Freeze-drying protectant for vibrio harveyi phage v-ydf132 and preservation method thereof

Abstract

The application discloses a freeze-drying protective agent of Vibrio harveyi phage V-YDF132 and a preservation method thereof. The freeze-drying protective agent contains inulin, skimmed milk powder, gelatin, L-glutamic acid sodium and water. The freeze-drying protective agent is mixed with a phage V-YDF132 solution, and then the mixture is frozen to a solid state, and after being subjected to freeze-drying, a phage V-YDF132 freeze-drying powder preparation is obtained. There are various preservation methods for phages, and the best preservation method is different for different phages. The application screens and prepares different phage freeze-drying protective agents and explores the protection effect of the freeze-drying protective agents on phages after freeze-drying, so as to lay a foundation for phage preservation. The phage powder preparation prepared from the freeze-drying protective agent formula P9 and the phage V-YDF132 has low requirements on the preservation conditions and can be long-term preserved without losing biological activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a freeze-drying protective agent of Vibrio harveyi phage V-YDF132 and a preservation method thereof. BACKGROUND

[0002] In recent years, phage as a new antibacterial means has attracted much attention from researchers at home and abroad. Phage has made many contributions in aquaculture, agriculture and food industry, and has broad application prospects. The activity of phage is easily affected by the external environment, so the preservation of phage is particularly important. At present, the practical preservation methods of phage at home and abroad mainly include suspension method, frozen liquid method and drying method. The properties of each phage are different, and improper preservation method will cause great loss.

[0003] Suspension method refers to directly preserving phage stock solution or adding protective agents to maintain the liquid state of phage. Protective agents can include glycerol, chloroform, SM buffer and calcium chloride, etc. It has been reported that the activity of Bacillus thuringiensis phage CP-51 culture solution preserved at room temperature is much higher than that preserved at 4℃. In addition, Xie Hujun (2003) added glycerol to the enrichment solution of Escherichia coli phage, and found that the best preservation effect was obtained at 4℃ for 1 month. Lerner (2016) et al. found that the three Achromobacter xylosoxidans phages could be preserved for 16 months by adding chloroform using suspension method. In addition, many reports have proved that adding SM buffer to phage stock solution can effectively preserve phage.

[0004] Frozen liquid method refers to preserving phage in low-temperature freezing conditions below-20℃. The preservation temperature varies, which can be dry ice (-70℃), ultra-low temperature refrigerator (-80℃) and liquid nitrogen (-196℃) and other methods for preservation. Protective agents such as glycerol or skimmed milk powder can be added to prevent phage from inactivating during the freezing process. Wagner (2017) found that the titer of 13 phages basically did not change after adding 15% glycerol to the suspension and storing at-80℃ for 4 years. Hubálek (2003) reported that various concentrations of DMSO can be used as protective agents.

[0005] The drying methods are mainly divided into freeze-drying method, vacuum drying method and spray drying method. The freeze-drying method (abbreviated as freeze-drying method) is to freeze the liquid by using the principle of ice sublimation, and then remove the water by sublimation under negative pressure to form a powder. In order to prevent the phage from being inactivated during the freezing process, various freezing protectants such as tryptone, skimmed milk and gelatin can be added. The phage after freeze-drying can be stored at different temperatures. Recent studies have shown that the phage powder prepared by freeze-drying has thermal stability and pH stability, and can also improve the stability of the phage in long-term storage (El et al., 2018). Charles (1959) reported that the staphylococcus phage was freeze-dried with skimmed milk as a protective agent, and stored at 4℃ and -20℃ for 8 months, and the phage still maintained high activity. The principle of vacuum drying method is to keep the phage from freezing and dry under high vacuum. If the dried phage is properly sealed and stored, the original titer and activity of the phage will be maintained for a long time, and the commonly used protective agents are skimmed milk powder, sugar, whey powder and gelatin. At the same time, it was found that the mycobacteriophage was vacuum dried, and the titer of the phage was only decreased by 1 order of magnitude after 28 weeks of storage at 4℃ and room temperature, and the storage effect at 4℃ was better than that at room temperature. Spray drying method uses traditional double-fluid nozzle to produce dry powder or granular phage products. Matinkhoo (2011) et al. reported that single phage or phage cocktail was spray dried to produce phage dry powder, and the titer was only reduced by 0.4-0.8 orders of magnitude.

[0006] In summary, there are various preservation methods for phage, and the best preservation method for different phage is different, so it is of great significance to select the suitable preservation method for subsequent practical application. SUMMARY

[0007] The purpose of the present application is to provide a freeze-drying protective agent for Vibrio harveyi phage V-YDF132 which has low requirements for storage conditions and can be stored for a long time without losing biological activity.

[0008] The freeze-drying protective agent for Vibrio harveyi phage V-YDF132 of the present application contains inulin, skimmed milk powder, gelatin, L-glutamic acid sodium and water.

[0009] Preferably, the freeze-drying protective agent for Vibrio harveyi phage V-YDF132 contains 15 g of inulin, 5.5 g of skimmed milk powder, 10 g of gelatin, 0.25 g of L-glutamic acid sodium per 100 ml, and the rest is purified water.

[0010] The second object of the present application is to provide a preservation method of the Vibrio harveyi bacteriophage V-YDF132, which is mixing the freeze-drying protective agent with the bacteriophage V-YDF132 solution, freezing the mixture to a solid state, and then freeze-drying the mixture to obtain the freeze-dried powder preparation of the bacteriophage V-YDF132.

[0011] Preferably, the mixing of the freeze-drying protective agent with the bacteriophage V-YDF132 solution is mixing them in a volume ratio of 1:2.

[0012] Preferably, the bacteriophage V-YDF132 solution has a concentration of 6.5x10 8 PFU / mL.

[0013] Preferably, the freezing of the mixture to a solid state is pre-freezing the mixture at -80℃ for 12 hours to form a solid state, and then starting the refrigeration function of the CHRSIT freeze-drying machine, and waiting for the cold trap temperature to drop below -40℃. The solidified mixture is then placed in the freeze-drying machine, and the freeze-drying machine is started in the vacuum mode, and the gas pressure in the cold trap reaches 0.37 mbar. After 18-24 hours of freeze-drying, the freeze-dried powder preparation of the Vibrio harveyi bacteriophage V-YDF132 is obtained.

[0014] There are various preservation methods for bacteriophages, and the best preservation method varies for different bacteriophages. The present application aims to lay a foundation for the preservation of bacteriophages by screening different freeze-drying protective agents for bacteriophages and exploring their protective effects on bacteriophages after freeze-drying. The bacteriophage powder preparation prepared by mixing the freeze-drying protective agent formula P9 obtained by screening with the bacteriophage V-YDF132 has low requirements for preservation conditions and can be stored for a long time without losing biological activity.

[0015] The Vibrio harveyi bacteriophage V-YDF132 of the present application was deposited at the Guangdong Microbial Culture Collection Center on July 5, 2022, and the deposit number is GDMCC No: 62599-B1. The bacteriophage is disclosed in the patent application number CN202210886269.9, with the invention name of a Vibrio harveyi bacteriophage V-YDF132 and its application.

[0016] The Vibrio harveyi YDF132 is preserved in the Laboratory of Aquatic Biomedicine, College of Oceanography, South China Agricultural University, and it is also disclosed in the patent application number CN202210886269.9, with the invention name of a Vibrio harveyi bacteriophage V-YDF132 and its application. The present applicant guarantees to provide it to the public for 20 years from the date of application. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1A lyophilized powder formulation of bacteriophage V-YDF132 with a lyoprotectant.

[0018] Figure 2 Bacteriophage titer determination of a lyophilized powder formulation of bacteriophage V-YDF132 at 37°C (a) and 4°C (b). DETAILED DESCRIPTION

[0019] The present application is further illustrated by the following description with reference to the accompanying drawings and specific examples. The examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0020] Example 1

[0021] I. Experimental Methods

[0022] 1. Preparation of bacteriophage V-YDF132

[0023] The bacteriophage in the bacteriophage lyophilizate is a bacteriophage lysate. The bacteriophage stock solution is expanded according to the method described in the Molecular Cloning: A Laboratory Manual, Third Edition, with slight modifications. 500 μL of Vibrio harveyi YDF132 in the logarithmic phase is added to 500 mL of LB liquid medium, and the mixture is cultured at 37°C with shaking; 50 mL of bacteriophage V-YDF132 solution is added, and the mixture is further cultured on a shaker until the mixture becomes completely clear; the mixture is centrifuged at 4000 rpm for 10 min at 4°C, and the supernatant is filtered using a 0.22 μm filter to obtain bacteriophage YDF132 solution. Rnase A enzyme and Dnase I enzyme are added to the bacteriophage YDF132 solution to a final concentration of 1 μg / mL, and the mixture is placed at room temperature for 30 min; solid NaCl is added to a final concentration of 1 M, and the mixture is ice-bathed for 1 h; after centrifugation at 8300 rpm for 10 min at 4°C, the volume of the supernatant is determined; solid polyethylene glycol (PEG8000) is added to a final concentration of 10% (w / v), and the mixture is placed in an ice-water mixture overnight; the next day, the mixture is centrifuged at 12,000 rpm for 15 min at 4°C, and the precipitate is washed three times with SM buffer and resuspended in SM buffer. The bacteriophage titer of the resuspended solution after expansion is determined by double-layer plate method.

[0024] 2. Preparation of lyoprotectant for bacteriophage V-YDF132

[0025] The bacteriophage lyophilized powder formulation is a lyophilized mixture formed by lyophilizing a mixed solution of bacteriophage and lyoprotectant; wherein in the mixed solution, 100 mL of lyoprotectant is composed of the following components by mass fraction:

[0026] 1. Sucrose 30g, skim milk powder 5.5g, gelatin 10g, L-glutamate sodium 0.25g, and the rest is purified water.

[0027] 2. Trehalose 15g, skim milk powder 5.5g, gelatin 10g, L-glutamate sodium 0.25g, and the rest is purified water.

[0028] 3. Inulin 15g, skim milk powder 5.5g, gelatin 10g, L-glutamate sodium 0.25g, and the rest is purified water.

[0029] According to the weight of each component of the above three freeze-drying protectants, mix uniformly, autoclave, and obtain. Among them, the high pressure condition is 115℃ sterilization for 30min. The solution containing bacteriophage YDF132 (concentration is 6.5×10 8 PFU / mL) is mixed with each freeze-drying protectant in proportion to the volume ratio of 7:1, 4:1, 2:1, respectively, to prepare 9 kinds of mixed solutions, that is, 9 different formulations of this test:

[0030] P1: Sucrose 30g, skim milk powder 5.5g, gelatin 10g, L-glutamate sodium 0.25g, and the rest is purified water; mixed with bacteriophage solution in a volume ratio of 1:7.

[0031] P2: Sucrose 30g, skim milk powder 5.5g, gelatin 10g, L-glutamate sodium 0.25g, and the rest is purified water; mixed with bacteriophage solution in a volume ratio of 1:4.

[0032] P3: Sucrose 30g, skim milk powder 5.5g, gelatin 10g, L-glutamate sodium 0.25g, and the rest is purified water; mixed with bacteriophage solution in a volume ratio of 1:2.

[0033] P4: Trehalose 15g, skim milk powder 5.5g, gelatin 10g, L-glutamate sodium 0.25g, and the rest is purified water; mixed with bacteriophage solution in a volume ratio of 1:7.

[0034] P5: Trehalose 15g, skim milk powder 5.5g, gelatin 10g, L-glutamate sodium 0.25g, and the rest is purified water; mixed with bacteriophage solution in a volume ratio of 1:4.

[0035] P6: Trehalose 15g, skim milk powder 5.5g, gelatin 10g, L-glutamate sodium 0.25g, and the rest is purified water; mixed with bacteriophage solution in a volume ratio of 1:2.

[0036] P7: Inulin 15g, skim milk powder 5.5g, gelatin 10g, L-glutamate sodium 0.25g, and the rest is purified water; mixed with bacteriophage solution in a volume ratio of 1:7.

[0037] P8: Inulin 15g, skim milk powder 5.5g, gelatin 10g, L-glutamic acid sodium 0.25g, the rest is purified water; mixed with the phage solution at a volume ratio of 1:4.

[0038] P9: Inulin 15g, skim milk powder 5.5g, gelatin 10g, L-glutamic acid sodium 0.25g, the rest is purified water; mixed with the phage solution at a volume ratio of 1:2.

[0039] The mixed solution was pre-frozen at -80℃ for 12h to form a solid sample. The refrigeration function of the CHRSIT freeze dryer was turned on, and the cold trap temperature was reduced to below -40℃. The solidified sample was placed in the freeze dryer, and the vacuum mode of the freeze dryer was turned on. The gas pressure in the cold trap reached 0.37mbar. After 18-24h of freeze-drying, the Vibrio harveyi phage V-YDF132 lyophilized powder preparation was obtained.

[0040] 3Screening of the optimal lyophilization protective agent for phage V-YDF132

[0041] After freeze-drying, the phage titers in the nine lyophilized powders were immediately determined. The method for determining the titer of the phage lyophilized powder was as follows: 0.1g of lyophilized powder was dissolved in 1mL of SM buffer. After complete dissolution, the double-layer plate method was used to determine the phage titer, with Vibrio harveyi YDF132 as the host bacteria. Specifically, bottom-layer TCBS medium containing 2% and 1% agar and top-layer TCBS medium were prepared in advance. First, pour a layer of bottom-layer medium on a culture dish, and then, after solidification, pour the top-layer medium containing a relatively concentrated sensitive host and a certain volume of phage sample on the bottom-layer medium in a test tube, shake well, and immediately pour it on the bottom-layer medium to spread and solidify. Then, incubate at 37℃. After about 10h, count the plaques. The lyophilized powder was placed in a 37℃ constant-temperature incubator, and the phage titers in the nine lyophilized powders were recorded every 7d. According to the survival of the phage before and after lyophilization and after being placed in a 37℃ environment, the optimal lyophilization protective agent was determined. At the same time, the optimal lyophilized powder preparation was placed in a 4℃ environment, and the phage titer in the lyophilized powder was recorded every 3 months.

[0042] II. Experimental results

[0043] 1Preparation of lyophilization protective agent for Vibrio harveyi phage V-YDF132

[0044] After mixing the biologically active phage with a lyophilization protective agent, freeze-drying treatment was performed to form a phage lyophilized powder preparation. During the preparation process, it is very important to control the content ratio of the lyophilization protective agent and the phage to ensure that the protective agent can effectively protect the phage. In this study, the mixed solution of the nine lyophilization protective agents and the phage was freeze-dried to form nine lyophilized powder preparations (P1-P9) after freeze-drying treatment, as shown in Table 1.Figure 1 Therefore, the next step of screening the optimal lyophilization protectant can be carried out smoothly.

[0045] 2 Optimal lyophilization protectant of Vibrio harveyi phage V-YDF132

[0046] The above-mentioned lyophilized powder preparation is subjected to storage treatment at 37°C and 4°C, and the titer of the phage is determined periodically to determine the optimal lyophilization protectant among the nine lyophilized powder preparations. The phage lyophilized powder preparation prepared by the lyophilization protectant formula P9 and the phage V-YDF132 has a phage titer of 1 x 10 8.633 PFU / mL before lyophilization, and a phage titer of 1 x 10 8.123 PFU / mL after lyophilization, with a decrease of only 0.51 titers; and the phage titer in the lyophilized powder preparation is 1 x 10 7.226 PFU / mL after storage at 37°C for 21 days, with a decrease of 0.897 titers compared with the phage titer after lyophilization Figure 2 (a); and the phage titer in the lyophilized powder preparation is 1 x 10 7.616 PFU / mL after storage at 4°C for 9 months, with a decrease of 0.511 titers, and the survival rate of the phage is 93.7% Figure 2 (b). The above results show that the phage powder preparation prepared by the lyophilization protectant formula P9 and the phage V-YDF132 has low requirements for storage conditions and can be stored for a long time without losing biological activity.

[0047] P9: inulin 15 g, skim milk powder 5.5 g, gelatin 10 g, L-glutamic acid sodium 0.25 g, and the rest is purified water The lyophilized powder preparation of the phage improves the transportation and storage convenience of the lyophilized powder preparation of the phage and improves the practical applicability of the lyophilized powder preparation of the phage.

Claims

1. A method for preserving Vibrio harveyi bacteriophage V-YDF132, characterized in that, The process involves mixing a lyophilization protectant with a phage V-YDF132 solution, freezing the sample to a solid state, and then freeze-drying it to obtain a lyophilized phage V-YDF132 powder formulation. The lyophilization protectant and the phage V-YDF132 solution are mixed at a volume ratio of 1:

2. The lyophilization protectant consists of 15 g of inulin, 5.5 g of skim milk powder, 10 g of gelatin, 0.25 g of sodium L-glutamate, and the remainder being purified water per 100 ml.

2. The preservation method according to claim 1, characterized in that, The bacteriophage V-YDF132 solution has a concentration of 6.5 × 10⁻⁶. 8 PFU / mL.

3. The preservation method according to claim 1, characterized in that, The process of first freezing the sample to a solid state and then freeze-drying it to obtain the phage V-YDF132 lyophilized powder formulation involves pre-freezing the mixed solution at -80℃ for 12 hours to form a solid sample, then turning on the CHRSIT freeze dryer's cooling function until the cold trap temperature drops below -40℃. The solidified sample is then placed in the freeze dryer, which is then turned on in vacuum mode, with the pressure inside the cold trap reaching 0.37 mbar. After freeze-drying for 18-24 hours, the Vibrio harveyi phage V-YDF132 lyophilized powder formulation is obtained.

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