, thereby increasing profits.

[0021] 2. This product is used in prokaryotic expression systems to effectively inhibit phage contamination of cells, significantly improve the efficiency and quality of scientific research and production, and provide researchers with a more relaxed fermentation environment.

[0022] 3. The synergistic effect of carboxymethylated seabuckthorn polysaccharide, citric acid, sodium citrate and oxalic acid contained in the phage inhibitor can significantly enhance its inhibitory effect on phage. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Figure 1 is a graph showing the inhibitory effects of different concentrations of phage inhibitors on Escherichia coli phage T4. Numbers 1-8 in the figure correspond to numbers 1-8 in Table 1.

[0024] Figure 2 This is a graph showing the inhibitory effects of different concentrations of phage inhibitors on Escherichia coli λ phage. Numbers 1-8 in the figure correspond to numbers 1-8 in Table 2.

[0025] Figure 3 It is a bar graph showing the inhibition rate of different phage inhibitors on Escherichia coli phage T4;

[0026] Figure 4It is a bar graph showing the inhibition rate of different phage inhibitors on Bacillus subtilis phage. DETAILED DESCRIPTION

[0027] The following describes the embodiments of the present invention through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. The details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other unless they conflict.

[0028] Example 1

[0029] A phage inhibitor comprises 6 mg / L carboxymethylated seabuckthorn polysaccharide, 0.18 mg / L citric acid, 0.23 mg / L sodium citrate, 0.12 mg / L sodium D-gluconate, 0.4 mg / L dihydroxyethylglycine, 0.22 mg / L sodium hexametaphosphate, and 0.08 mg / L oxalic acid; and the pH value is adjusted to 7.2 with sodium hydroxide.

[0030] Example 2

[0031] A phage inhibitor comprises 3 mg / L carboxymethylated seabuckthorn polysaccharide, 0.22 mg / L citric acid, 0.27 mg / L sodium citrate, 0.08 mg / L sodium D-gluconate, 0.6 mg / L dihydroxyethylglycine, 0.18 mg / L sodium hexametaphosphate, and 0.12 mg / L oxalic acid; and the pH value is adjusted to 7.4 with sodium hydroxide.

[0032] Example 3

[0033] A phage inhibitor comprises 4 mg / L carboxymethylated seabuckthorn polysaccharide, 0.2 mg / L citric acid, 0.25 mg / L sodium citrate, 0.1 mg / L sodium D-gluconate, 0.5 mg / L dihydroxyethylglycine, 0.2 mg / L sodium hexametaphosphate, and 0.1 mg / L oxalic acid; and the pH value is adjusted to 7.4 with sodium hydroxide.

[0034] The preparation method of the bacteriophage inhibitor of this embodiment is as follows: 4 g of carboxymethylated seabuckthorn polysaccharide, 0.2 g of citric acid, 0.25 g of sodium citrate, 0.1 g of sodium D-gluconate, 0.5 g of dihydroxyethylglycine, and 0.2 g of sodium hexametaphosphate are weighed according to the ratio of Example 3, and the mixture is filled to 800 mL with pure water. 1 mL of 10% oxalic acid aqueous solution is added, and the pH is adjusted to approximately 7 with solid sodium hydroxide while stirring. The pH is then adjusted to 7.4 with 0.5 M sodium hydroxide. After the pH stabilizes, water is added to bring the volume to 1 L. For use, a corresponding culture medium or water is selected as needed, and a 1:999 dilution is performed. 1 mL of the solution after the volume is diluted with culture medium or water to 800 mL. The pH is then adjusted to 7.4 with 0.5 M sodium hydroxide. After the pH stabilizes, culture medium or water is added to bring the volume to 1 L. This yields the bacteriophage inhibitor of this embodiment.

[0035] The carboxymethylated seabuckthorn polysaccharide used in Examples 1-3 was prepared by the following method:

[0036] 400 mg of seabuckthorn polysaccharide was dissolved in 80 mL of 4 mol / L NaOH solution. 100 mL of 2 mol / L monochloroacetic acid was then slowly added. The reaction temperature was fixed at 60°C and the reaction was heated in a water bath for 3 h. After completion, the reaction solution was cooled to room temperature and the pH was adjusted to 7.0 with glacial acetic acid. The filtrate was collected after filtration and dialyzed against distilled water in a dialysis bag (8-14 kDa) for 96 h. The solution in the dialysis bag was vacuum concentrated and freeze-dried to obtain carboxymethylated seabuckthorn polysaccharide. The dialysis bag MD55 (8000-14000D) was purchased from Beijing Solebau Technology Co., Ltd.

[0037] The preparation method of sea buckthorn polysaccharide is as follows: 100g of dried sea buckthorn fruit is crushed, passed through a 40-mesh sieve, and 10 times the volume of petroleum ether is added and refluxed and extracted twice, each time for 1 hour, until the fat-soluble substances therein are removed, filtered, and the filter residue is dried to constant weight for use; the pretreated sea buckthorn filter residue is weighed, distilled water is added according to a material-liquid ratio of 1:10 (g / mL), ultrasonically assisted extraction is performed at 70°C (ultrasonic power 180W) for 1 hour, and extracted twice, the extracts are combined and concentrated to 100ml, 4 times the volume of anhydrous ethanol is added, and the extract is allowed to stand at 4°C for 12 hours to produce precipitation, the precipitate is collected by centrifugation, redissolved in water, and then vacuum-freeze-dried to obtain crude sea buckthorn polysaccharide; and the protein in the crude sea buckthorn polysaccharide is removed by Sevag solution to obtain sea buckthorn polysaccharide. The specific method for removing protein from crude seabuckthorn polysaccharides using Sevag solution is as follows: weigh crude seabuckthorn polysaccharides and dissolve them in distilled water to obtain a 0.1g / ml crude polysaccharide solution. The crude polysaccharide solution is then mixed with Sevag solution in a 1:3 volume ratio. The mixture is stirred thoroughly for 30 minutes, then centrifuged at 4000 rpm for 10 minutes. The supernatant is collected and repeated five times. The supernatants are combined, evaporated and concentrated to remove organic reagents, and then vacuum freeze-dried to obtain seabuckthorn polysaccharides. The Sevag solution is prepared by mixing chloroform and n-butanol in a 4:1 ratio and stored in the dark. Seabuckthorn dried fruit is produced in Altay, Xinjiang.

[0038] Comparative Example 1

[0039] This comparative example is similar to Example 3, except that the carboxymethylated seabuckthorn polysaccharide is removed.

[0040] Comparative Example 2

[0041] This comparative example is similar to Example 3, except that citric acid is removed.

[0042] Comparative Example 3

[0043] This comparative example is similar to Example 3, except that sodium citrate is removed.

[0044] Comparative Example 4

[0045] This comparative example is similar to Example 3, except that oxalic acid is removed.

[0046] Comparative Example 5

[0047] This comparative example is similar to Example 3, except that carboxymethylated seabuckthorn polysaccharide is replaced with seabuckthorn polysaccharide. The preparation method of seabuckthorn polysaccharide refers to the preparation method of seabuckthorn polysaccharide in Example 3.

[0048] Experiment 1 Inhibitory effect of different concentrations of phage inhibitors on Escherichia coli phage

[0049] 1. Experimental materials: E. coli BL21 (CICC 23796), E. coli K12 (CICC 10424), bacteriophage T4, and λ phage. E. coli BL21 (CICC 23796) and E. coli K12 (CICC 10424) were purchased from China Industrial Culture Collection Administration Center; bacteriophage T4 was E. coli phage T4 SHBCC D24645, and λ phage was E. coli λ phage SHBCC D24685. E. coli phage T4 SHBCC D24645 and E. coli λ phage SHBCC D24685 were purchased from Shanghai Ruichu Biotechnology Co., Ltd.

[0050] LB liquid medium: Weigh 10 g of tryptone, 5 g of yeast extract powder, and 10 g of NaCl, dissolve them thoroughly in 900 ml of distilled water, and then make up to 1000 ml with distilled water. Adjust the pH to 7.4 with 10 mol / l NaOH. Sterilize in an autoclave at 121°C for 20 min. Cool and store at room temperature until ready for use.

[0051] Phage inhibitor 1X, 2X, 4X, 6X, 8X, 10X configurations: The culture medium used in Example 3 was LB medium. The specific method was as follows: 4 g of carboxymethylated seabuckthorn polysaccharide, 0.2 g of citric acid, 0.25 g of sodium citrate, 0.1 g of sodium D-gluconate, 0.5 g of dihydroxyethylglycine, and 0.2 g of sodium hexametaphosphate were weighed, and the mixture was filled to 800 mL with purified water. 1 mL of 10% oxalic acid aqueous solution was added, and the pH was adjusted to approximately 7 with solid sodium hydroxide while stirring continuously. The pH was then adjusted to 7.4 with 0.5 M sodium hydroxide. After the pH stabilized, water was added to make the volume 1 L to obtain the phage inhibitor mother solution. 1 mL, 2 mL, 4 mL, 6 mL, 8 mL, and 10 mL of the phage inhibitor stock solution were respectively added to 800 mL of LB liquid medium, and the pH was adjusted to 7.4 with 0.5 M sodium hydroxide. After the pH stabilized, LB medium was added to make the volume 1 L, thus obtaining LB medium containing different concentrations of phage inhibitors (1X, 2X, 4X, 6X, 8X, 10X), 1X, 2X, etc., i.e., 1 times, 2 times, ... and so on, i.e., the phage inhibitor concentration of Example 3, 2 times the phage inhibitor concentration of Example 3, ... and so on.

[0052] 2. Experimental methods:

[0053] Evaluation method: The effect of phage inhibitors in inhibiting phages can be determined by measuring the OD600 value of the bacterial solution using a spectrophotometer: when Escherichia coli is infected with a certain concentration of phage, if the OD600 value of the group with phage inhibitors added is significantly larger than that of the group without phage inhibitors, then there is an inhibitory effect, otherwise the opposite is true.

[0054] (1) Verify the inhibitory effect of phage inhibitors on phage T4 (virulent phage)

[0055] The experimental design is shown in Table 1. E. coli BL21 cultured to the logarithmic phase was transferred to LB medium containing different concentrations of phage inhibitors (1X, 2X, 4X, 6X, 8X, 10X), and then phage T4 (final titer of 10 7 pfu / mL), E. coli BL21 was used as a positive control, and phage T4 was added to E. coli BL21 as a negative control. The cells were cultured in a shake flask at 37°C for 18 h, and the OD600 value was measured. The results are shown in Table 3 and Figure 1 shown.

[0056] Table 1

[0057]

[0058] Note: √ in the table means addition.

[0059] (2) Verification of the inhibitory effect of phage inhibitors on λ phage (temperate phage)

[0060] The experimental design is shown in Table 2. E. coli K12 cultured to the logarithmic phase was transferred to LB medium containing different concentrations of phage inhibitors (1X, 2X, 4X, 6X, 8X, 10X), and then λ phage (final titer of 10 7 pfu / mL), E. coli BL21 was used as a positive control, and phage T4 was added to E. coli BL21 as a negative control. The cells were cultured at 37°C for 18 h, and the OD600 value was measured. The results are shown in Table 4 and Figure 2 shown.

[0061] Table 2

[0062]

[0063] Note: √ in the table means addition.

[0064] Table 3

[0065]

[0066] Note: √ in the table means addition.

[0067] From Table 3 and Figure 1It can be seen that compared with the positive group, the OD values ​​of the experimental group were all lower than those of the positive group, indicating that the simultaneous addition of phage inhibitors and phage T4 had no overall effect on the growth of E. coli BL21; compared with the negative control, it was observed that the OD values ​​of the experimental groups with different concentration gradients of phage inhibitors added were significantly higher than those of the negative control, indicating that the phage inhibitors had a significant inhibitory effect on phage T4. The experiment proved that phage inhibitors had a significant effect on virulent phages, indicating that the addition of phage inhibitors can indeed effectively inhibit the infection of bacteria by phage T4.

[0068] Table 4

[0069]

[0070] Note: √ in the table means addition.

[0071] From Table 4 and Figure 2 It can be seen that compared with the positive group, the OD values ​​of the experimental group were all lower than those of the positive group, indicating that the simultaneous addition of phage inhibitors and λ phage had no overall effect on the growth of E. coli K12; comparing the positive and negative controls, it was observed that the OD values ​​of the experimental groups with the addition of different concentration gradients of phage inhibitors were significantly higher than those of the negative control, indicating that the phage inhibitors had a significant inhibitory effect on λ phage, indicating that the phage inhibitors had a significant effect on temperate phages, indicating that the addition of phage inhibitors can indeed effectively inhibit the invasion of λ phage into bacteria.

[0072] Experiment 2 Effects of different phage inhibitors on phage T4

[0073] 1. Materials: E. coli BL21 (CICC 23796), E. coli phage T4 SHBCC D24645;

[0074] LB liquid medium: weigh trypsin

[0075] Dissolve 10 g of peptone, 5 g of yeast extract powder, and 10 g of NaCl in 900 ml of distilled water, then dilute to 1000 ml with distilled water. Adjust the pH to 7.4 with 10 mol / l NaOH. Sterilize in an autoclave at 121°C for 20 min. Cool and store at room temperature for later use.

[0076] When used for phage titer determination, the bottom solid culture medium is the above liquid culture medium with 1.5% agar added; the upper solid culture medium is the above liquid culture medium with 0.7% agar added.

[0077] Preparation of phage inhibitors corresponding to Examples 1-3 and Comparative Examples 1-5: Using Example 3 as an example, corresponding phage inhibitor mother solutions were prepared. The phage inhibitor mother solution of Example 3 was prepared by weighing 4 g of carboxymethylated seabuckthorn polysaccharide, 0.2 g of citric acid, 0.25 g of sodium citrate, 0.1 g of sodium D-gluconate, 0.5 g of dihydroxyethylglycine, and 0.2 g of sodium hexametaphosphate, adding purified water to 800 mL, then adding 1 mL of a 10% aqueous oxalic acid solution. While stirring continuously, the pH was adjusted to approximately 7 with solid sodium hydroxide, and then to 7.4 with 0.5 M sodium hydroxide. After the pH stabilized, water was added to 1 L to obtain the phage inhibitor mother solution. The preparation methods for the phage inhibitor mother solutions corresponding to Examples 1-2 and Comparative Examples 1-5 were similar to those for the phage inhibitor mother solution of Example 3.

[0078] 2. Experimental methods:

[0079] The phage titer was determined using a double-layer agar plate method. 10 mL of the bottom solid culture medium was poured into the plate, shaken, condensed, and set aside. A phage inhibitor (the stock solution of the phage inhibitors from Examples 1-3 and Comparative Examples 1-5, added in an amount of 0.1% of the test tube solution system), 1 mL of phage T4 solution (preferably to achieve a final dilution concentration of 260-300 PFU / mL in the test tube solution system), and 3 mL of E. coli BL21 suspension (OD600 of approximately 3.0) were added to sterile empty test tubes. The three were gently mixed and allowed to stand for 15 minutes to obtain a mixture. 500 μL of the mixture was added to 5 mL of the top solid culture medium insulated in a 45°C constant temperature water bath. The phage inhibitor was replaced with sterile LB medium as a control group. The mixture was slowly inverted to mix, then quickly poured onto the bottom solid culture medium and shaken to cover the entire plate. After cooling, the plate was placed in a 37°C incubator and cultured overnight (usually 16 hours). Plaque units (PFU) were counted, and one PFU was counted as one virus with infectious activity. Each group of experiments was repeated 5 times, and the inhibition rate (IR) of the phage inhibitor on the phage was calculated. The results are shown in Table 5 and Figure 3 As shown;

[0080] The inhibition rate (IR) formula is: IR (%) = (1-N / N0) × 100, where: the value of N is the titer of the added different phage inhibitors (Examples 1-3, Comparative Examples 1-5), and the value of N0 is the titer of the phage in the control group.

[0081] Table 5

[0082]

[0083] From Table 5 and Figure 3It can be seen that the inhibition rate of Examples 1-3 on bacteriophages is significantly higher than that of Comparative Examples 1-5, which indicates that the synergistic effect of carboxymethylated seabuckthorn polysaccharide, citric acid, sodium citrate and oxalic acid can significantly improve their inhibitory effect on Escherichia coli phages.

[0084] Experiment 3 Effects of different phage inhibitors on Bacillus subtilis phage

[0085] 1. Experimental materials: Bacillus subtilis CICC 10732, Bacillus subtilis phage (Bio-67330, original number BSNP01); Bacillus subtilis CICC 10732 was purchased from China Industrial Culture Collection Administration Center of Microorganisms, and Bacillus subtilis phage BSNP01 was purchased from Beijing Biobowei Biotechnology Co., Ltd.

[0086] 2. Nutrient broth medium: Weigh 5.0 g of peptone, 3 g of beef extract powder, and 5.0 g of NaCl, fully dissolve them in 900 ml of distilled water, and then dilute to 1000 ml with distilled water. Adjust the pH to 7.4 with 10 mol / l NaOH, sterilize in an autoclave at 121°C for 20 min, cool, and store at room temperature for later use.

[0087] When used for phage titer determination, the bottom solid culture medium is the above liquid culture medium with 1.5% agar added; the upper solid culture medium is the above liquid culture medium with 0.7% agar added.

[0088] Example 3 and Preparation of Phage Inhibitors Corresponding to Comparative Examples 1-5: Using Example 3 as an example, corresponding phage inhibitor mother solutions were prepared. The phage inhibitor mother solution of Example 3 was prepared as follows: 4 g of carboxymethylated seabuckthorn polysaccharide, 0.2 g of citric acid, 0.25 g of sodium citrate, 0.1 g of sodium D-gluconate, 0.5 g of dihydroxyethylglycine, and 0.2 g of sodium hexametaphosphate were weighed, and the mixture was filled to 800 mL with purified water. 1 mL of a 10% aqueous oxalic acid solution was added, and the pH was adjusted to approximately 7 with solid sodium hydroxide while stirring. The pH was then adjusted to 7.4 with 0.5 M sodium hydroxide. After the pH stabilized, water was added to 1 L to obtain the phage inhibitor mother solution. The preparation methods for the phage inhibitor mother solutions corresponding to Comparative Examples 1-5 were similar to those for the phage inhibitor mother solution of Example 3.

[0089] 2. Experimental methods:

[0090] Phage titer was determined using a double-layer agar plate method. 10 mL of the bottom solid culture medium was poured into the plate, shaken, condensed, and set aside. A phage inhibitor (the stock solution of the phage inhibitor from Example 3 and Comparative Examples 1-5, added in an amount equal to 0.1% of the test tube solution system), 1 mL of Bacillus subtilis phage (preferably to achieve a final dilution concentration of 260-300 PFU / mL in the test tube solution system), and 3 mL of a Bacillus subtilis CICC 10732 suspension (OD600 of approximately 3.0) were added to sterile empty test tubes. The mixture was gently mixed and allowed to stand for 15 minutes to obtain a mixed solution. 500 μL of the mixed solution was added to 5 mL of the top solid culture medium insulated in a 45°C constant temperature water bath. The phage inhibitor was replaced with sterile nutrient broth as a control group. The mixture was slowly inverted to mix well, then quickly poured onto the bottom solid culture medium and shaken to cover the entire plate. After cooling, the plate was placed in a 37°C incubator and cultured overnight (usually 16 hours). Plaque units (PFU) were counted, and one PFU was counted as one virus with infectious activity. Each group of experiments was repeated 5 times, and the inhibition rate (IR) of the phage inhibitor on the phage was calculated. The results are shown in Table 6 and Figure 4 As shown;

[0091] The inhibition rate (IR) formula is: IR (%) = (1-N / N0) × 100, where: the value of N is the titer of the added different phage inhibitors (Example 3, Comparative Examples 1-5), and the value of N0 is the titer of the phage in the control group.

[0092] Table 6

[0093]

[0094] From Table 6 and Figure 4 It can be seen that the inhibition rate of Example 3 on phage is significantly higher than that of Comparative Examples 1-5, which shows that the synergistic effect of carboxymethylated seabuckthorn polysaccharide, citric acid, sodium citrate and oxalic acid can significantly improve their inhibitory effect on Bacillus subtilis phage.

[0095] The above-described embodiments merely represent specific implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A bacteriophage inhibitor, characterized in that: It is composed of the following ingredients: carboxymethylated seabuckthorn polysaccharide, citric acid, sodium citrate, sodium D-gluconate, dihydroxyethylglycine, sodium hexametaphosphate, oxalic acid, and sodium hydroxide; The preparation comprises, by weight, 30-60 parts of carboxymethylated seabuckthorn polysaccharide, 1.8-2.2 parts of citric acid, 2.3-2.7 parts of sodium citrate, 0.8-1.2 parts of sodium D-gluconate, 4-6 parts of dihydroxyethylglycine, 1.8-2.2 parts of sodium hexametaphosphate, and 0.8-1.2 parts of oxalic acid. The amount of sodium hydroxide is added according to the pH value when the solution is prepared.

2. A bacteriophage inhibitor according to claim 1, characterized in that: The preparation method of the carboxymethylated seabuckthorn polysaccharide comprises the following steps: weighing 400 mg of seabuckthorn polysaccharide and dissolving it in 80 mL of a 4 mol / L NaOH solution; then slowly adding 100 mL of monochloroacetic acid with a concentration of 2 mol / L; fixing the reaction temperature at 60° C. and heating the reaction in a water bath for 3 hours; cooling the reaction solution to room temperature after completion of the reaction; adjusting the pH of the reaction solution to 7.0 with glacial acetic acid; filtering and collecting the filtrate, placing the filtrate in a dialysis bag, and dialyzing the solution with distilled water for 96 hours; vacuum concentrating the solution in the dialysis bag, and freeze-drying the solution to obtain the carboxymethylated seabuckthorn polysaccharide.

3. A bacteriophage inhibitor according to claim 2, characterized in that: The preparation method of sea buckthorn polysaccharide is as follows: first, 100 g of sea buckthorn dried fruit is crushed, passed through a 40-mesh sieve, and 10 times the volume of petroleum ether is added and refluxed for extraction twice, each time for 1 hour, and then filtered, and the filter residue is dried to a constant weight for later use; Then, the pretreated seabuckthorn residue was weighed, and distilled water was added at a material-liquid ratio of 1g:10mL. Ultrasonic-assisted extraction was performed at 70℃ for 1h, and the extraction was repeated twice. The extracts were combined and concentrated to 100ml, and 4 times the volume of anhydrous ethanol was added. The mixture was allowed to stand at 4℃ for 12h to produce precipitation. The precipitate was collected by centrifugation, redissolved in water, and then vacuum-freeze-dried to obtain crude seabuckthorn polysaccharide. Finally, the protein in the crude seabuckthorn polysaccharide was removed with Sevag solution to obtain seabuckthorn polysaccharide.

4. The bacteriophage inhibitor according to claim 3, characterized in that: The Sevag solution is prepared by mixing chloroform and n-butanol in a ratio of 4:1 and stored in the dark.

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