Application of D-tryptophan synergistic low-temperature plasma

Abstract

The invention discloses an application of D-tryptophan synergetic low-temperature plasma, and the D-tryptophan synergetic low-temperature plasma can generate a killing effect on escherichia coli, pseudomonas fluorescens, staphylococcus aureus, shewanella volvacea and bacterial suspensions of the shewanella volvacea with the concentration of 107 CFU / mL under the condition that the concentration of the D-tryptophan is relatively low, so that the D-tryptophan synergetic low-temperature plasma can be used for killing the bacterial suspensions of the escherichia coli, the pseudomonas fluorescens, the staphylococcus aureus, the shewanella volvacea. And the sterilization method is high in safety, simple and convenient to operate, short in time consumption and low in cost.

Description

Technical Field

[0001] The invention relates to the fields of food and medicine, and in particular to an application of D-tryptophan in coordination with low-temperature plasma. Background Art

[0002] One of the main reasons for food spoilage is microbial contamination. These microorganisms convert proteins, polysaccharides, lipids, etc. in food into nutrients for growth and reproduction, which are consumed and degraded, changing the sensory qualities of food such as appearance and smell, and ultimately causing food spoilage, posing a safety hazard to humans, and causing environmental pollution and waste of resources. Among them, the microorganisms that dominate the spoilage process are called specific spoilage microorganisms. For example, the main specific spoilage microorganisms in aquatic products, such as Shewanella and Pseudomonas, cause food spoilage by regulating their own growth metabolism and product production. Therefore, it is extremely important to study a method that can effectively kill and inhibit bacteria.

[0003] Plasma is an emerging non-thermal sterilization and preservation technology. Plasma is formed by ionizing the gas in a certain space through a high-voltage electric field, thereby producing active nitrogen oxides. These active nitrogen oxides will damage bacterial cell membranes, DNA, related enzyme activities, etc., causing serious oxidative damage and ultimately leading to bacterial death. It has the advantages of high sterilization efficiency, no chemical residues, and no pollution, and has great application potential in the field of food sterilization. At present, the main plasmas are: dielectric barrier discharge plasma, jet plasma, corona discharge plasma, etc. However, under low frequency conditions, the plasma sterilization effect will be significantly weakened.

[0004] The Chinese invention patent with application publication number CN 109675195 A discloses a sterilization method based on plasma-assisted auxiliary agent. The sterilization method comprises the following steps: (1) preparation of auxiliary agent: placing persulfate in ultrapure water, stirring to dissolve the persulfate, then adding glycerol and acetic acid and continuing to stir and mix to obtain an auxiliary agent, the pH value of which ranges from 3.4 to 6.8; (2) adding the auxiliary agent to the infected biological sample, and then placing it in a plasma generator for plasma discharge treatment. The sterilization method can treat fungal infections of the skin and nails, but is not suitable for application in the field of food sterilization with high safety requirements. Summary of the invention

[0005] In order to solve the above problems, the present invention provides an application of D-tryptophan in conjunction with low-temperature plasma, which can be applied to various fields such as food sterilization.

[0006] The technical solution of the present invention to solve the above problems is as follows: The present invention provides application of D-tryptophan in cooperation with low-temperature plasma in food antibacterial and sterilization. D-tryptophan is a natural material with high safety and can be used in the field of food sterilization.

[0007] Plasma refers to a mixture of substances including ions, electrons, free radicals and neutral molecules produced by injecting enough energy into a gas to partially or completely ionize it. It is generally electrically neutral and is also called the "fourth state" of matter. Generally speaking, people use temperature to distinguish plasmas, and the temperature here usually refers to the electron temperature. High-temperature plasma is generally used for nuclear fusion, and the temperature produced reaches millions of Kelvin. Low-temperature plasma refers to plasma with charged particle energy in the order of several eV. In this case, the reaction is mainly participated by free electrons and valence electrons, and does not involve nuclear reactions and X-rays, but it is also sufficient to induce the dissociation and ionization of compounds. The temperature of ions and gases can be close to room temperature.

[0008] Preferably, the concentration of D-tryptophan is not less than 2.5 mmol / L.

[0009] More preferably, the concentration of the D-tryptophan solution is 2.5-40 mmol / L.

[0010] Preferably, the bacteria is at least one of Shewanella balticola, Shewanella putrefaciens, Pseudomonas fluorescens, Staphylococcus aureus, and Escherichia coli.

[0011] Preferably, the concentration of the bacteria is 10 1 ~10 8 CFU / mL.

[0012] Preferably, D-tryptophan and the object to be treated are prepared into a solution and a bacterial suspension, respectively, and then the solution and the bacterial suspension are mixed at a volume ratio of (0.5-1.5):(8.5-9.5), and then treated with low-temperature plasma.

[0013] Preferably, the low-temperature plasma is a high-voltage electric field low-temperature plasma.

[0014] Preferably, the high-voltage electric field low-temperature plasma is manufactured using high-voltage electric field low-temperature plasma equipment; the medium of the high-voltage electric field low-temperature plasma is air.

[0015] Preferably, the peak voltage generated by the high-voltage electric field low-temperature plasma device is 80-160 kV, the action time is greater than 20 s, the distance between the dielectric barrier discharge plates of the high-voltage electric field low-temperature plasma device is 20-35 mm, and the device operating frequency is 15-75 Hz.

[0016] Further preferably, the peak voltage generated by the high-voltage electric field low-temperature plasma device is 100-160 kV, the action time is greater than 90-120 s, the distance between the dielectric barrier discharge plates of the high-voltage electric field low-temperature plasma device is 25-30 mm, and the device operating frequency is 40-60 Hz.

[0017] The present invention also provides other applications of D-tryptophan in combination with low-temperature plasma, such as in vitro bactericidal and antibacterial effects, instrument sterilization and disinfection, etc.

[0018] The present invention has the following beneficial effects: (1) The present invention uses D-tryptophan in combination with low-temperature plasma to kill bacteria. The results show that the method has a significant bactericidal effect and has the characteristics of broad-spectrum bactericidal. In the presence of a low D-tryptophan concentration, the method can kill bacteria with a concentration of 10 7 CFU / mL of bacterial suspensions of Escherichia coli, Pseudomonas fluorescens, Staphylococcus aureus, Shewanella balticola, and Shewanella balticola all produced killing effects; (2) The D-tryptophan used in the present invention is a natural material with high safety and can be used in the field of food sterilization with wide applicability; (3) The sterilization method of the present invention using D-tryptophan in combination with low-temperature plasma is simple to operate, short in time and low in cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The sterilization effect diagram of Examples 1 to 5. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than all 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.

[0021] It should be noted that the experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0022] If the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased from the market.

[0023] Example 1 S1, pick Escherichia coli ( E. coli) into a conical flask containing 100 mL of LB medium, shake culture overnight at 180 rpm and 37 °C, centrifuge the medium at 6000 rpm for 10 min, wash twice with saline and resuspend, and adjust the concentration of the bacterial suspension to 10 8 CFU / mL or so; S2. Weigh a certain amount of D-tryptophan using an analytical balance, dissolve it with sterile deionized water, and perform ultrasonic treatment until the dissolution is complete. The concentration of the D-tryptophan solution is 2.5 mM. S3, divided into four groups of experiments: Blank group: CK (no treatment); Single control group (single dielectric barrier discharge plasma treatment group): single dielectric barrier discharge plasma treatment (DBD); Single control group (D-tryptophan treatment group): 2.5 mM D-tryptophan treatment (D-Trp); Experimental group: dielectric barrier discharge plasma + D-tryptophan treatment (DBD+D-Trp); S4, take 200 μL of bacterial suspension and 1.8 mL of D-tryptophan solution (i.e., the volume ratio of bacterial suspension to D-tryptophan solution is 1:9) and mix them evenly into a 35 mm sterile culture dish without a cover. Sterile deionized water was used instead of D-tryptophan in the blank group and the single dielectric barrier discharge plasma treatment group. S5. Move the culture dish into a 650 mL fresh-keeping box and seal the box. S6, experimental group and single dielectric barrier discharge plasma treatment group: the fresh-keeping box was placed in a high-voltage electric field low-temperature plasma device, and the parameters were set as follows: peak voltage 110 kV, the distance between the dielectric barrier plates was 28 mm, the working frequency was 50 Hz, and the working time was 90 s to complete the sterilization; S7. Perform ten-fold gradient dilution of the treated bacterial solution, apply 100 μL of the appropriate gradient dilution solution to the plate, and culture at 37°C for 48 h to count the colonies.

[0024] Example 2 It is basically the same as Example 1, except that: S1. Pick out Pseudomonas fluorescens ( P.fluorescens ) into a conical flask containing 100 mL of LB medium, shake culture overnight at 180 rpm and 30°C, centrifuge the medium at 6000 rpm for 10 min, wash twice with saline and resuspend, and adjust the concentration of the bacterial suspension to 10 8 CFU / mL or so; S7. Perform ten-fold gradient dilution of the treated bacterial solution, apply 100 μL of the appropriate gradient dilution solution to the plate, and culture at 30°C for 48 h to count the colonies.

[0025] Example 3 It is basically the same as Example 1, except that: S1. Pick Staphylococcus aureus ( S. aureus ) into a conical flask containing 100 mL of LB medium, shake culture overnight at 180 rpm and 37°C, centrifuge the medium at 6000 rpm for 10 min, wash twice with saline and resuspend, and adjust the concentration of the bacterial suspension to 10 8 CFU / mL or so.

[0026] Example 4 It is basically the same as Example 1, except that: S1. Pick up Baltic Shewanella ( S.baltica ) into a conical flask containing 100 mL of LB medium, shake culture overnight at 180 rpm and 30°C, centrifuge the medium at 6000 rpm for 10 min, wash twice with saline and resuspend, and adjust the concentration of the bacterial suspension to 10 8 CFU / mL or so; S6, experimental group and single dielectric barrier discharge plasma treatment group: the fresh-keeping box was placed in a high-voltage electric field low-temperature plasma device, and the parameters were set as follows: peak voltage 100 kV, the distance between the dielectric barrier plates was 28 mm, the working frequency was 50 Hz, and the working time was 90 s to complete the sterilization; S7. Perform ten-fold gradient dilution of the treated bacterial solution, apply 100 μL of the appropriate gradient dilution solution to the plate, and culture at 30°C for 48 h to count the colonies.

[0027] Example 5 It is basically the same as Example 1, except that: S1. Pick a single colony of S. putrefaciens into a conical flask containing 100 mL of LB medium and culture it overnight at 180 rpm and 30°C. Centrifuge the medium at 6000 rpm for 10 min, wash it twice with saline and resuspend it. Adjust the concentration of the bacterial suspension to 10 8 CFU / mL or so; S7. Perform ten-fold gradient dilution of the treated bacterial solution, apply 100 μL of the appropriate gradient dilution solution to the plate, and culture at 30°C for 48 h to count the colonies.

[0028] The bactericidal effects of the experimental group, the blank group and the two single control groups in the above embodiment are as follows: Figure 1 As shown, according to Figure 1 It can be seen that compared with the single dielectric barrier discharge plasma sterilization treatment (D-Trp) and the single D-tryptophan sterilization treatment (D-Trp), the D-tryptophan synergistic low-temperature plasma sterilization method (DBD+D-Trp) of the present invention has a significant sterilization effect. 7 The bacterial suspensions of Escherichia coli, Pseudomonas fluorescens, Staphylococcus aureus, Shewanella balticola, and Shewanella balticola all produced killing effects.

Claims

1. Application of D-tryptophan and low-temperature plasma in food antibacterial and sterilization.

2. The use according to claim 1, characterized in that: The concentration of the D-tryptophan is not less than 2.5 mmol / L.

3. The use according to claim 1, characterized in that: The bacteria is at least one of Shewanella balticola, Shewanella putrefaciens, Pseudomonas fluorescens, Staphylococcus aureus and Escherichia coli.

4. The use according to claim 1, characterized in that: The concentration of the bacteria was 10 1 ~10 8 CFU / mL.

5. The use according to claim 1, characterized in that: D-tryptophan and the object to be treated are respectively prepared into a solution and a bacterial suspension, the solution and the bacterial suspension are mixed at a volume ratio of (0.5-1.5):(8.5-9.5), and then treated with low-temperature plasma.

6. The use according to claim 1, characterized in that: The low-temperature plasma is a high-voltage electric field low-temperature plasma.

7. The use according to claim 6, characterized in that: The high-voltage electric field low-temperature plasma is manufactured using high-voltage electric field low-temperature plasma equipment; the medium of the high-voltage electric field low-temperature plasma is air.

8. The use according to claim 7, characterized in that: The peak voltage generated by the high-voltage electric field low-temperature plasma equipment is 80-160 kV, the action time is greater than 20s, the distance between the dielectric barrier discharge plates of the high-voltage electric field low-temperature plasma equipment is 20-35 mm, and the equipment operating frequency is 15-75 Hz.

9. Application of D-tryptophan synergistically with low-temperature plasma in in vitro bactericidal and antibacterial effects.

10. Application of D-tryptophan synergistic with low-temperature plasma in sterilization and disinfection of instruments.

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