Staphylococcus aureus bacteriophage and application thereof

Abstract

The invention provides a staphylococcus aureus phage and application of the staphylococcus aureus phage, the staphylococcus aureus phage is staphylococcus aureus phage GRNSAP30, and the preservation number of the staphylococcus aureus phage is CCTCC (China Center for Type Culture Collection) NO: M2024537. The phage GRNSAP30 disclosed by the invention has a cleaning effect on staphylococcus aureus of vegetable, fruit or meat packaging materials, and can be used for effectively reducing the bacterium loading amount of the staphylococcus aureus in food packaging materials and reducing the food poisoning rate caused by staphylococcus aureus pollution; the invention provides a safe biological scavenger without toxic and side effects for resisting food poisoning caused by grape balls.

Description

Technical Field

[0001] The present invention relates to the technical field of bacteriophages, in particular to a bacteriophage of Staphylococcus aureus and application thereof. Background Art

[0002] Food packaging materials play a key role in the storage and transportation of food. They can protect food from physical damage and environmental pollution. However, food packaging materials themselves may become carriers of microbial contamination, thus affecting the safety and quality of food. Among them, Staphylococcus aureus is a common pathogen that can not only grow and multiply on the surface of packaging materials, but also produce heat-resistant enterotoxins, leading to food poisoning. Traditional sterilization methods, such as heat treatment and chemical disinfection, although effective, may change the sensory and nutritional properties of food, and excessive use of chemical disinfectants can cause environmental pollution and drug-resistant strains. Therefore, the development of an efficient, safe and environmentally friendly way to remove Staphylococcus aureus, especially for its application in food packaging materials, has important practical significance and scientific research value.

[0003] Bacteriophages are a type of virus that specifically infects and kills bacteria. Since they were discovered in the early 20th century, they have been widely studied by scientists. Bacteriophages have a high degree of host specificity and can selectively infect target bacteria without affecting humans, animals, plants, and beneficial microorganisms. Therefore, they are considered to be a potential green antibacterial agent. In recent years, bacteriophages have been widely used in the fields of food industry, medical care, environmental protection, etc. to prevent and control bacterial infections. However, the application of bacteriophages to food packaging materials to combat Staphylococcus aureus contamination is still in the initial exploration stage. Therefore, further research and development of technologies that can stably and effectively fix bacteriophages on food packaging materials, and their application methods in actual production, are important current research directions. This will not only enhance the antibacterial properties of food packaging materials, but also provide new solutions for food safety. Summary of the invention

[0004] In view of this, the present invention provides a bacteriophage of Staphylococcus aureus and application thereof, wherein the bacteriophage removes Staphylococcus aureus in food packaging materials.

[0005] The technical solution of the present invention is implemented as follows: On the one hand, the present invention provides a phage of Staphylococcus aureus, wherein the phage of Staphylococcus aureus is Staphylococcus aureus phage GRNSAP30 (hereinafter referred to as "phage GRNSAP30"), which was sent to the China Center for Type Culture Collection on March 21, 2024 for preservation, and the preservation address is: Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M2024537. The collection center identified phage GRNSAP30 on March 28, 2024, and the result was a survival state.

[0006] On the other hand, the present invention also provides a use of Staphylococcus aureus phage in food packaging materials.

[0007] On the basis of the above technical solution, preferably, the Staphylococcus aureus phage GRNSAP30 is used to kill Staphylococcus aureus, Staphylococcus lentus or Staphylococcus xylinum on the surface of food packaging materials.

[0008] On the basis of the above technical solution, preferably, the food packaging material is made of metal, glass or plastic.

[0009] In yet another aspect, the present invention further provides a bactericide, comprising Staphylococcus aureus phage GRNSAP30.

[0010] On the basis of the above technical solution, preferably, the bactericide is a spray or a rinse agent.

[0011] The phage of Staphylococcus aureus and its application of the present invention have the following advantages compared with the prior art:

[0012] Beneficial effects:

[0013] The Staphylococcus aureus phage GRNSAP30 of the present invention has a scavenging effect on Staphylococcus aureus in vegetable, fruit or meat packaging materials, can effectively reduce the bacterial load of Staphylococcus aureus in food packaging materials, and reduce the rate of food poisoning caused by Staphylococcus aureus contamination, thus providing a safe biological scavenger with no toxic side effects for resisting food poisoning caused by Staphylococcus.

[0014] Compared with the existing phages, the Staphylococcus aureus phage GRNSAP30 of the present invention has the following advantages: (1) High fermentation titer: When the concentration of phage GRNSAP30 is 2.5×10 5 PFU / mL, host bacteria concentration is 3.3×10 8 CFU / mL, when the fermentation temperature was 30℃, the fermentation titer of bacteriophage GRNSAP30 reached the highest level of 5.6×10 11 PFU / mL). ⑵Good high temperature resistance: the activity remains basically unchanged below 55℃, and still has a high titer after 12h in a 65℃ water bath. ⑶Broad spectrum of phage lysis: 34 out of 36 strains of Staphylococcus aureus can be lysed, with a lysis rate of 85.0%; 8 out of 15 strains of Staphylococcus aureus can be lysed, with a lysis rate of 53.3%; 2 out of 5 strains of Staphylococcus aureus can be lysed, with a lysis rate of 40%, which is highly practical. DETAILED DESCRIPTION

[0015] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] The formula of the TSB liquid culture medium used in the present invention is: 17g of casein pancreatic digest, 3g of soy flour papain digest, 5g of sodium chloride, 2.5g of dipotassium hydrogen phosphate, 2.5g of glucose, 1000mL of purified water, pH 7.3±0.2, and it is used after being sterilized by pressure steam at 121°C.

[0017] The formula of the TSB semi-solid culture medium used in the present invention is: 17g of casein pancreatic digest, 3g of soy flour papain digest, 5g of sodium chloride, 2.5g of dipotassium hydrogen phosphate, 2.5g of glucose, 7.5g of agar, 1000mL of purified water, pH 7.3±0.2, and it is used after being sterilized by pressure steam at 121°C.

[0018] The SM buffer used in the present invention has a formula of: 5.8 g sodium chloride, 2 g magnesium sulfate, 50 mL 1 mol / L Tris-HCl, 0.25 g gelatin, 1000 mL distilled water, pH 7.3±0.2, and is used after being sterilized by pressure steam at 121°C.

[0019] 1. Isolation and purification of bacteriophage GRNSAP30

[0020] 16 water samples were collected from a chicken farm in Jiangxi Province where Staphylococcus aureus was found. Each sample was 25 mL. The water sample was centrifuged at 10,000 rpm for 30 min, and 20 mL of the supernatant was filtered and sterilized. 10 mL of the filtrate was mixed with 10 mL of 2×TSB liquid culture medium and 1 mL of Staphylococcus aureus culture medium in the logarithmic phase (10 8 cfu / mL) and cultured overnight at 37℃180rpm to enrich phages. Centrifuge the sample enrichment solution at 10000rpm for 15min, take the supernatant and sterilize it through a 0.22μm microporous filter membrane to obtain a filtrate containing phages. Take 1mL of the filtrate and mix it evenly with 500μL of its host Staphylococcus aureus solution, let it stand for 15min to allow it to adsorb to the bacterial surface. Add 7mL of TSB semi-solid agar medium cooled to 50℃ to the above mixture, mix well and immediately spread it on the solidified NA plate. After the agar solidifies, invert and culture it at 37℃ for 8-12h to observe the growth of phage plaques. On the double-layer plate where plaques are formed, pick up large and transparent plaques with a sterile pipette tip, desorb them by shaking in 1mL SM buffer, and sterilize them through a 0.22μm microporous filter to obtain a phage filtrate. Inoculate the filtrate in 10mL TSB liquid culture medium, add 100μL of host Staphylococcus aureus culture activated overnight and mix well, culture overnight at 37℃ and 180rpm, centrifuge at 5000rpm for 10min, take the supernatant and pass it through a 0.22μm microporous filter, and observe the morphology of plaques using the double-layer plate method. After repeating the operation 3-5 times, plaques of the same shape and size can be obtained, the center of the plaque is transparent, and the diameter is about 1mm. The phage was sent to the China Center for Type Culture Collection for preservation on March 21, 2024, and its classification name is Staphylococcus aureus phage GRNSAP30. The address is Wuhan University, Wuhan, China. The preservation number is CCTCC NO: M2024537.

[0021] 2. Determination of the optimal fermentation conditions for bacteriophage GRNSAP30

[0022] A single colony of the host Staphylococcus aureus was picked and inoculated into a test tube containing 3 mL of TSB culture medium. The culture was shaken at 180 rpm at 37 ° C for 12 hours to obtain a host bacterial suspension. The bacterial suspension was transferred to 10 mL of TSB culture medium at a ratio of 1:100 and cultured at 37 ° C and 180 rpm to the early logarithmic period. The phage GRNSAP30 and its host Staphylococcus aureus were diluted to a certain concentration and counted, mixed and cultured in different proportions, and TSB liquid culture medium was added to make the total volume of each tube the same. Four copies were prepared for each concentration ratio and cultured at 180 rpm for 12 hours at 25 ° C, 28 ° C, 32 ° C and 37 ° C respectively. After the culture was completed, the fermentation broth was centrifuged at 6000g for 10 minutes and the supernatant was collected. The supernatant was passed through a 0.22 μm microporous filter membrane and counted by double-layer plate method. Each point was cultured in duplicate tubes and the average value was taken. The condition that produced the highest phage titer was the optimal fermentation condition. The experiment was repeated 3 times and the results are shown in Table 1.

[0023] Table 1 The titer of phage GRNSAP30 at different infection multiplicity

[0024]

[0025] As shown in Table 1, when the concentration of phage GRNSAP30 was 2.5×10 5 PFU / mL, host bacteria concentration is 3.3×10 8 CFU / mL, when the fermentation temperature was 30℃, the fermentation titer of bacteriophage GRNSAP30 reached the highest level of 5.6×10 11 PFU / mL.

[0026] 3. Determination of temperature resistance of bacteriophage GRNSAP30

[0027] Take several sterile 50mL centrifuge tubes, add 20mL TSB to each tube and place it in a constant temperature water bath at the corresponding temperature. After the temperature is balanced, add 1mL of pure phage culture solution to each tube and act at 4℃, 25℃, 37℃, 45℃, 55℃, 65℃, 75℃, and 85℃ for 1h, 2h, 12h, 24h, 48h, and 72h, respectively. After the action time is over, take out the sample tube and immediately cool it in a room temperature water bath. After appropriate dilution, use the double-layer plate method to determine the phage titer. Each point is cultured in duplicate tubes to take the average value. The experiment was repeated 3 times. The results are shown in Table 2.

[0028] Table 2 The titer of phage GRNSAP30 after storage at different temperatures for different time periods

[0029]

[0030] As shown in Table 2, the activity of bacteriophage GRNSAP30 remained basically unchanged at a temperature below 55°C, and it still had a high titer after being placed in a 65°C water bath for 12 hours. It had good stability at 4-55°C and could be stored for a long time.

[0031] IV. Phage GRNSAP30 lysis spectrum

[0032] Phage GRNSAP30 lysis range test on Staphylococcus aureus: the titer is 7.2×10 9 PFU / mL Staphylococcus phage GRNSAP30 stock solution, 10 times diluted to 10 -4 , take the original solution and 10 -3 and 10 -4 The lysis spectrum of phage was determined by the spotting method at three concentrations.

[0033] 40 chicken-derived Staphylococcus aureus monoclonals, 15 slow Staphylococcus monoclonals, and 5 wood Staphylococcus monoclonals isolated from clinical sites were selected and inoculated into centrifuge tubes containing 3 mL TSB, and cultured at 37 ° C 180 rpm for 12 hours to obtain bacterial suspensions of each strain. 1 mL of bacterial suspension was mixed with TSB semi-solid medium and spread on the prepared TSA plate, and 5 μL of phage culture solution and dilution solution were dripped on the plate. After natural air drying, culture at 37 ° C for 12-16 hours, and the results were observed. The experiment was repeated three times, and the results are shown in Table 3.

[0034] Table 3 Results of lysis spectrum determination of Staphylococcus phage GRNSAP30

[0035]

[0036]

[0037] Note: “-” indicates no lysis; “+” indicates lysis with blurred lysis spots; “++” indicates lysis with clear lysis spots.

[0038] As shown in Table 3, the phage stock solution can lyse 36 of the 40 strains of Staphylococcus aureus, and 10 -3 and 10 -4 The diluent can lyse 34 of 36 strains of Staphylococcus aureus (lysis rate is 85.0%), 8 of 15 strains of Staphylococcus lentus (lysis rate is 53.3%), and 2 of 5 strains of Staphylococcus xylinus (lysis rate is 40%). Through lysis spectrum determination, it was found that the phage has a wide lysis spectrum and has great application potential in preventing and controlling food contamination caused by Staphylococcus.

[0039] V. Experimental study on the effect of bacteriophage GRNSAP30 on reducing the bacterial load of Staphylococcus aureus in metal cans, glass bottles and polyethylene plastic food packaging materials

[0040] Pick a single colony of the host Staphylococcus aureus and inoculate it into a test tube containing 3 mL of TSB culture medium. Incubate it in a shaker at 37°C at 180 rpm for 12 h to obtain a host bacterial suspension. Adjust the host bacterial concentration to 5 × 10 8 cfu / mL, 500 μL of bacterial solution was added to the three materials, and after drying, it was placed at 4°C, 25°C and 37°C. After 24 hours, the bacterial colonization on the materials was calculated by plate count method. The titer of the experimental group was 7.2×10 9 PFU / mL phage GRNSAP30 stock solution 200uL, the control group without phage was tested for bacterial load on the packaging material after 24h. The experiment was repeated three times, and the results are shown in Table 4.

[0041] Table 4 Reduction of bacterial load of food packaging materials by Staphylococcus phage GRNSAP30

[0042]

[0043] The results in Table 4 show that bacteriophage GRNSAP30 can effectively reduce the Staphylococcus aureus load in metal cans, glass bottles and polyethylene plastic food packaging materials, and can be used as a bactericide for metal cans, glass bottles and polyethylene plastic food packaging materials, and the bactericide is a spray or rinse agent.

[0044] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A bacteriophage of Staphylococcus aureus, characterized in that: The Staphylococcus aureus phage is Staphylococcus aureus phage GRNSAP30, and its deposit number is CCTCC NO: M2024537.

2. Use of the bacteriophage of Staphylococcus aureus as claimed in claim 1 in food packaging materials.

3. The use of a Staphylococcus aureus phage in food packaging materials as claimed in claim 2, characterized in that: The Staphylococcus aureus phage GRNSAP30 is used to kill Staphylococcus aureus, Staphylococcus lentus or Staphylococcus xylinum on the surface of food packaging materials.

4. The use of a Staphylococcus aureus phage in food packaging materials as claimed in claim 2, characterized in that: The food packaging material is made of metal, glass or plastic.

5. A bactericide, characterized in that: The invention comprises the Staphylococcus aureus phage GRNSAP30 as claimed in claim 1.

6. A fungicide according to claim 5, characterized in that: The bactericide is a spray or a rinse agent.

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