Method for preparing bacterial shadow and application thereof

Abstract

The present invention relates to a method for preparing bacterial ghosts, comprising forming a non-thermal plasma by gas discharge, and treating bacteria using the non-thermal plasma, wherein the gas is nitrogen. The present invention also relates to bacterial ghosts and compositions containing bacterial ghosts.

Description

Technical Field

[0001] The invention relates to the preparation and application fields of bacterial ghosts. Background Art

[0002] Bacterial ghosts refer to empty bacterial shells that do not contain bacterial cell contents such as nucleic acids and cytoplasm. They can be prepared by forming pores in the inner and outer membranes of bacteria through biological, physical, or chemical methods to allow the contents to flow out. Because bacterial ghosts contain many natural immune stimulatory factors, they can effectively activate innate immunity and adaptive immunity. At the same time, bacterial ghosts can also serve as immune adjuvants and carriers to assist different antigens in producing immune protection. Therefore, it is of great significance to prepare bacterial ghosts with complete bacterial cell membrane surface antigen characteristics to induce the body's humoral and cellular immune responses and enhance mucosal immune responses.

[0003] Since bacterial ghosts are produced by forming transmembrane channels on the bacterial surface to expel cytoplasmic contents, the composition of the cell membrane and cell wall directly influences the formation of transmembrane channels. Currently, the main methods for producing bacterial ghosts are genetic engineering and chemical methods. Genetic engineering methods involve cloning the lytic gene E into an expression control system to achieve regulated expression of the lytic gene. The E protein can induce the fusion of the inner and outer membranes of Gram-negative bacteria, thereby forming a specific transmembrane pore structure. Cellular contents, including nucleic acids and ribosomes, escape through this pore, leaving only an empty bacterial shell without any contents, thus forming a Gram-negative bacterial ghost. However, many bacteria have significant restrictive genetic barriers. For example, the expression control system for the lytic gene E cannot be introduced into the bacteria or cannot be recognized and activated for expression, making it difficult to produce bacterial ghosts through genetic engineering. In addition, using chemical methods to produce bacterial ghosts can lead to problems such as reagent residues, which are not conducive to medical applications. Therefore, there is an urgent need in the art for a method for producing bacterial ghosts that can overcome the above-mentioned shortcomings of the existing technology. Summary of the Invention

[0004] The present invention provides a physical preparation method for producing bacterial ghosts using non-thermal plasma. Specifically, the inventors discovered that by regulating the gas type, gas flow rate, voltage, frequency, and other conditions of a gas discharge system to generate non-thermal plasma with appropriate energy, and using it to treat bacteria in an appropriate growth stage for an appropriate period of time, bacterial ghosts can be generated. Specifically, the non-thermal plasma treatment can cause the bacteria to form pores on their outer shells, through which the bacterial contents flow out, thereby generating bacterial ghosts. The efficiency of bacterial ghost generation and / or preparation is affected by the above-mentioned preparation conditions. For example, non-thermal plasma with too low energy cannot form pores on the bacterial outer shells, while non-thermal plasma with too high energy directly shatters the bacteria and cannot preserve the basic shape of the bacterial outer shell. By testing different combinations of conditions (such as the gas type, gas flow rate, voltage, frequency, treatment time, bacterial growth stage, etc. of the discharge system), the inventors discovered for the first time an effective method for producing bacterial ghosts, especially Gram-positive bacterial ghosts, using non-thermal plasma.

[0005] The beneficial effects of the present invention include (1) wide applicability to bacteria and unaffected by bacterial restrictive genetic barriers; (2) no problem of chemical reagent residues, and the bacterial ghosts prepared by the present invention are safe and non-toxic, suitable for use in the medical field; and (3) simple operation and short processing time. Furthermore, the present invention has achieved for the first time a physical method for preparing lactic acid bacteria ghosts. Lactic acid bacteria are probiotics. As Gram-positive bacteria, they do not contain endotoxins and can be cultured facultatively anaerobicly. Therefore, they are particularly suitable for use in the medical field, especially on humans or animals.

[0006] The method of preparing bacterial ghosts of the present invention comprises forming a non-thermal plasma by discharging a gas, preferably nitrogen, and treating bacteria with the non-thermal plasma. In some embodiments, the voltage used to form the non-thermal plasma is from about 3,500 to about 12,000 volts, preferably from about 5,000 to about 12,000 volts, more preferably from about 6,000 to about 12,000 volts, more preferably from about 6,500 to about 12,000 volts, more preferably from about 7,000 to about 12,000 volts, more preferably from about 7,500 to about 12,000 volts, more preferably from about 7,500 to about 11,500 volts, more preferably from about 7,500 to about 11,000 volts, more preferably from about 7,500 to about 10,500 volts, more preferably from about 7,500 to about 10,000 volts, more preferably from about 8,000 to about 10,000 volts, and more preferably from about 8,000 to about 9,500 volts.

[0007] In some embodiments, the frequency of the current used to form the non-thermal plasma is from about 10,000 to about 25,000 Hz, preferably from about 11,000 to about 25,000 Hz, more preferably from about 12,000 to about 25,000 Hz, more preferably from about 13,000 to about 25,000 Hz, more preferably from about 14,000 to about 25,000 Hz, more preferably from about 15,000 to about 25,000 Hz, more preferably from about 16,000 to about 25,000 Hz, more preferably from about 17,000 to about 25,000 Hz, more preferably from about 18,000 to about 25,000 Hz, more preferably from about 19,000 to about 25,000 Hz, more preferably from about 20,000 to about 25,000 Hz, Preferably, it is about 18,500 to about 25,000 Hz, more preferably about 19,000 to about 25,000 Hz, more preferably about 19,000 to about 24,500 Hz, more preferably about 19,500 to about 24,500 Hz, more preferably about 20,000 to about 24,500 Hz, more preferably about 20,500 to about 24,500 Hz, more preferably about 21,000 to about 24,500 Hz, more preferably about 21,000 to about 24,500 Hz, more preferably about 21,000 to about 24,000 Hz, more preferably about 21,000 to about 23,500 Hz, more preferably about 21,000 to about 23,000 Hz.

[0008] In some embodiments, the gas flow rate used to form the non-thermal plasma is about 1 to about 10 liters per minute, more preferably about 1 to about 8 liters per minute, more preferably about 1 to about 7 liters per minute, more preferably about 1 to about 6 liters per minute, more preferably about 2 to about 6 liters per minute, more preferably about 2 to about 5.5 liters per minute, more preferably about 2 to about 5 liters per minute, more preferably about 2 to about 4.5 liters per minute, more preferably about 2 to about 4 liters per minute, more preferably about 2.5 to about 3.5 liters per minute, more preferably about 2.75 to about 3.25 liters per minute, and more preferably about 3 liters per minute.

[0009] In some embodiments, the bacteria to be treated are present in a bacterial suspension. In some embodiments, the concentration of the bacterial suspension is about 1×10 6 - Approximately 1×10 7 CFU / ml.

[0010] In some embodiments, the bacteria are treated with non-thermal plasma for a period of time of about 15 to about 30 seconds, more preferably about 15 to about 28 seconds, more preferably about 15 to about 25 seconds, more preferably about 16 to about 24 seconds, more preferably about 17 to about 23 seconds, more preferably about 18 to about 22 seconds, more preferably about 19 to about 21 seconds, and more preferably about 20 seconds.

[0011] In some embodiments, the bacteria used to generate bacterial ghosts are Gram-negative bacteria or Gram-positive bacteria. In some embodiments, the bacteria used to generate bacterial ghosts are Gram-positive bacteria. In some embodiments, the bacteria used to generate bacterial ghosts are lactic acid bacteria (Lactobacillales). In some embodiments, the lactic acid bacteria used to generate bacterial ghosts are Lactococcus or Lactobacillus. In some embodiments, the lactococcus used to generate bacterial ghosts is Lactococcus lactis. In some embodiments, the lactobacillus used to generate bacterial ghosts is Lactobacillus lactis.

[0012] In some embodiments, the non-thermal plasma may be formed by a jet discharge method or a dielectric barrier discharge method.

[0013] In some embodiments, the position of the ejector in the jet discharge method can be anywhere between the highest and lowest suitable positions (inclusive); wherein the highest suitable position is when the ejector's distal end is approximately 20 mm above the liquid surface of the bacterial suspension, and the lowest suitable position is when the ejector's distal end is approximately 15 mm below the liquid surface of the bacterial suspension. Preferably, the highest suitable position is when the ejector's distal end is approximately 15 mm above the liquid surface of the bacterial suspension, and the lowest suitable position is when the ejector's distal end is approximately 10 mm below the liquid surface of the bacterial suspension. More preferably, the highest suitable position is when the ejector's distal end is approximately 10 mm above the liquid surface of the bacterial suspension, and the lowest suitable position is when the ejector's distal end is approximately 5 mm below the liquid surface of the bacterial suspension.

[0014] In some embodiments, in the dielectric barrier discharge method, the container containing the bacterial suspension is placed between two electrode plates, and the upper electrode plate is about 1 to about 3 mm above the liquid surface of the bacterial suspension.

[0015] In some embodiments, the method of the present invention further comprises processing the bacterial ghosts to remove residual DNA.

[0016] In some embodiments, the method of the present invention further comprises freeze-drying the obtained bacterial ghosts.

[0017] In another aspect, the present invention provides bacterial ghosts prepared by the method of the present invention. The bacterial ghosts prepared by the method of the present invention can be used alone as a vaccine adjuvant, can be used in combination with other known adjuvants, or can be used as a carrier for carrying therapeutic biomolecules.

[0018] In another aspect, the present invention provides a composition comprising bacterial ghosts prepared by the method of the present invention, and the composition may further comprise a pharmaceutically acceptable carrier. Detailed Description of the Invention

[0020] Plasma or gas discharge plasma is a mixture of partially ionized gases composed of reactive species such as electrons, charged ions, free radicals, excited molecules, photons and atoms. Electron collisions play a key role in the generation of these reactive species. Artificial plasmas can be generated by applying energy to a dielectric gas or fluid. Plasma can be generated using any energy that can ionize a gas, such as microwaves, radio frequencies, electric or electromagnetic fields, thermal energy, light energy, radioactivity and X-rays. Plasma can be divided into thermal plasma and non-thermal plasma based on the thermodynamic equilibrium between electrons and ions, where non-thermal plasma is also called low temperature plasma. Thermal plasma is in a state of thermodynamic equilibrium, where the energy and temperature of the electrons are approximately equal to the energy and temperature of the ions. Therefore, thermal plasma has a temperature as high as 10 4 K, and has a high ion population (degree of ionization). In contrast, non-thermal plasmas are in a state of thermodynamic non-equilibrium with low ionization, where electrons have a relative high ionization rate of 10 4 The non-thermal plasma is kept at a low temperature, which is on the same order of magnitude as the K, while the temperature of heavy particles (ions and neutrals) is close to room temperature. Therefore, the non-thermal plasma is kept at a low temperature as a whole and can be safely used to process heat-sensitive materials.

[0021] Non-thermal plasma can be obtained through gas discharge. Gas discharge occurs when an electric current flows through a gas medium due to the ionization of the gas. Methods for forming non-thermal plasma are well known to those skilled in the art and include jet discharge (non-thermal atmospheric pressure plasma jet), dielectric barrier discharge, glow discharge, corona discharge, radio frequency discharge, sliding arc discharge, atmospheric pressure glow discharge, sub-atmospheric pressure glow discharge, and the like. The basic discharge form of the jet discharge method is dielectric barrier discharge. The jet device used for the jet discharge method is typically configured with two electrodes (e.g., a combination of a needle electrode and a ring electrode, or a combination of two ring electrodes). Single-electrode configurations also exist (e.g., a needle electrode and a virtual ground electrode). The distance between the electrodes is typically between several millimeters and more than ten millimeters (e.g., 2 mm to 19 mm). In dielectric barrier discharge, non-thermal plasma is generated between two electrodes separated by an insulating dielectric. The insulating dielectric can cover both electrodes, one electrode, or be placed between the two electrodes.

[0022] Treating bacteria using a non-thermal plasma refers to contacting the non-thermal plasma with the bacteria, wherein the contact may be that the distal end of the non-thermal plasma torch contacts the liquid surface of the bacterial suspension, and / or the non-thermal plasma torch is partially or completely immersed in the bacterial suspension, and / or a non-thermal plasma is formed in the bacterial suspension.

[0023] The bacteria of the present invention include Gram-negative bacteria and Gram-positive bacteria, and can be any naturally occurring bacteria or bacteria modified by any genetic engineering technology. For example, in order to reduce the DNA residue of the generated ghost, a nuclease gene under the control of an inducible promoter can be engineered into the bacterial cell, and the expression of the nuclease gene can be induced after the bacterial culture is completed and before the bacterial ghost is produced to destroy the bacterial genomic DNA (Escherichia coli Ghost Production by Expression of Lysis Gene E and Staphylococcal Nuclease Applied and Environmental Microbiology Oct 2003, 69 (10) 6106-6113). Lactic acid bacteria (Lactobacillales or lactic acid bacteria) are Gram-positive bacteria and are a general term for bacteria that can produce large amounts of lactic acid using fermentable carbohydrates. Lactic acid bacteria are probiotics and include Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, Bifidobacterium, and Pediococcus.

[0024] Bacterial ghost (BG) (also known as bacterial exuvia) refers to the empty bacterial shell that does not contain bacterial cell contents such as nucleic acids and cytoplasm.

[0025] Methods for further treating bacterial ghosts to remove residual DNA are known in the art, such as treating biological products with beta-propiolactone to destroy the structure of DNA and inactivate the DNA (Inactivation of DNA by beta-propiolactone. Biologicals: journal of the International Association of Biological Standardization 23 (1995) 207–11; Development of awhole cell pneumococcal vaccine: BPL inactivation, cGMP production, and stability. Vaccine 32 (2014) (1113–20)); ion exchange chromatography, protamine precipitation; enzymatic hydrolysis (DNaseI, non-restriction endonuclease, etc.).

[0026] "Adjuvant" refers to an auxiliary substance that is injected into the body together with an antigen or in advance and can enhance the body's immune response to the antigen or change the type of immune response. The specific substances included are well known to those skilled in the art, for example: Toll-like receptor (TLR) agonists, aluminum salts, calcium phosphate, oil-in-water emulsions, Freund's adjuvant, inactivated bacteria, cytokines IL-1, IL-2, IL-12, etc.

[0027] "Vaccine" refers to a biological or pharmaceutical preparation containing an antigen. The preparation may be a composition that, in addition to the antigen, may also contain other ingredients such as an adjuvant and a pharmaceutically acceptable carrier. The vaccines of the present invention include preventive vaccines and therapeutic vaccines. Preventive vaccines can provide acquired immunity against specific pathogenic microorganisms, diseases, tumors or cancers before infection with pathogenic microorganisms, or before the occurrence of diseases, tumors or cancers; therapeutic vaccines can treat or prevent the worsening of the infection, disease, tumor or cancer after infection with pathogenic microorganisms, or after the occurrence of a certain disease, tumor or cancer. Treatment includes complete cure and relief of some or all symptoms. In some embodiments, the vaccine of the present invention is a freeze-dried product, or a product to be injected obtained by resuspending the freeze-dried product in a pharmaceutically acceptable solution or carrier. The freeze-dried product of the present invention may also include the use of a pharmaceutically acceptable carrier or solution during preparation.

[0028] "Pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0029] "Pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, formulation aid (such as a lubricant, talc, magnesium stearate, calcium stearate or zinc stearate or stearic acid). Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, For example, propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (9) ethanol; (20) pH buffer solutions (such as PBS, etc.); (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other non-toxic compatible substances used in pharmaceutical preparations. The vaccine of the present invention can be present in various dosage forms, including liquid dosage forms, lyophilized dosage forms, oral dosage forms, etc., and the route of administration can be reasonably selected according to the corresponding dosage form, such as oral administration, intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, nasal administration, etc. The dosage and number of administrations of the vaccine of the present invention can be determined according to the specific conditions of the subject.

[0030] "About" in this article means a range of ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1% or ±0.5% of the stated value, including the endpoints of the range and any value within the range.

[0031] As used herein, “include,” “comprising,” and “having” mean that the substance, composition, system, or method at least contains, but is not limited to, the described structure, ingredient, element, component, feature, or step; “include,” “comprising,” and “having” are intended to indicate the presence of the described structure, ingredient, element, component, feature, or step, but do not exclude the presence of any other structure, ingredient, element, component, feature, or step. In this article, when a compound is mentioned as containing, including, or having one or more structures, it should be understood that it also covers compounds composed of this or these structures; when a product or composition is mentioned as containing, including, or having one or more ingredients, it should be understood that it also covers products or compositions composed of this or these ingredients; when a method is mentioned as containing, including, or having one or more steps, it should be understood that it also covers methods composed of this or these steps.

[0032] As used herein, the term "and / or" is intended to include any possible combination of one or more of the listed items. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be more fully understood through the following detailed description taken in conjunction with the accompanying drawings, in which like elements are numbered in a similar manner and have similar features or functions:

[0034] Figure 1 : Schematic diagram of non-thermal plasma discharge system. Figure 1 AB: Two exemplary configurations of jet discharge reactors. The gas supply system 1 supplies gas to the gas regulating system 2, which adjusts the gas flow rate, flow rate, ratio of different gases and other parameters as needed and then introduces the gas into the ejector 4 ( Figure 1 A) or ejector 12 ( Figure 1 B), 3 represents the direction of gas flow. The gas supply system 1 can supply different gases such as N2, He, H2, Ar, O2, air, CO2, CO, etc. The bacterial suspension 8 can be directly added to the reaction vessel 11, or it can be placed in another container 8a and then introduced into the reaction vessel 11 through a conduit. The treated bacterial suspension can be collected directly or discharged to another container 8b through a conduit. The power supply system 9 applies a high voltage to the two electrodes. The power supply regulation system 10 adjusts the voltage, frequency, and current to the optimal combination range. The power supply system 9 also includes a feedback display and recording system for feedback display and recording changes in various parameters. Figure 1 The ejector 4 in A comprises a housing (or pipe) 6, a high-voltage electrode 5, a grounded metal electrode 7, and a cavity within the housing or pipe through which gas passes. The distal end 4a of the ejector can be between 20 mm and 10 mm below the surface of the bacterial suspension. Figure 1The ejector 12 in Figure B comprises a dielectric housing (or tube) 15, a first annular electrode 13, a second annular electrode 14, and a cavity within the housing or tube through which gas passes. The distal end 12a of the ejector can be positioned between 20 mm above the surface of the bacterial suspension and 10 mm below the surface. Figure 1 C: Example configuration of a dielectric barrier discharge reactor. Gas supply system 1 supplies gas to gas conditioning system 2, which adjusts various gas parameters as needed before directing the gas between first electrode plate 16 and second electrode plate 17. The first electrode plate can be positioned 1-3 mm above the surface of the bacterial suspension.

[0035] Figure 2 : Scanning electron micrograph of Lactococcus lactis without non-thermal plasma treatment.

[0036] Figure 3 : Scanning electron micrograph of Lactococcus lactis after treatment with the conditions of experimental group (2).

[0037] Figure 4 : Scanning electron micrograph of Lactococcus lactis after treatment with the conditions of control group (1).

[0038] Figure 5 : Scanning electron micrograph of Lactococcus lactis after treatment with the conditions of control group (2).

[0039] Figure 6 : The results of solid plate culture of Lactococcus lactis after the conditions of experimental group (2) were as follows ( Figure 6 right side), Figure 6 The left side is the blank control. DETAILED DESCRIPTION

[0040] The technical solutions of the present invention are further described in detail below through examples and in conjunction with the accompanying drawings. However, the present invention is not limited to the following examples. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and details, and these are all considered to fall within the scope of protection of the present invention.

[0041] Example:

[0042] 1. Experimental Materials

[0043] 1. Non-thermal plasma discharge system.

[0044] 2. Microbial strains: Lactococcus lactis and Lactobacillus lactis.

[0045] 3. Microbial culture medium

[0046] Prepare MRS medium:

[0047] Add 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 20.0 g of glucose, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 1.0 g of Tween-80, 0.4 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.29 g of manganese sulfate, 20.0 g of calcium carbonate, and 15.0 g of agar to distilled water in sequence, and add the distilled water to 1000 mL. Adjust the pH to 6.3, stir, heat, boil for 2 min, and sterilize at 121°C and 0.1 MPa for 30 min.

[0048] 2. Experimental steps

[0049] 1. Cultivate the lactic acid bacteria to the exponential growth phase, collect the cells by centrifugation, rinse with PBS buffer, and then resuspend the cells in sterilized distilled water to adjust the cell concentration to 1×10 6 -1×10 7 CFU / ml.

[0050] 2. Place the bacteria obtained in step 1 into a non-thermal plasma reactor, use nitrogen as the excitation gas, a gas flow rate of 1-10 liters / minute, a power supply of 30-300 watts, a voltage of 3000-12000 volts, a frequency of 12000-25000 Hz, and a treatment time of 5-60 seconds depending on the amount of bacteria.

[0051] Experimental group (1): The lactic acid bacteria were Lactococcus lactis, the discharge voltage was 6000-12000 V, the frequency was 12000-25000 Hz, the gas flow rate was 2-4 L / min, and the treatment time was 15-30 seconds.

[0052] Experimental group (2): Lactobacillus lactis, discharge voltage 8000-9500V, frequency 21000-23000Hz, gas flow rate 3L / min, treatment time 20s. Figure 3 shown.

[0053] Experimental group (3): Lactobacillus lactis, discharge voltage 6000-13000 V, frequency 12000-25000 Hz, gas flow rate 2-4 L / min, treatment time 15-30 s,

[0054] Experimental group (4): The lactic acid was Lactobacillus lactis, the discharge voltage was 9000-10000 V, the frequency was 21000-23000 Hz, the gas flow rate was 3 L / min, and the treatment time was 15 seconds.

[0055] Comparative group (1): Lactobacillus lactis, discharge voltage 12000-13000V, frequency 22000-23000Hz, gas flow rate 3L / min, treatment time 20s. Figure 4 shown.

[0056] Comparative group (2): Lactobacillus lactis, discharge voltage 3000-3200V, frequency 22000-23000Hz, gas flow rate 3L / min, treatment time 20s. Figure 5 shown.

[0057] Comparative group (3): The lactic acid bacteria were Lactobacillus lactis, the discharge voltage was 12000-13000 V, the frequency was 22000-23000 Hz, the gas flow rate was 3 L / min, and the treatment time was 15 seconds.

[0058] Comparative group (4): The lactic acid bacteria were Lactobacillus lactis, the discharge voltage was 3000-3200 V, the frequency was 22000-23000 Hz, the gas flow rate was 3 L / min, and the treatment time was 15 seconds.

[0059] Negative control group (1): Lactobacillus lactis was used and was not treated with non-thermal plasma. Figure 2 shown.

[0060] Negative control group (2): Lactobacillus lactis was used and was not treated with non-thermal plasma.

[0061] 3. Centrifuge the treated mixture, collect the bacterial pellet, rinse the pellet with PBS buffer, and then resuspend it with sterile distilled water.

[0062] 4. Observe bacterial shadows using a scanning electron microscope: After the bacteria (bacterial shadows) are freeze-dried, the samples are sprayed with an ion sputtering device and then observed and photographed using a scanning electron microscope.

[0063] 5. To verify that there are no live bacteria after the treatment, culture the treated bacteria on a solid plate. The specific operation method is as follows:

[0064] 1) Bacteria: Take 1 ml of Lactococcus in the logarithmic growth phase and centrifuge at 10,000 rpm. Remove the supernatant and wash twice with 1 ml of sterile PBS. Centrifuge at 10,000 rpm and remove the supernatant. Resuspend the cells in sterile PBS and adjust the cell concentration to an absorbance of OD600 of 0.2. This will serve as a blank control. Treat the bacterial shadow suspension in the same manner and set aside.

[0065] 2) Culture medium preparation: Dissolve MRS solid culture medium in distilled water, sterilize under high pressure at 115°C for 30 min, remove from the container, cool to 60°C, and place 20 ml of the culture medium on a sterile plate. Allow the culture to cool to room temperature and solidify completely before use.

[0066] 3) Inoculation: 100 μl of each of the prepared Lactococcus lactis blank control and bacterial shadow suspension was placed on a solid plate, evenly spread with a sterile glass spreader, and then sealed with a sealing film.

[0067] 4) Cultivation: The inoculated solid culture medium was placed in a mold incubator, and the temperature of the incubator was adjusted to 37.0° C. and the relative humidity to 60%. After culturing for 72 hours, the growth status of the colonies was observed.

[0068] 6. Determine the amount of DNA in the bacterial ghosts: Enzymatically hydrolyze the bacterial cells obtained after centrifugation in step 3. After a certain period of enzymatic hydrolysis, elute the DNA using an adsorption column to obtain DNA fragments. Determine the DNA concentration using a microplate reader.

[0069] 3. Experimental Results

[0070] 1. Electron microscope photos of the treated bacteria

[0071] Compared with the negative control group (1)( Figure 2 ) Compared with the experimental group (2), the holes on the surface of the Lactococcus lactis cells after treatment were very obvious, but the basic shape of the cell shell was retained ( Figure 3 ). On the contrary, when the non-thermal plasma treatment conditions are not suitable, for example, when the discharge field intensity is too high (comparison group (1)), the bacterial cells are broken and bacterial shadows cannot be formed ( Figure 4 For example, when the discharge field strength is too low (comparison group (2)), the bacteria cannot form holes on their surface and cannot generate bacterial shadows ( Figure 5 ).

[0072] Compared with the negative control group (2), the holes on the surface of the Lactobacillus lactis cells in the experimental group (4) after treatment were very obvious, but the basic shape of the cell shell was retained. In contrast, when the non-thermal plasma treatment conditions were not suitable, for example, when the discharge field intensity was too high (comparison group (3)), the cell body was broken and no bacterial shadow was formed. When the discharge field intensity was too low (comparison group (4)), the cell body could not form holes on its surface and no bacterial shadow was generated.

[0073] 2. The bacteria after treatment under the experimental group conditions do not contain viable bacteria

[0074] like Figure 6 As shown, there were no live Lactococcus lactis after treatment under the conditions of experimental group (2) ( Figure 6 right side), Figure 6 The left side is the blank control.

[0075] 3. DNA content in treated bacterial cells

[0076] The DNA content in the cells of Lactococcus lactis after treatment is shown in Table 1.

[0077] Table 1: DNA content in Lactococcus lactis cells compared to negative control

[0078]

Claims

1. A method for preparing bacterial ghosts, comprising forming a non-thermal plasma by gas discharge, and treating bacteria with the non-thermal plasma, wherein the gas is nitrogen and the bacteria are Lactococcus lactis ( Lactococcus lactis ) or Lactobacillus lactis ( Lactobacillus lactis ); wherein the voltage used to form the non-thermal plasma is 8000-10000 volts; wherein the power supply frequency for forming the non-thermal plasma is 21000-23000 Hz; The gas flow rate for forming the non-thermal plasma is 2-4 liters per minute; The concentration of the bacterial suspension was 1×10 6 -1×10 7 CFU / ml; The time for treating bacteria with non-thermal plasma is 15-30 seconds.

2. The method of claim 1, further comprising freeze-drying the treated bacteria.

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