Use of cumin aldehyde in inhibiting klebsiella oxytoca

Abstract

The application of cumin aldehyde in inhibiting klebsiella quasimonile belongs to the technical field of food safety. In order to solve the technical problems that the existing plant extracts against klebsiella quasimonile have uneven bacteriostatic effect, insufficient stability and application prospect, etc., the application of cumin aldehyde in resisting klebsiella quasimonile is provided. The present application finds that the minimum bacteriostatic concentration and the minimum bactericidal concentration of cumin aldehyde to klebsiella quasimonile are both 1 muL / mL, which inhibits or kills klebsiella quasimonile by destroying the cell membrane and membrane protein of klebsiella quasimonile, changing the cell membrane permeability of klebsiella quasimonile, and inserting and combining with the DNA of klebsiella quasimonile. Cumin aldehyde is suitable for disinfection treatment of non-biological surfaces such as dairy processing equipment, storage tanks, pipelines and farm contact surfaces due to its strong bactericidal ability and membrane damage mechanism.

Description

Technical Field

[0001] This invention belongs to the field of food safety technology, specifically relating to the application of cucurbital in inhibiting Cronobacter sakazakii. Background Technology

[0002] Cronobacter (Cronobacter) formerly known as Enterobacter sakazakii ( Enterobacter sakazakii *Cronobacter* is a Gram-negative, rod-shaped, non-spore-forming facultative anaerobic bacterium belonging to the family Enterobacteriaceae. In 2008, it was reclassified as a new genus within the Enterobacteriaceae family, namely *Cronobacter*. This genus currently contains seven species, including *Cronobacter sakazakii*. Cronobacter sakazakii Kronobacter is the dominant species. It is widely distributed in nature and exists in water, soil, animal and human feces. The pathogen has been isolated from various food and environmental substrates and is closely related to infant formula (PIF) contamination.

[0003] Cronobacter sakazakii is an important foodborne opportunistic pathogen capable of surviving in various environments. Infants and young children are at high risk of infection, which primarily causes bacteremia, meningitis, and necrotizing enterocolitis, with a mortality rate as high as 40%-80%. However, this bacterium can also cause disease in all age groups. Cronobacter sakazakii exhibits strong resistance to high osmotic pressure and drying, as well as some antibiotic resistance, allowing it to adhere to and survive on the surfaces of equipment and utensils in food production facilities and farm environments, posing a threat to dairy product safety.

[0004] In traditional methods of sterilizing Cronobacter sakazakii, physical and chemical sterilization methods are commonly used. Common physical sterilization methods include dry heat, moist heat, radiation, and filtration. However, some physical methods may damage the structure and texture of food during sterilization, leading to nutrient loss and a decline in taste. Common chemical methods, such as synthetic food preservatives, may introduce new substances harmful to human health and increase environmental pollution. Therefore, to prevent harm from foodborne pathogens and the sterilization process itself, researchers are constantly developing new food sterilization methods that are more efficient, safer, and pollution-free than traditional methods.

[0005] Against this backdrop, consumers are increasingly turning their attention to safe, healthy, and sustainably produced natural food additives. Currently, research on novel sterilization methods against Cronobacter sakazakii is increasingly focusing on the application of natural food additives. Natural food additives are diverse, with those commonly used for food preservation including animal-derived chitosan, antimicrobial peptides, plant extracts, and organic acids produced by microbial metabolism. Among these additives, plant extracts have become a current research hotspot due to their antibacterial and preservative activities against various pathogenic microorganisms, as well as their safety and ease of extraction.

[0006] Currently, various plant extracts have been studied for their use in inhibiting Cronobacter sakazakii. However, although reported plant extracts have shown some antibacterial activity against Cronobacter sakazakii, their antibacterial effects vary, and some extracts have high MIC values ​​(such as Echinacea extract), limiting their practical application effectiveness. While some extracts, such as protocatechuic acid, have low MICs, their stability, safety, and large-scale application still require further investigation. Furthermore, most existing studies remain at the laboratory stage of activity evaluation, lacking sufficient validation of the applicability, safety, and industrial application prospects of plant extracts in actual food systems.

[0007] Cuminaldehyde is one of the main active ingredients extracted from cumin. Studies have shown that cuminaldehyde possesses good antioxidant, antibacterial, and insecticidal activities. Existing literature has confirmed that cuminaldehyde is effective against Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus Cuminaldehyde exhibits inhibitory effects. Mechanism-of-action studies have shown that cuminaldehyde can disrupt the cell membrane structure of Gram-positive bacteria (such as Staphylococcus aureus) through PI staining, nucleic acid and protein leakage experiments, and membrane protein fluorescence detection. Simultaneously, DNA fluorescence spectroscopy and agarose gel electrophoresis indicate that cuminaldehyde can also damage their DNA. However, the aforementioned antibacterial studies on cuminaldehyde have focused on Gram-positive bacteria; its inhibitory effect on Gram-negative bacteria (especially Cronobacter) and its specific mechanisms have not yet been reported. Given the significant differences in cell wall structure (outer membrane barrier) between Gram-negative and Gram-positive bacteria, the known activity of cuminaldehyde against Staphylococcus aureus cannot reasonably be extrapolated to its effectiveness against Cronobacter.

[0008] Therefore, there is an urgent need in this field to develop a new application scheme for plant extracts, specifically to explore the use of cuminaldehyde in the fight against Cronobacter sakazakii, in order to fill the gap in the antibacterial spectrum of cuminaldehyde in the existing technology, and to provide a safe and efficient new natural antibacterial strategy for the prevention and control of Cronobacter contamination in infant food and related production environments. Summary of the Invention

[0009] To address the existing limitations of cumin, where antibacterial research focuses solely on Gram-positive bacteria (such as Staphylococcus aureus) and there are no reports of its inhibitory effect on Cronobacter sakazakii, a Gram-negative bacterium, and the inconsistent antibacterial efficacy, instability, and limited application prospects of existing anti-Cronobacter sakazakii plant extracts, this invention provides the application of cumin in the treatment of Cronobacter sakazakii.

[0010] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide cuminaldehyde in the preparation of a formulation for inhibiting or killing Cronobacter sakazakii (… Cronobacter sakazakii Application in antibacterial agents.

[0011] In one embodiment of the present invention, the minimum inhibitory concentration of cumin against Cronobacter sakazakii is 1 μL / mL, and the minimum bactericidal concentration is 1 μL / mL.

[0012] In one embodiment of the present invention, the cuminaldehyde inhibits or kills Cronobacter sakazakii by disrupting the cell membrane of Cronobacter sakazakii.

[0013] In one embodiment of the present invention, the cuminaldehyde inhibits or kills Cronobacter sakazakii by disrupting the membrane proteins of Cronobacter sakazakii.

[0014] In one embodiment of the present invention, the cuminaldehyde inhibits or kills Cronobacter sakazakii by altering the cell membrane permeability of Cronobacter sakazakii.

[0015] In one embodiment of the present invention, the cuminaldehyde inhibits or kills Kronobacter sakazakii by intercalating and binding to its DNA.

[0016] The second objective of this invention is to provide an antibacterial agent with cuminaldehyde as the active ingredient for the inhibition or killing of Cronobacter sakazakii.

[0017] In one embodiment of the invention, the antibacterial agent further contains an acceptable carrier or excipient.

[0018] In one embodiment of the present invention, the concentration of cumin in the antibacterial agent is not less than 1 μL / mL.

[0019] In one embodiment of the present invention, the antibacterial agent is used for disinfection of the surfaces of equipment and utensils in food production facilities or farm environments.

[0020] The beneficial effects of this invention are: Compared with the prior art, the present invention has the following significant advantages: 1. Extremely high antibacterial activity and outstanding bactericidal efficacy. The minimum inhibitory concentration (MIC) of cucurbital against *Cronobacter sakazakii* is only 1 μL / mL, indicating that it can effectively inhibit the growth of this bacterium even at extremely low doses. More importantly, its minimum bactericidal concentration (MBC) is the same as its minimum inhibitory concentration (MIC), demonstrating that cucurbital not only has antibacterial effects but also significant bactericidal (sterilizing) efficacy, capable of directly killing pathogens while inhibiting bacterial proliferation. This is crucial for the thorough elimination of *Cronobacter sakazakii* contamination in food production facilities.

[0021] 2. Synergistic effect of multiple targets, unique and irreversible bactericidal mechanism. Cuminaldehyde exerts its antibacterial effect through a dual-core mechanism: on the one hand, it significantly alters cell membrane permeability by disrupting the cell membrane structure and function of membrane proteins in *Cronobacter sakazakii*, leading to the abnormal leakage of important intracellular substances (such as nucleic acids and proteins) and causing irreversible physical damage to the bacteria; on the other hand, natural compound A can interact with the DNA of *Cronobacter sakazakii* through intercalation, interfering with the normal replication and transcription of its genetic material. This multi-target synergistic effect of "physical damage to the cell membrane + DNA functional interference" not only greatly improves the bactericidal efficiency but also significantly reduces the risk of bacteria developing drug resistance, overcoming the shortcomings of traditional single-target antibiotics that easily induce drug resistance mutations.

[0022] 3. Suitable for surface sterilization in complex environments, with practical application value. Given that *Cronobacter sakazakii* can adhere to and survive for extended periods on the surfaces of equipment and containers in food production facilities and farm environments, cucurbitacin, with its potent bactericidal ability and membrane disruption mechanism, can effectively eliminate stubborn bacteria and bacteria existing in the form of biofilms attached to solid surfaces. It is suitable for disinfection of non-biological surfaces such as dairy processing equipment, storage tanks, pipelines, and farm contact surfaces, providing an efficient, reliable, and green antibacterial solution for improving the safety of dairy production. Attached Figure Description

[0023] Figure 1 Figure 1 shows the results of PI staining method for detecting the cell membrane disruption of different concentrations of cuminaldehyde on Cronobacter sakazakii. Figure 2 The image shows the detection results of nucleic acid leakage from Cronobacter sakazakii treated with different concentrations of cumin. Figure 3 The image shows the detection results of protein leakage in Cronobacter sakazakii treated with different concentrations of cumin; Figure 4 The graph shows the detection results of reactive oxygen species levels in Cronobacter sakazakii treated with different concentrations of cumin. Figure 5The image shows the detection results of phenylalanine in the membrane proteins of Cronobacter sakazakii treated with different concentrations of cumin; Figure 6 The image shows the detection results of tryptophan in the membrane proteins of Cronobacter sakazakii treated with different concentrations of cumin; Figure 7 The image shows the detection results of tyrosine in the membrane proteins of Cronobacter sakazakii treated with different concentrations of cumin; Figure 8 The images show the results of electron microscopy observation of the degree of cell membrane damage in *Cronobacter sakazakii* treated with different concentrations of cucurbital; where A is an electron micrograph of *Cronobacter sakazakii* without cucurbital treatment, B is an electron micrograph of *Cronobacter sakazakii* treated with 1 / 2 MIC cucurbital, C is an electron micrograph of *Cronobacter sakazakii* treated with MIC cucurbital, and D is an electron micrograph of *Cronobacter sakazakii* treated with 2 MIC cucurbital. Figure 9 The figure shows the detection results of β-galactosidase activity in Cronobacter sakazakii suspensions treated with different concentrations of cumin; Figure 10 The results of gel retardation analysis to determine whether cumin caused DNA fragmentation in Cronobacter sakazakii. Figure 11 The figure shows the results of DNA content determination to determine whether cumin directly affects DNA by degrading the DNA of Cronobacter sakazakii. Figure 12 The figure shows the results of DNA fluorescence spectroscopy determination of the interaction mode between cuminaldehyde and Cronobacter sakazakii DNA as an intercalation binding. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, embodiments obtained by other those skilled in the art without creative effort are all protected by this invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The strain used is Cronobacter sakazakii ATCC29544, and chlorpyrifos was purchased from McLean Company at a concentration of 97%. Other materials, reagents and instruments, unless otherwise specified, are conventional materials, reagents and instruments in the art and can be obtained by those skilled in the art through commercial channels.

[0026] Example 1: Determination of the antibacterial activity of cuminaldehyde against Cronobacter sakazakii This embodiment was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of cucurbital against *Cronobacter sakazakii*. The specific determination method is as follows: The MIC of cucurbital against *Cronobacter sakazakii* was determined using a serial two-fold dilution method. Cucurbital was serially diluted twofold with trypticase-soy broth (TSB) medium, with 1% Tween-80 added to promote emulsification, and the final bacterial concentration of *Cronobacter sakazakii* was adjusted to 10⁻⁶ using TSB medium. 6 CFU / mL, add 100 μL each of different concentrations of cuminaldehyde and bacterial culture to a 96-well plate, mix well, and incubate at 37℃ for 24 h. OD is measured using a microplate reader. 600 To assess bacterial growth, the lowest concentration at which no increase in OD value is observed is defined as the MIC. Based on the MIC results, the MBC is determined. 100 μL of bacterial suspension (treated with different concentrations of cuminaldehyde) is inoculated onto tryptone soy agar (TSA) medium, spread evenly, and incubated at 37°C for 24 h. The lowest treatment concentration at which no bacterial growth is observed is defined as the MBC.

[0027] Tests showed that the minimum inhibitory concentration (MIC) of cucurbitaldehyde against Cronobacter sakazakii was 1 μL / mL, which is the same as the minimum bactericidal concentration (MCC).

[0028] Example 2: Detection of the destructive effect of cuminaldehyde on the cell membrane of Cronobacter sakazakii using PI staining method The overnight cultured *Cronobacter sakazakii* culture was centrifuged, and the supernatant was removed. The culture was then resuspended in cumin solution diluted with PBS (concentrations of 1 / 2 MIC, MIC, and 2 MIC, with an equal volume of PBS without cumin (0 MIC) as a blank control) to its original volume and incubated at room temperature for 2 h. After treatment, the culture was centrifuged again, and the precipitate was washed three times with PBS. PI staining solution to a final concentration of 10 μM was added, and the culture was incubated in the dark for 30 min. After incubation, fluorescence spectrophotometers were used to excite the cells at an excitation wavelength of 485 nm, and the fluorescence spectrum in the range of 522-700 nm was measured. Changes in fluorescence intensity characterized the degree of damage to cell membrane integrity. PI is a DNA-intercalated fluorescent probe that cannot penetrate living cells. It can only penetrate the cell membrane and intercalate with intracellular DNA when the cell membrane is damaged, emitting fluorescence under excitation at a specific wavelength. Therefore, the difference in fluorescence intensity caused by the interaction between PI and DNA can quantify the degree of cell membrane damage.

[0029] Fluorescence spectroscopy results showed that the fluorescence intensity differed between the treatment group and the control group; the fluorescence intensity increased with increasing cumin concentration (0 MIC < 1 / 2 MIC < 1 MIC < 2 MIC), and the fluorescence intensity after cumin treatment was higher than that of the control group. Figure 1 This indicates that cucurbitacin can cause cell membrane damage, reduce cell membrane integrity, and increase cell membrane permeability, and the degree of damage is concentration-dependent.

[0030] Example 3: Detection of the destructive effect of cuminaldehyde on the cell membrane of Cronobacter sakazakii based on the leakage of nucleic acid protein contents. The overnight culture of *Cronobacter sakazakii* was centrifuged, and the supernatant was removed. The culture was then resuspended in cumin solution diluted with PBS (concentrations of 1 / 2 MIC, MIC, and 2 MIC, with an equal volume of PBS (0 MIC) as a blank control) to the original volume, and treated at room temperature for 2 h. After treatment, the supernatant was centrifuged and the OD was measured in a 96-well UV-Vis analysis plate. 260 OD 280 Value. OD 260 OD 280 The value reflects the leakage of nucleic acid and protein from the treated Cronobacter sakazakii.

[0031] Test results as follows Figure 2 and Figure 3 As shown, the leakage of nucleic acids and proteins increases with the increase of cucurbitaldehyde concentration, indicating that the cell membrane of Cronobacter sakazakii is damaged and cucurbitaldehyde alters its membrane permeability.

[0032] Example 4: Destructive effects of cuminaldehyde on the cell membrane or DNA of Cronobacter sakazakii based on reactive oxygen species detection. The overnight culture of *Cronobacter sakazakii* was centrifuged, and the supernatant was removed. The culture was then resuspended in cuminol solution diluted with PBS (concentrations of 1 / 2 MIC, MIC, and 2 MIC, with an equal volume of PBS without cuminol (0 MIC) as a blank control) to its original volume, and treated at room temperature for 2 h. After treatment, the culture was centrifuged again, and the precipitate was washed three times with PBS. The precipitate was then resuspended in PBS containing 10 μM DCFH-DA and treated in the dark for 30 min. Finally, the fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm was measured.

[0033] The results are as follows Figure 4 As shown, cucurbitacin treatment led to an increase in ROS (reactive oxygen species) levels in *Cronobacter sakazakii*, directly indicating that cucurbitacin inhibits or kills *Cronobacter sakazakii* by inducing oxidative stress. This typically means that critical structures of *Cronobacter sakazakii*, such as the cell membrane, DNA, or proteins, have suffered irreversible damage.

[0034] Example 5: Destructive effect of cuminaldehyde on membrane proteins of Cronobacter sakazakii based on membrane protein fluorescence detection Cell membrane proteins are important components of the cell membrane, participating in vital physiological activities such as intracellular and extracellular substance exchange and cell recognition. Therefore, it is necessary to study the effects of cuminaldehyde on the cell membrane proteins of *Cronobacter sakazakii*. Membrane proteins fluoresce under ultraviolet light because their structure contains three aromatic amino acids: phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). Certain substances can cause fluorescence quenching of these fluorescent amino acids. By detecting the fluorescence intensity of Phe, Trp, and Tyr, the destructive effect of cuminaldehyde on *Cronobacter sakazakii* membrane proteins can be analyzed. The specific method for detecting Phe, Trp, and Tyr is as follows: *Cronobacter sakazakii* culture cultured overnight was centrifuged at 10000xg for 1 min. After removing the supernatant, cuminaldehyde solution diluted with PBS (concentrations of 1 / 2 MIC, MIC, and 2 MIC, with an equal volume of PBS treatment group (0 MIC) without cuminaldehyde serving as a blank control) was added and the mixture was resuspended to its original volume and treated at room temperature for 2 h. After treatment, the sample was centrifuged again and washed three times with PBS. The treated liquid was added to a black microplate at a rate of 200 μL per well, and the spectra of excitation at 258 nm, emission at 300-500 nm, excitation at 280 nm, emission at 300-500 nm, excitation at 296 nm, and emission at 320-500 nm were measured.

[0035] like Figure 5 , Figure 6 , Figure 7As shown, with increasing cucurbitaldehyde concentration, the fluorescence intensity of Phe, Trp, and Tyr residues all decreased significantly, indicating that Phe, Trp, and Tyr residues are exposed outside the membrane, and more cucurbitaldehyde can interact with Phe, Trp, and Tyr residues, quenching their fluorescence. This result suggests that cucurbitaldehyde can bind to cell membrane proteins and thus alter the membrane function of Cronobacter sakazakii.

[0036] Example 6: Electron microscopy observation of the degree of cell membrane damage in *Cronobacter sakazakii* treated with different concentrations of calciferol The overnight culture of *Cronobacter sakazakii* was centrifuged, and the supernatant was removed. The culture was then resuspended in cuminol solution diluted with PBS (concentrations of 1 / 2 MIC, MIC, and 2 MIC, with an equal volume of PBS (0 MIC) as a blank control) to its original volume and incubated at room temperature for 2 h. After treatment, the culture was centrifuged again, and the precipitate was collected and resuspended in glutaraldehyde for fixation. The precipitate was washed three times with PBS, followed by gradient dehydration with 50%, 70%, 90%, and 100% ethanol. After freeze-drying, the precipitate was sputter-coated with gold on a conductive gel, adhered to the gel, and observed and photographed under a scanning electron microscope (SEM).

[0037] Depend on Figure 8 It can be seen that the surface of untreated Cronobacter sakazakii cells is smooth and rod-shaped. The surface of Cronobacter sakazakii cells treated with different concentrations of caloaldehyde is wrinkled. The surface of Cronobacter sakazakii cells treated with caloaldehyde at a concentration of 2 MIC is almost completely wrinkled, which proves the destructive effect of caloaldehyde on the cell membrane of Cronobacter sakazakii.

[0038] Example 7: Effect of cuminaldehyde on cell membrane permeability of Cronobacter sakazakii as determined by β-galactosidase assay The overnight cultured *Cronobacter sakazakii* suspension was centrifuged, and the supernatant was removed. The suspension was then resuspended in cuminol solution diluted with PBS (concentrations of 1 / 2 MIC, MIC, and 2 MIC, with an equal volume of PBS without cuminol (0 MIC) as a blank control) to the original volume, and treated at room temperature for 2 h. After treatment, the suspension was centrifuged again, and the precipitate was washed three times with PBS. 50 μL of 0.75 mol / L ONPG was added to 1 μL of the bacterial suspension, and the mixture was reacted in the dark for 10 min. The OD value was then measured at 420 nm using a microplate reader.

[0039] ONPG can be hydrolyzed by β-galactosidase into galactose and O-nitrophenol (ONPE), with ONPE exhibiting a UV absorption peak at 420 nm. Under normal conditions, ONPG penetrates the cell membrane extremely slowly. However, once the cell membrane permeability increases, a large amount of ONPG penetrates the cell membrane, resulting in a significant increase in UV intensity at 420 nm. The trend of β-galactosidase activity in bacterial cultures treated with different concentrations of cumin is shown in the figure. Figure 9 As shown, compared with the control group (0 MIC), the OD values ​​of the cucurbital treatment groups at concentrations of 1 / 2 MIC, 1 MIC, and 2 MIC were significantly higher. 420 The increases in all values ​​indicate that the permeability of the *Cronobacter sakazakii* cell membrane was affected by cuminaldehyde.

[0040] Example 8: Detection of the mechanism of action of cuminaldehyde on Cronobacter sakazakii DNA 1. Gel retardation analysis The overnight culture of *Cronobacter sakazakii* was centrifuged, and the supernatant was removed. The culture was then resuspended in cumin solution diluted with PBS (concentrations of 1 / 2 MIC, MIC, and 2 MIC, with an equal volume of PBS without cumin (0 MIC) as a blank control) to its original volume and incubated at room temperature for 2 h. DNA was then extracted from *Cronobacter sakazakii* using a DNA extraction kit. After incubation at 37°C for 30 min, 5 μL of DNA was added to 2 μL of 6× Loading Buffer, mixed well, loaded onto a 1% agarose gel for electrophoresis, and observed and photographed.

[0041] If DNA binds to calciform via shearing, the phosphodiester bonds in the DNA are damaged, causing breaks in both the single and double strands, resulting in typical ladder-like bands. Figure 10 As shown, with the increase of cucurbitacin concentration, the DNA bands did not differ significantly from the control group, and the migration distances were consistent with those of the control group. This indicates that cucurbitacin does not have the ability to cause DNA cleavage in Cronobacter sakazakii, and its interaction with DNA does not occur through shearing.

[0042] 2. DNA content determination The overnight cultured *Cronobacter sakazakii* culture was centrifuged, and the supernatant was removed. The culture was then resuspended in cumin solution diluted with PBS (concentrations of 1 / 2 MIC, MIC, and 2 MIC, with an equal volume of PBS without cumin (0 MIC) as a blank control) to its original volume and incubated at room temperature for 2 h. DNA was then extracted from the treated culture using a bacterial genomic DNA extraction kit. The extracted DNA solutions were then added to a micro-spectrophotometer to measure their DNA concentration.

[0043] DNA extraction from *Cronobacter sakazakii* treated with caloaldehyde revealed that, compared to the control group without caloaldehyde, the DNA content in the treatment groups with different amounts of caloaldehyde did not decrease. Figure 11 This indicates that cucurbitaldehyde does not directly affect DNA by degrading Sakazaki DNA.

[0044] 3. DNA fluorescence spectroscopy determination Genomic DNA was extracted from *Cronobacter sakazakii*. The genomic DNA solution was co-cultured with ethidium bromide (EB) solution at 37°C for 30 min to allow the DNA and EB to bind and form an EB-DNA complex. Subsequently, different concentrations of cucurbital were added to the EB-DNA complex to achieve final concentrations of 0 MIC, 1 / 2 MIC, 1 MIC, and 2 MIC, respectively. The mixture was then incubated again at 37°C in the dark for 30 min. Finally, the fluorescence spectra of the sample were detected at an excitation wavelength of 525 nm and an emission wavelength between 550 nm and 700 nm.

[0045] EB exhibits no significant fluorescence intensity in aqueous solution. However, its fluorescence intensity is significantly enhanced upon insertion into DNA base pairs. When another molecule with a similar binding mechanism to EB is present, it competitively binds to DNA, leading to fluorescence quenching. Figure 12 It can be concluded that the fluorescence intensity decreases with the increase of cuminaldehyde concentration, indicating that high concentrations of cuminaldehyde will replace EB in DNA base pairs to a greater extent, proving that the interaction mode of cuminaldehyde with Cronobacter sakazakii DNA should be intercalation binding.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Cuminaldehyde in the preparation of products used to inhibit or kill Cronobacter sakazakii ( Cronobacter sakazakii Application in antibacterial agents.

2. The application according to claim 1, characterized in that, The minimum inhibitory concentration (MIC) of the cumin against Cronobacter sakazakii is 1 μL / mL, and the minimum bactericidal concentration (MBC) is 1 μL / mL.

3. The application according to claim 1, characterized in that, The cuminaldehyde inhibits or kills Kronobacter sakazakii by disrupting the cell membrane of Kronobacter sakazakii.

4. The application according to claim 1, characterized in that, The cuminaldehyde inhibits or kills Kronobacter sakazakii by disrupting the membrane proteins of Kronobacter sakazakii.

5. The application according to claim 1, characterized in that, The cuminaldehyde inhibits or kills *Cronobacter sakazakii* by altering the cell membrane permeability of *Cronobacter sakazakii*.

6. The application according to claim 1, characterized in that, The cuminaldehyde inhibits or kills Kronobacter sakazakii by intercalating and binding to its DNA.

7. The application of an antibacterial agent with cuminaldehyde as the active ingredient in inhibiting or killing Cronobacter sakazakii.

8. The antibacterial agent according to claim 7, characterized in that, The antibacterial agent also contains an acceptable carrier or excipient.

9. The antibacterial agent according to claim 7, characterized in that, The concentration of cumin in the antibacterial activity is not less than 1 μL / mL.

10. The antibacterial agent according to claim 7, characterized in that, The antimicrobial agent is used for disinfection of the surfaces of equipment and utensils in food production facilities or farm environments.

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