Use of sanguinarine chloride in the preparation of foodborne pathogen growth inhibition and / or killing products

Abstract

The present application relates to the application of sanguinarine chloride in the preparation of foodborne pathogenic bacteria inhibition and / or sterilization products, and the monomer component sanguinarine chloride from bloodroot has low minimum inhibitory concentration and rapid sterilization efficiency for staphylococcus aureus, salmonella, diarrheal escherichia coli, listeria monocytogenes and cronobacter; more importantly, it has strong dual anti-membrane effect, which can not only inhibit the formation of early biofilm of pathogenic bacteria, but also deeply penetrate and dissolve the mature biofilm attached to the surface of the substrate. Based on the above antibacterial activity, sanguinarine chloride is prepared into a surface decontamination spray, and through carrier verification, the killing logarithm value of the above five kinds of pathogenic bacteria attached to the surface of stainless steel is all greater than or equal to 3.00, which meets the national disinfection qualified standard. The present application provides an efficient, broad-spectrum and natural source solution for the surface contamination control of foodborne pathogenic bacteria.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial technology, specifically relating to the application of sanguinarine chloride in the preparation of antibacterial and / or bactericidal products for foodborne pathogens. Background Technology

[0002] Bacterial contamination and the resulting foodborne illnesses, environmental cross-contamination, and medical device-related infections remain significant issues in food safety, public health, and healthcare. Staphylococcus aureus, Salmonella, diarrheal Escherichia coli, Listeria monocytogenes, and Kronobacter are five highly dangerous common pathogens. Staphylococcus aureus can produce enterotoxins, leading to food poisoning and various purulent infections; Salmonella and diarrheal Escherichia coli are common pathogens causing intestinal infections and foodborne diarrhea; Listeria monocytogenes can survive and multiply at low temperatures, posing a persistent threat to the safety of refrigerated ready-to-eat foods; and Kronobacter, in particular, can contaminate infant formula, posing a high risk to the health of newborns and infants.

[0003] More challengingly, besides existing in planktonic form, these pathogens can also adhere to the surfaces of food processing equipment, packaging materials, medical devices, and other biological or non-biological materials, further forming biofilms. Biofilms are complex structures composed of bacteria and their secreted extracellular polymers, enhancing their tolerance to adverse environmental conditions. Compared to planktonic bacteria, bacteria in biofilms typically exhibit higher tolerance to antimicrobial drugs, chemical disinfectants, and environmental stresses, leading to difficulty in removing contaminants, recurrent infections, and reduced effectiveness of disinfection treatments.

[0004] Currently, the control of these foodborne pathogens mainly relies on traditional antibiotics, chemical disinfectants, or some natural antibacterial substances. However, long-term use of antibiotics can easily lead to increased bacterial resistance, while chemical disinfectants may pose risks of irritation, corrosiveness, and residue. Although some existing natural antibacterial agents are relatively safe in origin, they often suffer from problems such as insufficient antibacterial spectrum, unstable activity, and limited ability to clear biofilms. Specifically, nisin, as the most widely used natural antimicrobial peptide, exhibits excellent antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus and Listeria monocytogenes. However, the lipopolysaccharide barrier of the outer membrane of Gram-negative bacteria prevents its binding to the cytoplasmic membrane, resulting in almost no direct antibacterial effect against Gram-negative pathogens such as Salmonella, Escherichia coli, and Cronobacter. Berberine, an isoquinoline alkaloid derived from traditional Chinese medicine, can inhibit the growth of Gram-positive bacteria such as Staphylococcus aureus at relatively high concentrations (MIC typically 32-256 μg / mL). However, its minimum bactericidal concentration (MBC) is often 4-8 times or even higher than the MIC, exhibiting a predominantly antibacterial rather than bactericidal effect, making it difficult to quickly and thoroughly eliminate target pathogens in practical applications. Regarding biofilm intervention, while tea polyphenols such as epigallocatechin gallate (EGCG) can inhibit early biofilm formation in Staphylococcus aureus at sub-inhibitory concentrations by interfering with quorum sensing signaling pathways, their ability to remove mature biofilms already attached to the matrix surface is very limited, failing to effectively dismantle their extracellular polymer matrix structure. While thymol can disrupt mature biofilms of Gram-positive bacteria such as Listeria monocytogenes at certain concentrations, it exhibits significantly weaker clearance efficacy against mixed biofilms formed by Gram-negative bacteria such as Salmonella on food contact surfaces. It struggles to achieve broad-spectrum disintegration of biofilms from multiple pathogenic bacteria. Therefore, developing novel active ingredients that are naturally derived, broad-spectrum, have good inhibitory effects on various common pathogens, and can simultaneously intervene in or eliminate bacterial biofilms has become an important research direction in the fields of food safety, antibiotic alternatives in aquaculture, environmental disinfection, and infection control. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects in the prior art and to provide the application of sanguinarine chloride in the preparation of antibacterial and / or bactericidal products for foodborne pathogens. Its low concentration can kill five kinds of high-risk foodborne pathogens with broad spectrum and high efficiency, and it also has the dual functions of blocking biofilm formation and penetrating and disintegrating mature biofilms.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Technical Solution 1: The application of sanguinarine chloride in the preparation of products for inhibiting and / or killing foodborne pathogens, wherein sanguinarine chloride is a broad-spectrum antibacterial / bactericidal agent.

[0007] As a further technical solution, the foodborne pathogens are selected from one or more of Staphylococcus aureus, Salmonella, diarrhea-causing Escherichia coli, Listeria monocytogenes, and Cronobacter.

[0008] As a further technical solution, the foodborne pathogens are foodborne pathogens that have not formed a biofilm and / or foodborne pathogens that have formed a biofilm.

[0009] As a further technical solution, the inhibition and / or sterilization of foodborne pathogens includes killing foodborne pathogens, preventing the formation of foodborne pathogen biofilms, and destroying the mature biofilms of foodborne pathogens.

[0010] As a further technical solution, the sanguisorbine chloride is plant-derived sanguisorbine chloride, and the product includes pharmaceuticals, antibacterial agents, and external cleaning and disinfection products; the dosage form of the product includes solutions, sprays, gels, emulsions, powders, or coating preparations.

[0011] As a further technical solution, the external disinfection product is used for disinfection and biofilm intervention treatment of the surfaces of food processing equipment, packaging materials, environmental objects, or medical devices.

[0012] Technical Solution Two: A bacteriostatic / bactericidal agent for foodborne pathogens, comprising sanguinarine chloride; wherein the foodborne pathogens are selected from one or more of Staphylococcus aureus, Salmonella, diarrheal Escherichia coli, Listeria monocytogenes, and Cronobacter.

[0013] Technical Solution 3: A composition for inhibiting / killing foodborne pathogens, comprising the aforementioned bacteriostatic / bacteriostatic agent for foodborne pathogens, and further comprising pharmaceutically or chemically acceptable excipients, wherein the pharmaceutically or chemically acceptable excipients include solvents, cosolvents, surfactants, and humectants.

[0014] As a further technical solution, the mass percentage of sanguinarine chloride is greater than 0.01%, preferably 0.01%-5%.

[0015] Technical Solution Four: A surface disinfectant spray for foodborne pathogens, comprising ≥8×10⁸ bacteria. -5 Sanhydramine chloride (preferably 8×10 g / mL) -5 -5×10 -4 (g / mL); the foodborne pathogens are selected from one or more of Staphylococcus aureus, Salmonella, diarrheal Escherichia coli, Listeria monocytogenes and Cronobacter.

[0016] As a further technical solution, the preparation method of the foodborne pathogen surface decontamination spray includes the following steps: (1) Dissolve sanguinarine chloride in anhydrous ethanol and stir to dissolve, thus preparing sanguinarine chloride ethanol mother liquor; (2) Add Tween-80 to the ethanol mother liquor of sanguisorbine chloride and stir continuously to mix it so that it can fully encapsulate the active ingredients as a non-ionic surfactant and form a stable solubilization system.

[0017] (3) Under continuous stirring, slowly add purified water, make up the volume, mix well and obtain a foodborne pathogen surface disinfection spray with uniform appearance and clear appearance.

[0018] The final concentration of sanguinarine chloride in the surface disinfectant spray for foodborne pathogens is greater than or equal to 500 μM.

[0019] Technical Solution 5: The application of sanguinarine chloride in the preparation of a drug for preventing and treating foodborne pathogen infections, wherein the foodborne pathogens are selected from one or more of Staphylococcus aureus, Salmonella, diarrheal Escherichia coli, Listeria monocytogenes, and Cronobacter.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention systematically demonstrates that sanguinarine chloride has significant inhibitory and bactericidal activity against five high-risk foodborne pathogens, including Gram-positive bacteria (Staphylococcus aureus, Listeria monocytogenes) and Gram-negative bacteria (Salmonella, Escherichia coli, and Cronobacter). The MIC range is 5-200 μM, and the MBC / MIC ratio is 1-2, indicating that it is a broad-spectrum bactericidal antibacterial agent rather than just having bacteriostatic effects.

[0021] 2. This invention confirms that sanguinarine chloride can not only inhibit or kill planktonic bacteria, but also inhibit the formation of various bacterial biofilms and destroy mature biofilms, thus expanding the application scenarios of sanguinarine chloride.

[0022] 3. The present invention prepares sanguinarine chloride into a surface disinfection spray. The quantitative bactericidal test of the carrier verifies that the log killing value of the above five foodborne pathogens attached to the stainless steel surface is ≥3.00, which meets the qualified standard stipulated by the national disinfection technical specifications, proving its convertibility from active ingredient to actual formulation.

[0023] 4. Sanguisorbine chloride is derived from natural plants that can be used as feed additives, which is in line with the industrial development trend of "reducing and replacing antibiotics" and green and safe industries, and has a very wide range of applications.

[0024] In summary, this invention, based on sanguinarine chloride derived from natural plants, establishes a systematic foodborne pathogen control solution that integrates broad-spectrum rapid sterilization, inhibition of biofilm formation, destruction of mature biofilms, and surface disinfection, providing a new technical approach and product form for microbial contamination control. Attached Figure Description

[0025] Figure 1 The graph shows the minimum inhibitory concentration (MIC) of sanguinarine chloride against foodborne pathogens. Figure 2 This is a plate inhibition effect diagram of sanguinarine chloride against foodborne pathogens; Figure 3 The graph shows the cell survival rate of foodborne pathogens after exposure to sanguinarine chloride. Figure 4 Figure 1 shows the anti-biofilm properties of different concentrations of sanguinarine chloride against Staphylococcus aureus. exist Figure 4 In the image, a: Crystal violet staining; b: Biofilm destruction rate; c: Biofilm inhibition rate; Figure 5 Figure 1 shows the anti-biofilm properties of different concentrations of sanguinarine chloride against Listeria monocytogenes. exist Figure 5 In the image, a: Crystal violet staining; b: Biofilm destruction rate; c: Biofilm inhibition rate; Figure 6 Figure 1 shows the anti-biofilm properties of different concentrations of sanguinarine chloride against Cronobacter. exist Figure 6 In the image, a: Crystal violet staining; b: Biofilm destruction rate; c: Biofilm inhibition rate; Figure 7 Figure 1 shows the anti-biofilm properties of different concentrations of sanguinarine chloride against diarrheal Escherichia coli. exist Figure 7 In the image, a: Crystal violet staining; b: Biofilm destruction rate; c: Biofilm inhibition rate; Figure 8 Figure 1 shows the anti-biofilm properties of different concentrations of sanguinarine chloride against Salmonella. exist Figure 8 In the image, a: Crystal violet staining; b: Biofilm destruction rate; c: Biofilm inhibition rate. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] 1. Sanguisorbine chloride: CAS No.: 5578 73 4; Molecular weight: 367.78, purchased from TargetMol, is an alkaloid derived from Sanguinaria Canadensis.

[0028] 2. Unless otherwise specified, the test reagents and consumables used in the following examples are all conventional biochemical reagents; unless otherwise specified, the experimental methods are all conventional methods.

[0029] 3. Unless otherwise specified, all raw materials used in this invention are commercially available.

[0030] Example 1: Determination of the minimum inhibitory concentration (MIC) of sanguinarine chloride against five pathogenic bacteria 1. The effect of sanguinarine chloride on Staphylococcus aureus was determined using the broth microdilution method. Staphylococcus aureus ATCC 26003) SA, Salmonella ( Salmonella Typhimurium (ATCC 14028) STYP, Enteroinvasive Escherichia coli Escherichia coli BNCC, BNCC 340975) EIEC, Listeria monocytogenes ( Listeria monocytogenes ATCC 13932) LM and Cronobacter ( Cronobacter sakazakii MIC of these 5 pathogenic bacteria (ATCC 51329) CS.

[0031] Five pathogenic bacteria were streaked and activated, and single colonies were picked and inoculated into LB broth. The cultures were incubated at 37°C with shaking until the logarithmic growth phase. The bacterial suspension concentration was then adjusted to approximately 1 × 10⁻⁶ using sterile culture medium. 6 CFU / mL. Different sanguinarine chloride concentration gradients were set up for strains with different sensitivities: for STYP, CS, and EIEC, the final concentration gradients were set to 0, 25, 50, 100, and 200 μM; for LM and SA, lower final concentration gradients were set to 0, 1.25, 2.5, 5, and 10 μM. Different concentrations of sanguinarine chloride solution were added to 96-well plates, and an equal volume of bacterial suspension was inoculated, resulting in a final bacterial concentration of approximately 5 × 10⁻⁶ CFU / mL. 5 CFU / mL. A bacterial culture without the drug was set up as a positive control, and a culture medium-only culture was set up as a negative control. The 96-well plates were incubated at 37°C for 18–24 h. The lowest drug concentration at which no bacterial growth was observed visually was taken as the MIC of the bacterium.

[0032] OD of sanguinarine against 5 pathogenic bacteria 600 The measurement results are as follows Figure 1 As shown, where, Indicates MIC concentration.

[0033] 2. From Figure 1 It can be seen that sanguinarine chloride has a significant, dose-dependent inhibitory effect on the growth of all the tested strains, as detailed below: (1) For Salmonella (STYP): When the concentration of sanguinarine chloride reached 100 μM, bacterial growth was significantly inhibited (OD). 600 (The absorbance decreased by more than half); when the concentration increased to 200 μM, the absorbance approached 0, so the MIC value of sanguinarine chloride against Salmonella was 200 μM.

[0034] (2) For Cronobacter (CS) and diarrheal Escherichia coli (EIEC): EIEC was significantly inhibited at a drug concentration of 25 μM; when the concentration reached 50 μM, the OD of both was significantly reduced. 600 The values ​​all dropped sharply to near 0, indicating that the MIC values ​​of sanguinarine chloride against these two common enteropathogenic bacteria were both 50 μM.

[0035] (3) For Listeria monocytogenes (LM) and Staphylococcus aureus (SA): These two bacteria showed extremely high sensitivity to sanguinarine chloride. At concentrations of 1.25 μM and 2.5 μM, bacterial biomass decreased dramatically in a stepwise manner; at a concentration of only 5 μM, both completely ceased to reproduce. Therefore, sanguinarine chloride had the lowest MIC values ​​for LM and SA, both at 5 μM.

[0036] To more intuitively demonstrate the antibacterial efficacy, the measured minimum inhibitory concentrations are summarized in Table 1: Table 1. Minimum inhibitory concentration (MIC) of sanguinarine chloride against five pathogenic bacteria. The data in Table 1 demonstrate that sanguinarine chloride derived from *Sanguinarium sarmentosum* exhibits excellent broad-spectrum antibacterial activity even at extremely low micromolar concentrations, showing particularly strong bactericidal potential against *Listeria monocytogenes* and *Staphylococcus aureus*, providing solid data support for its application as a highly effective antibacterial agent. Example 2: Antibacterial activity and bactericidal effect test of sanguinarine chloride against five pathogenic bacteria 1. To further verify the bactericidal activity of sanguinarine chloride, this embodiment uses the plate coating counting method to systematically determine the changes in the survival rate of five tested pathogenic bacteria after treatment with different concentrations of sanguinarine chloride.

[0037] Five pathogenic bacteria suspensions grown to the logarithmic growth phase were exposed to different concentrations of sanguinarine chloride solution. The concentration gradients for each strain were designed as follows: SA and LM groups: 0, 2.5, 5, 10, 20 μM; CS group: 0, 25, 50, 100, 200 μM; EIEC group: 0, 50, 100, 200, 400 μM; STYP group: 0, 100, 200, 400, 800 μM. After co-culturing at 37℃ for 12 h, 100 μL samples were plated, and additional samples were serially diluted and plated on LB agar plates. The plates were incubated upside down at 37℃ for 24 h, and the colony count (CFU) was recorded. Bacterial survival rate was calculated using the following formula: Bacterial survival rate (%) = (Colony count in treatment group / Colony count in control group) × 100%. The lowest concentration at which the colony count decreased by 99.9% was defined as MBC. The results of the antibacterial activity test of sanguinarine chloride against five pathogenic bacteria are as follows: Figure 2-3 As shown.

[0038] 2. From Figure 2-3 It can be seen that sanguinarine chloride exhibits potent antibacterial activity against all five pathogenic bacteria, as detailed below: (1) For Staphylococcus aureus (SA): As the concentration increases from 2.5 μM to 5 μM, the survival rate drops sharply to less than 10%; when the concentration reaches 10 μM, the bacterial survival rate approaches 0 and the number of colonies decreases by >99.9%; when the concentration increases to 20 μM, no colonies grow on the plate, achieving complete eradication.

[0039] (2) For Listeria monocytogenes (LM): the survival rate drops to about 50% at a concentration of 2.5 μM; when the concentration is only 5 μM, the survival rate drops sharply to close to 0, and the number of colonies decreases by >99.9%; when the concentration reaches 20 μM, the plate is completely sterile.

[0040] (3) For Cronobacter (CS): at a concentration of 25 μM, the survival rate drops to below 20%; when the concentration is increased to 100 μM, there are almost no viable bacteria on the plate, and the number of colonies is reduced by >99.9%.

[0041] (4) For diarrheal Escherichia coli (EIEC): the survival rate drops to about 25% at 50 μM treatment; when the concentration reaches 100 μM, the survival rate drops to a very low 0.05%, indicating that the number of colonies is reduced by >99.9%; concentrations of 200 μM and above can completely eliminate background colonies.

[0042] (5) For Salmonella (STYP): the survival rate dropped to about 25% at 100 μM treatment; when the concentration was increased to 200 μM, the survival rate dropped below 0.005%, achieving logarithmic killing and reducing the number of colonies by >99.9%; when the concentration reached 800 μM, no Salmonella survived on the plate.

[0043] In summary, the data from Examples 1 and 2 demonstrate that sanguinarine chloride is not simply a bacteriostatic agent, but a highly effective bactericide. Even at low concentrations, it can interrupt the proliferation of the aforementioned five high-risk pathogens at the source and rapidly and directly cause large-scale death of the pathogenic bacterial communities.

[0044] Experiment Example 3: The inhibitory effect of sanguinarine on biofilm formation and destruction of mature biofilms in five pathogenic bacteria. 1. Using crystal violet staining, the inhibitory effect of different concentrations of sanguinarine chloride on biofilm formation and its destructive effect on mature biofilms of five pathogenic bacteria were systematically evaluated. Results are shown in [Table missing]. Figure 3 .

[0045] (1) Experiment on inhibition of biofilm formation In 96-well plates, culture medium containing different concentrations of sanguinarine chloride and logarithmic-phase bacterial suspensions were added. The plates were incubated at 37°C for 24 h to allow mature biofilms to form at the bottom of the control wells. The supernatant was discarded, and the plates were gently washed with PBS to remove airborne bacteria. After fixing with methanol for 15 min, the plates were dried. Staining was performed with 0.1% crystal violet solution for 15 min, excess stain was washed away, and the plates were air-dried. The crystal violet was dissolved in 33% glacial acetic acid, and the OD was measured using a microplate reader. 572 Absorbance.

[0046] Biofilm inhibition rate (%) = [1 - (OD treatment group / OD control group)] × 100%.

[0047] (2) Experiment on destruction of mature biofilm First, an untreated logarithmic-phase bacterial suspension was added to 96-well plates and incubated at 37°C for 24 h to form a mature, dense biofilm. The supernatant was discarded, and airborne bacteria were gently washed away with PBS. Then, fresh culture medium containing different concentrations of sanguinarine chloride was added to each well, and the incubation continued for another 24 h. The medium was then discarded, washed, stained with crystal violet, and the OD was measured. 572 The steps are the same as above.

[0048] Mature biofilm destruction rate (%) = [1 - (OD-treated group / OD-untreated control group)] × 100%.

[0049] 2. From Figure 4-8 It can be known that: (1) Regarding Staphylococcus aureus (SA): Staphylococcus aureus is a Gram-positive pathogen widely distributed in clinical and food environments and readily forms stubborn biofilms, but it exhibits extremely high sensitivity to the drug of this invention. For example... Figure 4 As shown, sanguinarine chloride, at an extremely low concentration of only 5 μM, can achieve a film-forming inhibition rate of over 50% against Staphylococcus aureus; at a concentration of 20 μM, the inhibition rate approaches 100%. Even against the already dense and complex mature biofilm of Staphylococcus aureus, sanguinarine chloride still exhibits excellent matrix penetration. At a concentration of 10 μM, the destruction rate exceeds 60%; at a concentration of 20 μM, the destruction rate reaches approximately 80%, with a large amount of extracellular polymeric substances (EPS) effectively stripped and broken down.

[0050] For Listeria monocytogenes (LM): LM, as a pathogenic bacterium that readily forms stubborn biofilms, is extremely sensitive to the drug of this invention. For example... Figure 5 As shown, sanguinarine chloride only needs 10 μM to inhibit about 50% of Listeria monocytogenes biofilm formation; when the concentration is increased to 40 μM, the inhibition rate and destruction rate both reach extremely high levels, proving that sanguinarine chloride can effectively control Listeria monocytogenes biofilm contamination in cold chain food at low concentrations.

[0051] For Cronobacter CS: In the inhibition assay, as the concentration increased, the staining at the bottom of the well changed from deep blue to almost colorless. For example... Figure 6 As shown, at a concentration of 200 μM, the inhibition rate of Cronobacter was close to 100%. In the mature membrane destruction experiment, 200 μM sanguinarine chloride could still remove most of the matrix, with a destruction rate of over 80%, demonstrating strong penetration and stripping capabilities.

[0052] For diarrheal Escherichia coli EIEC: such as Figure 7 As shown, in the inhibition formation experiment, the bottom of the well in the 400 μM concentration group was completely transparent. In the mature membrane destruction experiment, 100-200 μM could significantly reduce the biofilm of diarrhea-causing Escherichia coli, and the destruction rate reached more than 80% at 400 μM, effectively solving the problem of difficult eradication of diarrhea-causing Escherichia coli contamination.

[0053] For Salmonella STYP: such as Figure 8 As shown, sanguinarine chloride also exhibits excellent dual anti-membrane effects against Salmonella. In the inhibition formation experiment, the 400 μM group showed a very strong intervention effect. In the mature membrane destruction experiment, 800 μM sanguinarine chloride could cause the originally extremely dense Salmonella biofilm to disintegrate, with a destruction rate of over 85%.

[0054] Experiment Example 4: Preparation and Surface Application of Antibacterial Surface Detergent Spray Based on Sanguisorbate Chloride 1. Preparation of surface decontamination spray Prepare the surface cleaning spray according to the following steps: (1) Weigh 0.2 g of sanguisorbine chloride and add it to 150 mL of anhydrous ethanol. Stir magnetically until completely dissolved to obtain a clear sanguisorbine chloride ethanol mother liquor.

[0055] (2) Add 10 mL of Tween-80 to the ethanol mother liquor of sanguisorbine chloride and stir continuously to mix it so that it can fully encapsulate the active ingredients as a non-ionic surfactant and form a stable solubilization system.

[0056] (3) Under continuous stirring, slowly add purified water to make up to 1000 mL, mix well and you will get a surface cleaning spray with a uniform and clear appearance.

[0057] The final concentration of sanguinarine chloride in the spray is approximately 544 μM. This concentration is related to the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each strain in Examples 1-3 as follows: It is approximately 108 times the MIC (5 μM) of Staphylococcus aureus (SA) and Listeria monocytogenes (LM); approximately 10 times the MIC (50 μM) of Cronobacter (CS) and Escherichia coli (EIEC); and 2.7 times the MBC (200 μM) of Salmonella (STYP). This concentration design ensures sufficient kill margin against all five tested pathogens.

[0058] 2. Quantitative bactericidal test of the carrier Referring to the principle of the carrier immersion method in GB / T 38502-2020 "Laboratory Sterilization Test Methods for Disinfectants" and with appropriate modifications, the specific steps are as follows: (1) Carrier preparation: Take a 304 stainless steel sheet with a specification of 2.0 cm × 2.0 cm, sterilize it under high pressure and set it aside for use to simulate a typical contact surface in the food processing environment.

[0059] (2) Bacterial contamination: Take suspensions of Staphylococcus aureus (SA), Listeria monocytogenes (LM), Cronobacter (CS), Escherichia coli (EIEC), and Salmonella (STYP) in the logarithmic growth phase (concentration approximately 5 × 10⁻⁶). 7 Take 50 μL of each of the CFU / mL values ​​and spread them evenly on the surface of a sterilized stainless steel sheet. Place the sheet in a biosafety cabinet at 37°C to air dry naturally, allowing the bacteria to adhere firmly to the carrier surface.

[0060] (3) Spraying treatment: Use a manual sprayer to evenly spray the surface disinfectant onto the contaminated carrier surface, so that the liquid film completely covers the carrier (about 0.5 mL / piece), and let it act at room temperature for 20 min.

[0061] (4) Control group treatment: Take the bacterial carrier prepared under the same conditions, spray an equal volume of sterile phosphate buffer (PBS, pH 7.2), and let it act for 20 min under the same conditions.

[0062] (5) Neutralization and elution: After the reaction time is over, immediately transfer the carrier into a test tube containing 5 mL of neutralizing agent (composed of PBS solution containing 3% Tween-80, 0.3% lecithin and 0.1% cysteine), shake and elute thoroughly for 1 min to neutralize the residual sanguisorbine activity and prevent the continuous bactericidal effect in the subsequent culture process from interfering with the accuracy of counting.

[0063] (6) Dilution and plate counting: Take the eluent and perform a 10-fold serial dilution. Take 100 μL of each and spread it evenly on LB agar plates. Incubate at 37℃ for 24-48 h and count the colony forming units (CFU).

[0064] (7) Calculation formula: Kill log value = lgN0 lgN t Kill rate (%) = (N0) N t ) / N0×100%; Where N0 represents the number of recovered bacteria from the control group vector (CFU / tablet), N t The amount of bacteria recovered from the experimental group carrier (CFU / tablet).

[0065] The results are shown in Table 2; Table 2. Killing effect of sanguisorbine chloride surface decontamination spray on five pathogenic bacteria on stainless steel carrier surfaces. Note: Each experiment was repeated 3 times in parallel. Data are expressed as mean ± standard deviation. "<10" indicates that no colonies were detected or only a single-digit number of colonies were detected after plating. The detection limit is 10 CFU / plate.

[0066] Table 2 shows that the sanguisorbent chloride surface decontamination spray, after 20 min at room temperature, exhibited a strong killing effect on all five high-risk pathogens attached to the stainless steel carrier surface. Among them: The kill log values ​​for Gram-positive bacteria (SA and LM) were both >4.68, with a kill rate >99.999%, and the carrier surface was basically sterile. The kill log values ​​against Gram-negative bacteria (CS, EIEC, and STYP) were all ≥3.25, with a kill rate >99.9%.

[0067] The above results confirm the practical application value of this invention in the fields of food processing and hygiene and contamination prevention.

[0068] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. The application of sanguinarine chloride in the preparation of antibacterial and / or bactericidal products for foodborne pathogens, characterized in that, The sanguinarine chloride is a broad-spectrum antibacterial / bactericidal agent.

2. The application as described in claim 1, characterized in that, The foodborne pathogens are selected from one or more of Staphylococcus aureus, Salmonella, diarrhea-causing Escherichia coli, Listeria monocytogenes, and Cronobacter.

3. The application as described in claim 1, characterized in that, The foodborne pathogens are foodborne pathogens that have not formed a biofilm and / or foodborne pathogens that have formed a biofilm.

4. The application as described in claim 1, characterized in that, The inhibition and / or sterilization of foodborne pathogens includes killing foodborne pathogens, preventing the formation of foodborne pathogen biofilms, and destroying mature foodborne pathogen biofilms.

5. The application as described in claim 1, characterized in that, The sanguisorbine chloride is a plant-derived sanguisorbine chloride, and the product includes pharmaceuticals, antibacterial agents, and external cleaning and disinfection products; the dosage forms of the product include solutions, sprays, gels, emulsions, powders, or coating preparations.

6. A bacteriostatic / bactericidal agent for foodborne pathogens, characterized in that, It includes sanguinarine chloride; the foodborne pathogens are selected from one or more of Staphylococcus aureus, Salmonella, diarrheal Escherichia coli, Listeria monocytogenes and Cronobacter.

7. A composition for inhibiting / killing foodborne pathogens, characterized in that, The product includes the foodborne pathogen inhibitor / bactericide as described in claim 5, and also includes pharmaceutically or chemically acceptable excipients, such as solvents, cosolvents, surfactants, and humectants.

8. The foodborne pathogen inhibitory / bactericidal composition as described in claim 7, characterized in that, The mass percentage of sanguinarine chloride is greater than 0.01%.

9. A surface disinfectant spray for foodborne pathogens, characterized in that, Including 8×10 -5 The concentration of sanguinarine chloride is g / mL; the foodborne pathogens are selected from one or more of Staphylococcus aureus, Salmonella, diarrheal Escherichia coli, Listeria monocytogenes, and Cronobacter.

10. The application of sanguinarine chloride in the preparation of drugs for preventing and treating foodborne pathogen infections, characterized in that, The foodborne pathogens are selected from one or more of Staphylococcus aureus, Salmonella, diarrhea-causing Escherichia coli, Listeria monocytogenes, and Cronobacter.

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