Phage inhibitor and application thereof

Abstract

The invention discloses a phage inhibitor and application thereof. The phage inhibitor is prepared from the following components in parts by weight: 30 to 60 parts of carboxymethylated hippophae rhamnoides polysaccharide, 1.8 to 2.2 parts of citric acid, 2.3 to 2.7 parts of sodium citrate, 0.8 to 1.2 parts of D-sodium gluconate, 4 to 6 parts of dihydroxyethyl glycine, 1.8 to 2.2 parts of sodium hexametaphosphate and 0.8 to 1.2 parts of oxalic acid, and the amount of sodium hydroxide is added according to the pH value during solution preparation. The product is designed into a mixed prefabricated product; or liquid with various concentrations can be prepared and diluted according to needs during use. When the product is applied to a prokaryotic expression system, the pollution of phage to cells can be effectively inhibited, the efficiency and quality of scientific research and production are remarkably improved, and a more leisurely fermentation environment is provided for scientific research personnel. Meanwhile, the carboxymethylated sea-buckthorn polysaccharide, the citric acid, the sodium citrate and the oxalic acid contained in the phage inhibitor have a synergistic effect, so that the inhibition effect of the phage inhibitor on the phage can be remarkably improved.

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] As a widely used gene expression technology, the prokaryotic expression system plays a key role in the production of proteins and enzymes, vaccine development, genetic engineering drug development and other scientific research fields. However, prokaryotes are often threatened by phage contamination, which not only leads to abnormal cell growth and death, but also seriously reduces production efficiency, affects product quality, and greatly increases the cost and time of R&D and production.

[0003] At present, common methods for treating phage contamination, such as building ultra-clean laboratories and using disinfectants, have many disadvantages, such as high cost, low efficiency, incomplete treatment, and delays in scientific research and production. The phage inhibitors developed in this project precisely target the pain point of phage contamination affecting scientific research. Only a trace amount of inhibitors needs to be added during cell growth to effectively inhibit phage contamination of cells, significantly improving the efficiency and quality of scientific research and production.

[0004] In view of the lack of products on the market that can effectively deal with bacteriophage 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 bacteriophage 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 bacteriophage 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, and the amount of sodium hydroxide is added according to the pH value when preparing the solution.

[0009] Preferably, the product of the bacteriophage inhibitor is designed as a mixed preformed product, or is configured as a liquid of various concentrations, which is diluted as needed when used. The mixed preformed product is a solid product of each component in the formula prepared in proportion.

[0010] Preferably, when the phage inhibitor is configured into a solution of 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 sodium hydroxide is used to adjust the pH to 7.2-7.4.

[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 with 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., the reaction is heated in a water bath for 3 hours, and 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, the filtrate is collected after filtration, and is placed in a dialysis bag (8-14 kDa) and dialyzed with distilled water for 96 hours, and the solution in the dialysis bag is vacuum concentrated and freeze-dried to obtain the carboxymethylated seabuckthorn polysaccharide.

[0014] Preferably, the preparation method of seabuckthorn polysaccharide is as follows: 100g of seabuckthorn dried fruit is crushed, passed through a 40-mesh sieve, and 10 times the volume of petroleum ether is added for reflux extraction 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 standby use; the pretreated seabuckthorn filter residue is weighed, distilled water is added according to a solid-liquid ratio of 1:10 (g / mL), ultrasonic (ultrasonic power 180W) assisted extraction is performed at 70°C for 1 hour, the extraction is performed twice, the extract is 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 a precipitate, the precipitate is taken by centrifugation, and the precipitate is redissolved in water and then vacuum-freeze-dried to obtain crude seabuckthorn polysaccharide; and the protein in the crude seabuckthorn polysaccharide is removed by Sevag solution to obtain seabuckthorn polysaccharide.

[0015] Further preferably, the specific method of removing the protein in the crude polysaccharide of sea buckthorn with Sevag solution to obtain sea buckthorn polysaccharide is as follows: weigh the crude polysaccharide of sea buckthorn and dissolve it in distilled water to obtain a 0.1g / ml crude polysaccharide solution, then mix it with the Sevag solution at a volume ratio of 1:3, stir it thoroughly for 30 minutes after mixing, then centrifuge it at 4000r / min for 10 minutes, collect the supernatant, and repeat the above operation 5 times; combine the supernatants, evaporate and concentrate to remove the organic reagent, and then perform vacuum freeze drying to obtain sea buckthorn polysaccharide. Wherein, the Sevag solution is prepared by mixing chloroform and n-butanol at a ratio of 4:1 and storing it 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 can significantly inhibit the adverse effects of lysogenic phages on the fermentation process and has two product forms. One is a high-concentration liquid additive with the advantages of low production cost and convenient transportation. The other is a mixed prefabricated product, which is characterized by the ability to achieve combined sales, thereby increasing profit levels.

[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 The figure is a graph showing the inhibitory effect of different concentrations of phage inhibitors on Escherichia coli phage T4, where 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 bacteriophage inhibitors on Escherichia coli λ phage, where 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 bacteriophage 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 by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways 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 without conflict.

[0028] Example 1

[0029] A bacteriophage 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 sodium hydroxide is used to adjust the pH to 7.2.

[0030] Example 2

[0031] A bacteriophage 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 sodium hydroxide is used to adjust the pH to 7.4.

[0032] Example 3

[0033] A bacteriophage 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 sodium hydroxide is used to adjust the pH to 7.4.

[0034] The preparation method of the bacteriophage inhibitor of this embodiment is as follows: according to the ratio of Example 3, 4g of carboxymethylated seabuckthorn polysaccharide, 0.2g of citric acid, 0.25g of sodium citrate, 0.1g of sodium D-gluconate, 0.5g of dihydroxyethylglycine, and 0.2g of sodium hexametaphosphate are weighed, pure water is added to 800mL, 1mL of 10% oxalic acid aqueous solution is added, and the pH is adjusted to about 7 with solid sodium hydroxide while stirring continuously, and then the pH is adjusted to 7.4 with 0.5M sodium hydroxide, and water is added to make the volume to 1L after the pH is stable. When using, the corresponding culture medium or water is selected as needed for 1:999 dilution, 1ml is taken from the solution after the volume is made, culture medium or water is added to 800mL, and then the pH is adjusted to 7.4 with 0.5M sodium hydroxide, and the culture medium or water is added to make the volume to 1L after the pH is stable, so as to obtain 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 weighed and dissolved in 80 mL of 4 mol / L NaOH solution, and then 100 mL of monochloroacetic acid with a concentration of 2 mol / L was slowly added, the reaction temperature was fixed at 60°C, and the reaction was heated in a water bath for 3 hours. After the reaction was completed, the reaction solution was cooled to room temperature, and the pH of the reaction solution was adjusted to 7.0 with glacial acetic acid. The filtrate was 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 was vacuum concentrated and freeze-dried to obtain carboxymethylated seabuckthorn polysaccharide. Dialysis bag MD55 (8000-14000D) was purchased from Beijing Solebow Technology Co., Ltd.

[0037] The preparation method of seabuckthorn polysaccharide is as follows: 100g of seabuckthorn dried fruit is crushed, passed through a 40-mesh sieve, and 10 times the volume of petroleum ether is added for reflux extraction 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 standby use; the pretreated seabuckthorn filter residue is weighed, distilled water is added according to a solid-liquid ratio of 1:10 (g / mL), and ultrasonic (ultrasonic power 180W) is used for assisted extraction at 70°C for 1 hour, and the extraction is performed twice, the extract is 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 a precipitate, the precipitate is obtained by centrifugation, and the precipitate is redissolved in water and then vacuum-freeze-dried to obtain the crude seabuckthorn polysaccharide; the protein in the crude seabuckthorn polysaccharide is removed by using a Sevag solution to obtain the seabuckthorn polysaccharide. The specific method of removing the protein from the crude polysaccharide of sea buckthorn with Sevag solution to obtain sea buckthorn polysaccharide is as follows: weigh the crude polysaccharide of sea buckthorn and dissolve it in distilled water to obtain a 0.1g / ml crude polysaccharide solution, then mix it with the Sevag solution in a volume ratio of 1:3, stir it thoroughly for 30 minutes after mixing, then centrifuge it at 4000r / min for 10 minutes, collect the supernatant, and repeat the above operation 5 times; combine the supernatant, evaporate and concentrate to remove the organic reagent, and then perform vacuum freeze drying to obtain sea buckthorn polysaccharide. The Sevag solution is prepared by mixing chloroform and n-butanol in a ratio of 4:1 and storing it in the dark. Sea buckthorn 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 Microbiological Culture Collection Center; bacteriophage T4 was Escherichia coli phage T4 SHBCC D24645, and λ phage was Escherichia coli λ phage SHBCC D24685. Escherichia coli phage T4 SHBCC D24645 and Escherichia coli λ phage SHBCC D24685 were purchased from Shanghai Ruichu Biotechnology Co., Ltd.

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

[0051] Configuration of phage inhibitor 1X, 2X, 4X, 6X, 8X, 10X: The culture medium used in Example 3 is LB culture medium, and the specific method is: weigh 4g carboxymethylated seabuckthorn polysaccharide, 0.2g citric acid, 0.25g sodium citrate, 0.1g sodium D-gluconate, 0.5g dihydroxyethylglycine, 0.2g sodium hexametaphosphate, add pure water to 800mL, add 1mL 10% oxalic acid aqueous solution, and adjust the pH to about 7 with solid sodium hydroxide while stirring continuously, and then adjust the pH to 7.4 with 0.5M sodium hydroxide. After the pH is stable, add water to make up to 1L to obtain the phage inhibitor mother solution. 1 mL, 2 mL, 4 mL, 6 mL, 8 mL, and 10 mL of the phage inhibitor mother solution were respectively added to 800 mL of LB liquid culture medium, and the pH was adjusted to 7.4 with 0.5 M sodium hydroxide. After the pH stabilized, LB culture medium was added to make up to 1 L, thereby obtaining LB culture medium containing phage inhibitors of different concentrations (1X, 2X, 4X, 6X, 8X, 10X), 1×, 2×, etc., i.e., 1 times, 2 times, ... and so on, i.e., the concentration of the phage inhibitor in Example 3, 2 times the concentration of the phage inhibitor in 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 with a spectrophotometer: when Escherichia coli is infected with a certain concentration of phage, if the OD600 value of the group with phage inhibitors is significantly larger than that of the group without phage inhibitors, there is an inhibitory effect, otherwise the opposite is true.

[0054] (1) Verification of 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 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 culture was carried out 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 have 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 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.coliK12; comparing the positive and negative controls, 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 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. Experimental materials: E.coli BL21 (CICC 23796), E. coli phage T4 SHBCC D24645;

[0074] LB liquid medium: weigh trypsin

[0075] Dissolve 10g of peptone, 5g of yeast extract powder and 10g of NaCl in 900ml of distilled water and make up to 1000ml with distilled water. Adjust the pH to 7.4 with 10mol / l NaOH. Sterilize in an autoclave at 121℃ for 20min. After cooling, 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] The phage inhibitors corresponding to Examples 1-3 and Comparative Examples 1-5 are configured as follows: Taking Example 3 as an example, the corresponding phage inhibitor mother solution is configured; the method for preparing the phage inhibitor mother solution of Example 3 is as follows: 4g carboxymethylated seabuckthorn polysaccharide, 0.2g citric acid, 0.25g sodium citrate, 0.1g sodium D-gluconate, 0.5g dihydroxyethylglycine, 0.2g sodium hexametaphosphate are weighed, pure water is added to 800mL, 1mL 10% oxalic acid aqueous solution is added, and the pH is adjusted to about 7 with solid sodium hydroxide while stirring continuously, and then the pH is adjusted to 7.4 with 0.5M sodium hydroxide, and after the pH is stable, water is added to make up to 1L to obtain the phage inhibitor mother solution. The method for preparing the phage inhibitor mother solution corresponding to Examples 1-2 and Comparative Examples 1-5 refers to the method for preparing the phage inhibitor mother solution of Example 3.

[0078] 2. Experimental methods:

[0079] The double-layer agar plate method was used to determine the phage titer. 10 mL of the bottom solid culture medium was poured into the plate, shaken, condensed, and set aside. In a sterile empty test tube, phage inhibitors (the mother solution of phage inhibitors in Examples 1-3 and Comparative Examples 1-5, the amount added was 0.1% of the test tube solution system), 1 mL of phage T4 solution (preferably 260-300 PFU / mL of the test tube solution system) and 3 mL of E. coli BL21 suspension (OD600 was about 3.0) were added respectively. The three were gently mixed and allowed to stand for 15 minutes to obtain a mixed solution. 500 ul of the mixed solution was added to 5 mL of the upper solid culture medium insulated in a 45°C constant temperature water bath. The phage inhibitor was replaced with a sterile LB culture medium as a control group. After slowly inverting and mixing, it was quickly poured onto the bottom solid culture medium and shaken to cover the plate with the upper culture medium. After cooling, the plate was placed in a 37°C incubator for incubation. After overnight incubation (generally 16 hours), the plaque units (PFU) were counted. 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 / N 0 )×100, where: the value of N is the potency of adding different phage inhibitors (Examples 1-3, Comparative Examples 1-5), N 0 The value is the titer of 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 the inhibition of Escherichia coli bacteriophages.

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

[0085] 1. Experimental materials: Bacillus subtilis CICC 10732, Bacillus subtilis bacteriophage (Bio-67330, original number BSNP01); Bacillus subtilis CICC 10732 was purchased from China Industrial Microbiological Culture Collection Administration Center, and Bacillus subtilis bacteriophage BSNP01 was purchased from Beijing Biobo 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, 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, and store at room temperature after cooling 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, configuration of phage inhibitors corresponding to comparative examples 1-5: Taking Example 3 as an example, the corresponding phage inhibitor mother solution is configured; the method for preparing the phage inhibitor mother solution of Example 3 is as follows: weigh 4g carboxymethylated seabuckthorn polysaccharide, 0.2g citric acid, 0.25g sodium citrate, 0.1g sodium D-gluconate, 0.5g dihydroxyethylglycine, 0.2g sodium hexametaphosphate, add pure water to 800mL, add 1mL 10% oxalic acid aqueous solution, and adjust the pH to about 7 with solid sodium hydroxide while stirring continuously, and then adjust the pH to 7.4 with 0.5M sodium hydroxide, and add water to 1L after the pH stabilizes, so as to obtain the phage inhibitor mother solution. The method for preparing the phage inhibitor mother solution corresponding to comparative examples 1-5 refers to the method for preparing the phage inhibitor mother solution of Example 3.

[0089] 2. Experimental methods:

[0090] The double-layer agar plate method was used to determine the phage titer. 10 mL of the bottom solid culture medium was poured into the plate, shaken, condensed, and set aside. Phage inhibitors (the mother solutions of phage inhibitors in Example 3 and Comparative Examples 1-5, the amount added was 0.1% of the test tube solution system), 1 mL of Bacillus subtilis phage (the final dilution concentration of the test tube solution system was preferably 260-300 PFU / mL) and 3 mL of Bacillus subtilis CICC 10732 suspension (OD600 was about 3.0) were added to sterile empty test tubes respectively. The three were gently mixed and allowed to stand for 15 minutes to obtain a mixed solution. 500 ul of the mixed solution was added to 5 mL of the upper solid culture medium insulated in a 45°C constant temperature water bath. The phage inhibitor was replaced with a sterile nutrient broth culture medium as a control group. After slowly inverting and mixing, it was quickly poured onto the bottom solid culture medium and shaken to cover the plate with the upper culture medium. After cooling, the plate was placed in a 37°C incubator for incubation. After overnight incubation (generally 16 hours), the plaque units (PFU) were counted. One PFU was counted as one virus with infectious activity. Each group of experiments was repeated 5 times, and the inhibition rate (IR: Inhibition Rate) 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 / N 0 )×100, where: the value of N is the potency of adding different phage inhibitors (Example 3, Comparative Examples 1-5), N 0 The value is the titer of 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 indicates that the synergistic effect of carboxymethylated seabuckthorn polysaccharide, citric acid, sodium citrate and oxalic acid can significantly improve the inhibition of Bacillus subtilis phage.

[0095] The above-mentioned embodiments only express the specific implementation of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A bacteriophage inhibitor, characterized in that: Contains carboxymethylated seabuckthorn polysaccharide, citric acid, sodium citrate, sodium D-gluconate, dihydroxyethylglycine, sodium hexametaphosphate, oxalic acid, and sodium hydroxide.

2. A bacteriophage inhibitor according to claim 1, characterized in that: The invention comprises 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 by weight, and the amount of sodium hydroxide is added according to the pH value when preparing the solution.

3. A bacteriophage inhibitor according to claim 2, characterized in that: Phage inhibitor products are designed as mixed pre-products; or they are formulated into liquids of various concentrations and diluted as needed when used.

4. A bacteriophage inhibitor according to claim 3, characterized in that: When the phage inhibitor is configured into a solution with the lowest concentration, it contains 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 D-gluconate, 0.4-0.6 mg / L dihydroxyethylglycine, 0.18-0.22 mg / L sodium hexametaphosphate, 0.08-0.12 mg / L oxalic acid, and the remainder is solvent; and sodium hydroxide is used to adjust the pH to 7.2-7.

4.

5. A bacteriophage inhibitor according to claim 4, characterized in that: It contains 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 sodium hydroxide is used to adjust the pH to 7.

4.

6. A bacteriophage inhibitor according to claim 4, characterized in that: The solvent is water or culture medium.

7. The bacteriophage inhibitor according to claim 1, characterized in that: 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., the reaction is heated in a water bath for 3 hours, and 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, the filtrate is collected after filtration, and the filtrate is put into a dialysis bag and dialyzed with distilled water for 96 hours, and the solution in the dialysis bag is vacuum concentrated and freeze-dried to obtain the carboxymethylated seabuckthorn polysaccharide.

8. A bacteriophage inhibitor according to claim 7, characterized in that: The preparation method of seabuckthorn polysaccharide is as follows: firstly, 100g of seabuckthorn dried fruit is crushed, passed through a 40-mesh sieve, 10 times the volume of petroleum ether is added, refluxed and extracted twice, each time for 1 hour, filtered, and the residue is dried to constant weight for later use; Then, the pretreated seabuckthorn residue was weighed, and distilled water was added at a solid-liquid ratio of 1:10 (g / mL). Ultrasonic-assisted extraction was performed at 70°C for 1 hour, and the extraction was repeated twice. The extracts were combined and concentrated to 100 ml, and 4 times the volume of anhydrous ethanol was added. The mixture was allowed to stand at 4°C for 12 hours to produce precipitation. The precipitate was centrifuged, redissolved in water, and then vacuum-freezed to obtain crude seabuckthorn polysaccharide. Finally, the protein in the crude seabuckthorn polysaccharide was removed with Sevag solution to obtain seabuckthorn polysaccharide.

9. A bacteriophage inhibitor according to claim 8, 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.

10. Use of the bacteriophage inhibitor according to any one of claims 1 to 9 in a prokaryotic expression system.

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