CN116694581A - Freeze-drying protective agent for vibrio harveyi phage V-YDF132 and preservation method thereof - Google Patents

Abstract

The invention discloses a freeze-drying protectant of Vibrio harveyi phage V-YDF132 and a preservation method thereof. It contains inulin, skimmed milk powder, gelatin, monosodium L‑glutamate and water. The above-mentioned lyoprotectant is mixed with the phage V-YDF132 solution, first frozen to a solid state to be frozen, and then freeze-dried to obtain the phage V-YDF132 freeze-dried powder preparation. There are many ways to store phages, and different phages have different best ways to store them. The present invention aims to lay the foundation for phage preservation by screening and preparing different phage freeze-drying protective agents and exploring their protective effects on phage after freeze-drying. The phage powder preparation prepared from the screened lyoprotectant formula P9 and phage V‑YDF132 has low requirements for storage conditions and can be stored for a long time without losing biological activity.

Description

Lyoprotectant of Vibrio harveyi phage V-YDF132 and preservation method thereof

technical field

The invention relates to the field of biotechnology, in particular to a freeze-drying protectant of Vibrio harveyi phage V-YDF132 and a preservation method thereof.

Background technique

In recent years, as a new antibacterial method, phages have attracted great attention from researchers at home and abroad. Phages have made many contributions in aquaculture, agriculture and food industries, and have 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 are mainly divided into suspension method, freezing liquid method and drying method. The nature of each phage is different, if the storage method is improper, it will cause great loss.

The suspension method refers to the direct preservation of the phage stock solution or adding a protective agent to keep the phage in a liquid form. The protective agent can be added to glycerol, chloroform, SM buffer, and calcium chloride. It has been reported that the culture solution of Bacillus thuringiensis phage CP-51 was stored at room temperature, and the activity of the phage was found to be much higher than that stored at 4°C. In addition, Xie Huijun (2003) added glycerol to the E. coli phage enrichment solution and found that the best effect was preserved at 4°C for 1 month. Lierna (2016) et al. found that chloroform was added to three xylose-oxidizing Achromobacter phages, which could be preserved for 16 months by the suspension method. In addition, many reports have proved that adding SM buffer to the phage stock solution can effectively preserve the phage.

The frozen solution method refers to the preservation of phages in low-temperature freezing conditions of -20°C and below. The storage temperature is different, and it can be stored in dry ice (-70°C), ultra-low temperature refrigerator (-80°C) and liquid nitrogen (-196°C). Protective agents such as glycerin or skimmed milk powder can be added to prevent phage from freezing. inactivated. Wagner (2017) found that adding 15% glycerol to 13 phage suspensions stored at -80°C for up to 4 years did not change the titer of phages. Hubálek (2003) reported that various concentrations of DMSO can be used as a protective agent.

Drying methods are mainly divided into freeze drying, vacuum drying and spray drying. The freeze-drying method (abbreviated as the freeze-drying method) uses the principle of ice crystal sublimation to freeze the liquid, and then uses negative pressure to sublimate to form a powder after removing water. To prevent the inactivation of phage during freezing, various cryoprotectants such as tryptone, skimmed milk and gelatin can be added. Freeze-dried phage can be stored at different temperatures. Studies in recent years have shown that phage powder prepared by freeze-drying has thermal stability and pH stability, and can improve the stability of phage in long-term storage (El et al., 2018). Charles (1959) reported that using skim milk as a protective agent, staphylococcal phages could still maintain high activity after freeze-drying at 4°C and -20°C for 8 months. The principle of the vacuum drying method is to keep the phage from freezing and dry under reduced pressure under high vacuum. If dried and sealed properly, the bacteriophage will maintain its original potency and vitality for a long time. At present, commonly used protective agents include skim milk powder, sugar, whey powder and gelatin. At the same time, a study found that the titer of mycobacteriophages was only reduced by 1 order of magnitude after vacuum-drying mycobacteriophages at 4°C and room temperature for 28 weeks, and the preservation effect at 4°C was better than that at room temperature. The spray drying method uses a conventional two-fluid nozzle to produce dry powdered or granulated phage products. Matinkhoo (2011) reported that the titer of dry phage powder was only reduced by 0.4 to 0.8 orders of magnitude when a single phage or phage cocktail was spray-dried.

To sum up, there are various storage methods for phages, and different phages have different optimal storage methods. Therefore, it is of great significance to select a suitable storage method for subsequent practical applications.

Contents of the invention

The purpose of the present invention is to provide a lyoprotectant for Vibrio harveyi phage V-YDF132 which has low requirements on preservation conditions and can preserve for a long time without losing biological activity.

The lyoprotectant of Vibrio harveyi phage V-YDF132 of the present invention contains inulin, skimmed milk powder, gelatin, L-sodium glutamate and water.

Preferably, the lyoprotectant of 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 sodium L-glutamate, and the balance is purified water per 100 ml.

The second object of the present invention is to provide a preservation method of Vibrio harveyi phage V-YDF132, which is to mix the above-mentioned lyoprotectant with the phage V-YDF132 solution, first freeze the sample to be frozen until solid, and then After freeze-drying, the phage V-YDF132 freeze-dried powder preparation is obtained.

Preferably, the mixing of the lyoprotectant and the phage V-YDF132 solution is based on a volume ratio of 1:2.

Preferably, the concentration of the phage V-YDF132 solution is 6.5×10 ⁸ PFU/mL.

Preferably, the sample to be frozen is first frozen to a solid state, and then freeze-dried to obtain the phage V-YDF132 freeze-dried powder preparation. The mixed solution is pre-frozen at -80°C for 12 hours to form a solid sample to be frozen, and the CHRSIT freeze is turned on. Dryer refrigeration function, when the temperature of the cold trap drops below -40°C; put the solidified sample to be frozen into the freeze dryer, the freeze dryer turns on the vacuum mode, the air pressure in the cold trap reaches 0.37mbar, freeze-dry for 18-24 hours Afterwards, the Vibrio harveyi phage V-YDF132 freeze-dried powder preparation is prepared.

There are many ways to store phages, and different phages have different best ways to store them. The present invention aims to lay the foundation for phage preservation by screening and preparing different phage freeze-drying protective agents and exploring their protective effects on phage after freeze-drying. The phage powder preparation prepared from the screened lyoprotectant formula P9 and phage V-YDF132 requires low storage conditions and can be stored for a long time without losing biological activity.

The virulent Vibrio harveyi bacteriophage V-YDF132 of the present invention was deposited in the Guangdong Microbial Culture Collection Center on July 05, 2022, and the preservation number is GDMCC No: 62599-B1. The phage is disclosed in the patent application number CN202210886269.9, the invention name is a patent of Vibrio harveyi phage V-YDF132 and its application.

Vibrio harveyi YDF132 is preserved in the laboratory of the Department of Aquatic Biomedicine, Ocean College, South China Agricultural University, and it is also disclosed in the patent application number CN202210886269.9, the invention name is a Vibrio harvelii phage V-YDF132 and its application patent , the applicant undertakes to make it available to the public within 20 years from the filing date.

Description of drawings

Fig. 1 is the lyophilized powder preparation prepared by phage V-YDF132 and lyoprotectant.

Fig. 2 is the phage titer determination of phage V-YDF132 freeze-dried powder preparation at 37°C (a) and 4°C (b).

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention 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.

Example 1

1. Experimental method

1 Preparation of phage V-YDF132

The phage in the phage freeze-dried reagent is a phage lysate. Refer to the method described in the "Molecular Cloning Experiment Guide, Third Edition" to expand the phage stock solution, and the specific operation method is slightly modified. Take 500 μL of Vibrio harveyi YDF132 in the logarithmic phase and add it to 500 mL of LB liquid medium, and culture it with shaking at 37°C; add 50 mL of phage V-YDF132 solution and continue culturing on the shaker until the mixture becomes completely clear; mix the The liquid was centrifuged at 4000 rpm for 10 min at 4°C, and the supernatant was filtered with a 0.22 μm filter membrane to obtain a phage YDF132 liquid. Add RNase A enzyme and DNase I enzyme to the phage YDF132 solution to a final concentration of 1 μg/mL, and place at room temperature for 30 minutes; add solid NaCl to a final concentration of 1 M, and ice-bath for 1 hour after the NaCl dissolves; centrifuge at 4°C, 8300rpm After 10 minutes, measure the volume of the supernatant; add solid polyethylene glycol (PEG8000) to a final concentration of 10% (w/v), dissolve and place in an ice-water mixture overnight; the next day at 4 ° C, 12,000 rpm high speed After centrifugation for 15 min, the precipitate after centrifugation was washed 3 times with SM buffer, and SM buffer was added to resuspend the precipitate. The titer of phage in the resuspension after expansion culture was determined by double-layer plate method.

2 Preparation of phage V-YDF132 lyoprotectant

The phage lyophilized powder preparation is a lyophilized mixture formed by lyophilization of a mixed solution of phage and lyoprotectant; wherein, in the mixed solution, every 100mL lyoprotectant consists of the following components in parts by mass:

1. Sucrose 30g, skimmed milk powder 5.5g, gelatin 10g, L-sodium glutamate 0.25g, the rest is purified water.

2. Trehalose 15g, skimmed milk powder 5.5g, gelatin 10g, L-sodium glutamate 0.25g, the balance is purified water.

3. Inulin 15g, skimmed milk powder 5.5g, gelatin 10g, L-sodium glutamate 0.25g, the rest is purified water.

Weigh according to the weight of each component of the above three freeze-drying protective agents, mix uniformly, and sterilize under high pressure to obtain the product. Among them, the high-pressure condition is sterilized at 115° C. for 30 minutes. The solution containing bacteriophage YDF132 (concentration: 6.5×10 ⁸ PFU/mL) was mixed with each lyoprotectant in volume ratios of 7:1, 4:1, and 2:1 respectively to prepare 9 kinds of mixed solutions, namely 9 different formulations for this trial:

P1: 30g sucrose, 5.5g skimmed milk powder, 10g gelatin, 0.25g sodium L-glutamate, and purified water as the rest; mix with phage solution at a volume ratio of 1:7.

P2: 30g of sucrose, 5.5g of skimmed milk powder, 10g of gelatin, 0.25g of sodium L-glutamate, and the rest is purified water; mix with phage solution at a volume ratio of 1:4.

P3: 30g of sucrose, 5.5g of skimmed milk powder, 10g of gelatin, 0.25g of sodium L-glutamate, and the rest is purified water; mix with phage solution at a volume ratio of 1:2.

P4: Trehalose 15g, skimmed milk powder 5.5g, gelatin 10g, L-sodium glutamate 0.25g, the rest is purified water; mix with phage solution at a volume ratio of 1:7.

P5: Trehalose 15g, skimmed milk powder 5.5g, gelatin 10g, L-sodium glutamate 0.25g, the rest is purified water; mix with phage solution at a volume ratio of 1:4.

P6: Trehalose 15g, skimmed milk powder 5.5g, gelatin 10g, L-sodium glutamate 0.25g, the rest is purified water; mix with phage solution at a volume ratio of 1:2.

P7: Inulin 15g, skimmed milk powder 5.5g, gelatin 10g, L-sodium glutamate 0.25g, the rest is purified water; mix with phage solution at a volume ratio of 1:7.

P8: Inulin 15g, skimmed milk powder 5.5g, gelatin 10g, L-sodium glutamate 0.25g, the rest is purified water; mix with phage solution at a volume ratio of 1:4.

P9: Inulin 15g, skimmed milk powder 5.5g, gelatin 10g, L-sodium glutamate 0.25g, the rest is purified water; mix with phage solution at a volume ratio of 1:2.

The mixed solution was pre-frozen at -80°C for 12 hours to become a solid sample to be frozen. Turn on the refrigeration function of the CHRSIT freeze dryer, and wait until the temperature of the cold trap drops below -40°C; put the solidified sample to be frozen into the freeze dryer, turn on the vacuum mode of the freeze dryer, and the air pressure in the cold trap reaches 0.37mbar, freeze-dry After 18-24 hours, the freeze-dried powder preparation of Vibrio harveyi phage V-YDF132 is ready.

3 Screening of the best lyoprotectant for phage V-YDF132

After the lyophilization was completed, the phage titers in the 9 lyophilized powders were immediately determined. The method for determining the potency of the phage freeze-dried powder is to dissolve 0.1 g of the freeze-dried powder in 1 mL of SM buffer, and then use the double-layer plate method to measure the potency of the phage after it is completely dissolved. The host bacteria is Vibrio harveyi YDF132. Specifically, the bottom TCBS medium and the upper TCBS medium containing 2% and 1% agar are respectively prepared in advance. First pour a layer of flat plate on the petri dish with the bottom medium, after it solidifies, then melt and cool it to below 45°C, add a thicker sensitive host and a certain volume of the upper medium of the phage sample to be tested, and put it in the test tube After shaking well, immediately pour it on the bottom medium and spread it flat to wait for coagulation, and then keep it warm at 37°C. Plaques can be counted after about 10 hours. The lyophilized powder was placed in a constant temperature incubator at 37°C, and the phage titers in the 9 lyophilized powders were recorded every 7 days. The best lyoprotectant was determined according to the survival status of the phage before lyophilization, after lyophilization and in a 37°C environment. At the same time, the best lyophilized powder preparation was placed in a 4°C environment, and the phage titer in the lyophilized powder was recorded every 3 months.

2. Experimental results

1 Preparation of Vibrio harveyi phage V-YDF132 lyoprotectant

The phage with biological activity is mixed with a lyoprotectant, and then lyophilized to form a phage lyophilized powder preparation. During the preparation process, it is very important to control the content ratio of the lyoprotectant to the phage to ensure that the protectant can effectively protect the phage. The mixed solutions formed by nine kinds of lyoprotectants and phage in this study were all prepared into nine kinds of lyophilized powder preparations (P1-P9) after lyophilization, as shown in Figure 1, so the next step can be carried out smoothly Screening of optimal lyoprotectants.

2 The best lyoprotectant for Vibrio harveyi phage V-YDF132

The above-mentioned lyophilized powder preparations were stored at 37° C. and 4° C., and the phage titer was measured regularly to determine the best lyoprotectant among the nine lyophilized powder preparations. The phage titer of the phage lyophilized powder preparation prepared by the lyoprotectant formula P9 and phage V-YDF132 was 1×10 ^8.633 PFU/mL before lyophilization, and the phage titer in the lyophilized powder preparation after lyophilization was 1×10 ^8.123 PFU/mL, only a drop of 0.51 titers; the phage titer in the lyophilized powder preparation was 1×10 ^7.226 PFU/mL after storage at 37°C for 21 days, which was comparable to the phage titer after lyophilization The ratio decreased by 0.897 titers (Fig. 2a); the phage titer in the lyophilized powder preparation was 1×10 ^7.616 PFU/mL after being stored at 4°C for 9 months, a decrease of 0.511 titers, and the phage survival rate was 93.7 % (Fig. 2b). The above results indicate that the phage powder preparation prepared by lyoprotectant formula P9 and phage V-YDF132 requires low storage conditions and can be stored for a long time without losing biological activity.

P9: Inulin 15g, skimmed milk powder 5.5g, gelatin 10g, L-sodium glutamate 0.25g, and the balance is purified water. applicability.

Claims (6)

1. A lyoprotectant for Vibrio harveyi phage V-YDF132, characterized in that it contains inulin, skimmed milk powder, gelatin, L-sodium glutamate and water. 2. The lyoprotectant according to claim 1, characterized in that, the lyoprotectant of the Vibrio harveyi phage V-YDF132 contains 15g of inulin per 100ml, 5.5g of skimmed milk powder, and 10g of gelatin , L-sodium glutamate 0.25g, and the balance is purified water. 3. a preservation method of Vibrio harveyi phage V-YDF132, is characterized in that, the lyoprotectant of claim 1 or 2 is mixed with the phage V-YDF132 solution, first frozen to the solid state to be frozen sample, After freeze-drying, the phage V-YDF132 freeze-dried powder preparation is obtained. 4. The preservation method according to claim 3, wherein the mixing of the lyoprotectant and the phage V-YDF132 solution is based on a volume ratio of 1:2. 5. The preservation method according to claim 3 or 4, characterized in that the concentration of the phage V-YDF132 solution is 6.5×10 ⁸ PFU/mL. 6. The preservation method according to claim 3, characterized in that, the first freezing to a solid state to be frozen sample, and then freeze-drying to obtain the phage V-YDF132 freeze-dried powder preparation is carried out at -80 ° C with the mixed solution Pre-freezing treatment for 12 hours to solidify the sample to be frozen, turn on the refrigeration function of the CHRSIT freeze dryer, wait for the temperature of the cold trap to drop below -40°C; put the solidified sample to be frozen into the freeze dryer, and turn on the vacuum mode of the freeze dryer , the air pressure in the cold trap reaches 0.37mbar, and freeze-dried for 18-24 hours to obtain a freeze-dried powder preparation of Vibrio harveyi phage V-YDF132.