Pyracantha fortuneana extract with bacteriostatic effect and application thereof in food storage and preservation

Abstract

The application belongs to the technical field of separation and extraction and biological preservation, and relates to application of a pear fruit extract with bacteriostatic action in food storage and preservation. The pear fruit extract is an ethanol extract of pear fruit roots, which is extracted by chloroform and concentrated by a rotary evaporator under reduced pressure. The pear fruit extract shows good bacteriostatic activity on various gram-positive and gram-negative bacteria, and the minimum inhibitory concentration (MIC) is 4 mg / mL. The pear fruit extract contains various compounds with bacteriostatic activity, such as organic acids, aldehydes, phenols, ketones, esters and amides, and the bacteriostatic activity of 2,6-dimethoxybenzoquinone is the strongest, with the MIC of 0.0625 mg / mL for gram-positive bacteria and the MIC of 0.125 mg / mL for gram-negative bacteria. The pear fruit extract with broad-spectrum and high-efficiency antibacterial activity can be prepared into a biological preservative for food storage and preservation.

Description

Technical Field

[0001] This invention belongs to the field of separation, extraction and biological preservation technology, and relates to a pear extract with antibacterial properties and its application in food storage and preservation. Background Technology

[0002] During food production, transportation, and storage, contamination by spoilage bacteria and pathogenic microorganisms can easily lead to food spoilage, seriously threatening global food security. Therefore, developing efficient food antimicrobial and preservative technologies is crucial for controlling microbial contamination. Although chemical preservatives are widely used, their safety remains controversial. In contrast, natural antimicrobial agents, due to their high safety and significant antimicrobial effects, have become a hot research topic in the field of food storage and preservation. Plant-derived antimicrobial agents are abundant, but in the current GB 2760-2024 "National Food Safety Standard for the Use of Food Additives," only cinnamaldehyde, a plant-derived preservative, is explicitly permitted for use. Therefore, exploring more plant-derived antimicrobial agents is a future development trend. However, current research on plant-derived preservatives is limited, and there is an urgent need to discover more and more efficient plant-derived preservatives. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides the application of pear extract with antibacterial properties in food storage and preservation. The pear extract provided by this invention exhibits good broad-spectrum antibacterial activity.

[0004] The technical solution provided by this invention is as follows:

[0005] The application of pear extract in food storage and preservation, wherein the pear extract is obtained by chloroform extraction of ethanol extract of pear root and concentration under reduced pressure using a rotary evaporator.

[0006] Furthermore, the preparation method of the pear extract includes the following steps:

[0007] The root of *Pyrus pyrifolia* was pulverized and sieved to obtain root powder. The root powder was mixed evenly with ethanol solution and extracted with ultrasonic assistance. The ultrasonic extract was filtered under reduced pressure, the filtrate was collected, and concentrated under reduced pressure using a rotary evaporator to obtain a concentrate. The concentrate was extracted with chloroform, the chloroform extract was collected, and the extract was evaporated to dryness using a rotary evaporator to obtain *Pyrus pyrifolia* extract.

[0008] Furthermore, the pear extract includes organic acids, aldehydes, phenols, ketones, esters, and amides.

[0009] Furthermore, the pear extract includes 2-methylcitric acid, coniferaldehyde, 2,6-dimethoxybenzoquinone, eugenol, cinnamamide, 5,7-dihydroxychromone, 4-hydroxy-3-(3-methylbut-2-enyl)benzoic acid, ryegrass lactone, vanillin, vanillic acid, ethyl 3,4-dihydroxybenzoate, benzoic acid, hesperidin, indole-3-carboxaldehyde, sagerol, naringenin chalcone, and 3,4-dihydrocoumarin.

[0010] Furthermore, the pear extract includes 2,6-dimethoxybenzoquinone.

[0011] Furthermore, the pear extract is used for antibacterial purposes.

[0012] Furthermore, the bacteria include Escherichia coli, Salmonella typhimurium, Bacillus cereus, and Staphylococcus aureus.

[0013] Furthermore, the pear extract is used to delay the logarithmic growth phase of bacteria, increase bacterial cell membrane permeability, and induce leakage of intracellular components.

[0014] Furthermore, the pear extract is used to disrupt the integrity of bacterial cell walls, inhibit biofilm formation, promote the clearance of mature bacterial biofilms, induce oxidative damage in bacteria, reduce bacterial cell metabolic activity, inhibit bacterial SDH activity, and interfere with bacterial TCA cycles.

[0015] Furthermore, the pear extract can be used to prepare a biological preservative.

[0016] Furthermore, the biopreservative prepared from the pear extract can be used for food storage and preservation.

[0017] The present invention also provides a biological preservative, which includes pear extract, wherein the pear extract is obtained by chloroform extraction of ethanol extract of pear root and concentration under reduced pressure using a rotary evaporator.

[0018] Beneficial effects

[0019] Pyrus pyrifolia extract exhibits good antibacterial activity against various Gram-negative and Gram-positive bacteria, including Escherichia coli, Salmonella typhimurium, Bacillus cereus, and Staphylococcus aureus, with a minimum inhibitory concentration (MIC) of 4 mg / mL. It possesses a broad antibacterial spectrum and high antibacterial activity. However, there are currently no research reports on the antibacterial activity of Pyrus pyrifolia. Based on its good antibacterial performance and the research gap, the chloroform extract (TE) of Pyrus pyrifolia was selected as a candidate extract to explore novel antibacterial substances. Through silica gel column chromatography separation, antibacterial activity detection, and UPLC-MS analysis of the TE, 27 compounds with relative contents higher than 1% were identified in the fraction with high antibacterial activity. Among them, 2,6-dimethoxybenzoquinone not only showed the most prominent antibacterial effect but also had a relatively high content in the extract. Verification by comparison with standards confirmed it as the key antibacterial active ingredient in Pyrus pyrifolia extract.

[0020] 2,6-Dimethoxybenzoquinone is a key antibacterial active ingredient in pear extract, but its antibacterial mechanism remains largely unknown. To fill this gap, *Staphylococcus aureus* and *Escherichia coli* were selected as model strains to investigate its antibacterial mechanism. Studies on the effect of 2,6-dimethoxybenzoquinone on bacterial growth curves revealed that its inhibitory effect on bacterial growth was dose-dependent. A concentration of 2,6-dimethoxybenzoquinone at 1 / 2 MIC delayed the logarithmic growth phase of bacteria, while at concentrations of 1 MIC and 2 MIC, bacterial growth almost ceased. Detection of nucleic acid leakage and extracellular relative conductivity showed that 2,6-dimethoxybenzoquinone can disrupt bacterial cell membranes, affecting their permeability and integrity. Increased extracellular AKP content confirmed that 2,6-dimethoxybenzoquinone can damage bacterial cell walls. Crystal violet staining experiments showed that this substance can inhibit and scavenge bacterial biofilms, and both inhibitory and scavenging abilities increased with increasing concentration. Agarose gel electrophoresis results showed that 2,6-dimethoxybenzoquinone does not exert its antibacterial effect by directly binding to bacterial genomic DNA. Furthermore, this substance can dose-dependently reduce bacterial cell metabolic activity, induce oxidative damage, and inhibit SDH activity. Attached Figure Description

[0021] Figure 1 Flowchart for the separation of the main antibacterial component from the chloroform extract of Pyrus pyrifolia root;

[0022] Figure 2 The results of column chromatography-dispersive TLC analysis of the chloroform extract of Pyrus pyrifolia root;

[0023] Figure 3 The results are from TLC analysis of the Fr.2-6 column chromatography.

[0024] Figure 4 The results are from TLC analysis of the Fr.2-7 column chromatography.

[0025] Figure 5The figures show the total ion chromatograms by UPLC-MS. A represents the TIC chromatogram of the combined components of Fr.2-6 in negative ion mode; B represents the TIC chromatogram of the combined components of Fr.2-6 in positive ion mode; C represents the TIC chromatogram of the combined components of Fr.2-7 in negative ion mode; and D represents the TIC chromatogram of the combined components of Fr.2-7 in positive ion mode.

[0026] Figure 6 The chromatograms of the standard and the chloroform extract of Pyrus pyrifolia root are shown in the liquid chromatography.

[0027] Figure 7 The effect of 2,6-dimethoxybenzoquinone on the growth curves of Staphylococcus aureus (A) and Escherichia coli (B);

[0028] Figure 8 Changes in the extracellular relative conductivity of Staphylococcus aureus (A) and Escherichia coli (B) after treatment with 2,6-dimethoxybenzoquinone;

[0029] Figure 9 Changes in extracellular nucleic acid content of Staphylococcus aureus (A) and Escherichia coli (B) after treatment with 2,6-dimethoxybenzoquinone;

[0030] Figure 10 The extracellular alkaline phosphatase activity of Staphylococcus aureus (A) and Escherichia coli (B) after treatment with 2,6-dimethoxybenzoquinone;

[0031] Figure 11 The effect of 2,6-dimethoxybenzoquinone on biofilm formation in Staphylococcus aureus (A) and Escherichia coli (B);

[0032] Figure 12 The effect of 2,6-dimethoxybenzoquinone on the removal of biofilms from Staphylococcus aureus (A) and Escherichia coli (B);

[0033] Figure 13 The effect of 2,6-dimethoxybenzoquinone on the genomic DNA of Staphylococcus aureus (left) and Escherichia coli (right);

[0034] Figure 14 The effect of 2,6-dimethoxybenzoquinone on the metabolic activity of Staphylococcus aureus (A) and Escherichia coli (B) cells;

[0035] Figure 15 The effect of 2,6-dimethoxybenzoquinone on intracellular ROS in Staphylococcus aureus (A) and Escherichia coli (B);

[0036] Figure 16The effect of 2,6-dimethoxybenzoquinone on the SDH activity of Staphylococcus aureus (A) and Escherichia coli (B). Detailed Implementation

[0037] Example 1

[0038] Extraction of the chloroform portion of pear root

[0039] The following steps were followed to extract 2 kg of *Pyrus pyrifolia* root. The chloroform extract was concentrated under reduced pressure using a rotary evaporator to obtain 7.2157 g of *Pyrus pyrifolia* root chloroform extract.

[0040] The dried plant sample was pulverized using a high-speed grinder and passed through a 40-mesh sieve. 20 g of plant powder was weighed and placed in an Erlenmeyer flask, 200 mL of 80% ethanol solution was added, and the mixture was shaken thoroughly. Ultrasonic extraction (40 kHz, 99%) was performed for 60 min, followed by filtration under reduced pressure. The residue was collected, and the extraction was repeated three times under the same conditions. The filtrates were combined and concentrated to 100 mL under reduced pressure using a rotary evaporator. The mixture was then extracted twice each with 100 mL of petroleum ether (60–90 °C), chloroform, ethyl acetate, and water-saturated n-butanol, and the two extracts were combined. The four organic phases and the aqueous phase after extraction were rotary evaporated to complete dryness. The weight of the obtained extract was weighed, and it was dissolved in dimethyl sulfoxide to a concentration of 200 mg / mL. The solution was collected in a brown reagent bottle to obtain stock solutions of extracts from the polar fractions of the plant.

[0041] Chloroform (trichloromethane extract, TE), as a weakly polar solvent, is mainly used to extract steroids, free alkaloids, and certain aglycones. The chloroform extract of *Pyrus pyrifolia* root showed an inhibition rate exceeding 80% against both Gram-positive and Gram-negative bacteria. The minimum inhibitory concentrations (MICs) of the petroleum ether, chloroform, and ethyl acetate extracts of *Pyrus pyrifolia* root against *Escherichia coli* were 4 mg / L, 4 mg / L, and 4 mg / L, respectively; the MICs against *Salmonella typhimurium* were 4 mg / L, 8 mg / L, and 8 mg / L, respectively; and the MICs against *Staphylococcus aureus* were 4 mg / L, 8 mg / L, and 4 mg / L, respectively. These results indicate that *Pyrus pyrifolia* root extract possesses broad-spectrum antibacterial activity, with the chloroform extract showing the best effect. Therefore, the chloroform extract of *Pyrus pyrifolia* root was ultimately selected.

[0042] Example 2

[0043] The separation flow chart of the main antibacterial component of the chloroform extract of Pyrus pyrifolia root is as follows: Figure 1 As shown:

[0044] The active components of *Pyrus pyrifolia* root were separated using a method that tracks antibacterial activity. First, TE from *Pyrus pyrifolia* root was initially separated using 100-200 mesh silica gel, followed by gradient elution using a petroleum ether-ethyl acetate-methanol system. The experiment employed wet column packing and dry sample loading, with the specific steps as follows:

[0045] Column packing: Weigh 150 g of 100-200 mesh silica gel into a beaker, add an appropriate amount of petroleum ether, and mix thoroughly to form a slurry. Slowly pour the slurry into a 50×400 mm glass chromatography column, gently tapping the column to allow it to settle evenly. After the silica gel has completely settled, keep the liquid level slightly above the silica gel layer.

[0046] Sample loading: Take 7.2157 g of *Pyrus pyrifolia* root TE sample, dissolve it in 2 mL of chloroform by sonication, and then mix it thoroughly with 100-200 mesh silica gel at a 1:1 mass ratio. Evaporate the solvent in a 40 °C water bath to obtain the mixed silica gel. Add the treated sample evenly to the top of the column and cover with a thin layer of quartz sand for protection.

[0047] Elution: Gradient elution was performed sequentially with petroleum ether:ethyl acetate (V / V) = 50:1, 25:1, 10:1, 1:1, 1:5, 1:10, 1:25, 0:1 and ethyl acetate:methanol (V / V) = 20:1, 10:1, 1:1, 0:1. The volume of each eluent ratio was 300 mL, and each 100 mL fraction was collected as a fraction.

[0048] Thin-layer chromatography (TLC) detection: The eluted fractions were detected using TLC. Petroleum ether, ethyl acetate, and methanol were prepared into mixed solvents with varying proportions according to the eluent ratio, and gradient development was performed using the developing solvents in ascending polarity. Specifically, a less polar mixed solvent was first used for the initial development of the eluted fractions. For fractions that did not show spots on the TLC plate under these developing solvent conditions, a more polar developing solvent was used for the second development. This process was repeated until all components in the fractions could be well separated and detected on the TLC plate. When the developing solvent migrated to 1 cm from the top of the TLC plate, it was removed, the solvent was evaporated, and the spot distribution was observed under a 254 nm or 365 nm UV lamp. Fractions with similar migration values ​​under the same developing solvent conditions were combined.

[0049] Antibacterial activity assay of eluted fractions: The combined fractions were rotary evaporated until completely dry, the weight of the obtained components was weighed, and a 100 mg / mL component stock solution was prepared by dissolving in methanol and the antibacterial rate was determined. The yield of each component was calculated according to formula (1):

[0050] Yield (%) = ×100% (1)

[0051] In the formula, The mass (g) of each component The TE weight of *Pyrus pyrifolia* root is 7.2157 g.

[0052] Component 2-6 silica gel column chromatography separation and antibacterial activity detection

[0053] The active component Fr.2-6 was separated twice using a 200-300 mesh silica gel and a 30×400 mm chromatography column. Silica gel was weighed at a Fr.2-6 to silica gel mass ratio of 1:100 and packed using a wet packing method. The packing and loading methods were the same as in 3.1.5.1, with a silica gel to Fr.2-6 mass ratio of 1:1. After loading, a gradient elution was performed sequentially with a mixture of petroleum ether:ethyl acetate (V / V) = 1:0, 10:1, 8:1, 6:1, 4:1, 3:1, 2.5:1, 2:1, 1:1, 1:2, 1:4, 1:10, and 0:1, and methanol. Each eluent volume was 60 mL, and every 30 mL was collected as a fraction. The combined fractions were rotary evaporated to dryness and then dissolved in methanol to prepare a 100 mg / mL stock solution. Based on the yield ratio of each component in Fr.2-6, the solution was diluted with LB liquid medium to the corresponding concentration working solution (with 2 mg / mL as the 100% relative concentration standard) for the determination of the antibacterial rate. The yield of each component was calculated according to formula (2):

[0054] Yield (%) = ×100% (2)

[0055] In the formula, The mass (g) of each component The mass of components 2-6 is 0.4829 g.

[0056] Components 2-7 were separated by silica gel column chromatography and their antibacterial activity was detected.

[0057] After sample loading, gradient elution was performed sequentially using petroleum ether:ethyl acetate (V / V) ratios of 5:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:7, 1:10, 1:15, 1:20, 1:50 and ethyl acetate:methanol (V / V) ratios of 50:1, 10:1, 3:1, 1:1, 1:3, 1:10. Each eluent ratio had a volume of 120 mL, and each 40 mL fraction was collected as a separate fraction. The yield of each component was calculated according to formula (3).

[0058] Yield (%) = ×100% (3)

[0059] In the formula, The mass (g) of each component The mass of components 2-7 is 0.5735 g.

[0060] Experimental results:

[0061] The chloroform extract of *Pyrus pyrifolia* root was eluted by 100-200 mesh silica gel column chromatography using a petroleum ether-ethyl acetate-methanol solvent system, yielding 37 fractions. These 37 fractions were then spotted onto a silica gel plate for thin-layer chromatography, developed in a thin-layer developing tank, and finally visualized using a thin-layer chromatography imaging system. The results are shown below. Figure 2 As shown, fractions with similar specific migration values ​​under the same developing solvent conditions were combined to obtain 18 components.

[0062] Table 1. Antibacterial rate of column chromatography fractions from chloroform extract of Pyrus pyrifolia root.

[0063]

[0064] As shown in Table 1, the 18 obtained components were numbered, their separation yields were calculated, and their antibacterial activities were determined. The results showed that most components exhibited antibacterial effects against *Escherichia coli*, but there were significant differences among the components. Only Fr.2-6 and Fr.2-7 showed antibacterial rates exceeding 60%, with Fr.2-7 exhibiting the highest antibacterial activity at 93.31±1.22%. For *Salmonella typhimurium*, the overall antibacterial effect of all components was not as good as against *Escherichia coli*, with only Fr.2-6 and Fr.2-7 showing relatively high antibacterial rates of 62.39±3.09% and 72.84±3.41%, respectively. For *Bacillus cereus* and *Staphylococcus aureus*, the antibacterial activity of all components was higher than that against Gram-negative bacteria, with Fr.2-6, Fr.2-7, Fr.2-8, Fr.2-9, and Fr.2-10 all showing antibacterial rates above 90%. Overall, there is no necessary correlation between the isolation yield and the antibacterial rate. For example, the yield of Fr.2-16 was 31.75%, but its antibacterial rate against all four bacteria was less than 40%; the isolation yield of Fr.2-13 was 9.71%, with inhibition rates against Gram-negative bacteria all below 10% and inhibition rates against Gram-positive bacteria all below 50%. Furthermore, the antibacterial activity of the components from the early and late stages of isolation against the four strains was lower than that of the components from the middle stage, and the yield of the components from the early stage was generally lower. This may be because the less polar eluent used in the early stage has poor solubility for the chloroform extract components, while the eluent used in the middle stage has a similar polarity to chloroform, which can better dissolve the antibacterial active components that were originally soluble in chloroform, allowing these active components to be successfully eluted from the column chromatography packing material, thus ensuring the effective collection of active components. In contrast, the excessively polar eluent used in the later stage may dissolve some non-target chemical components that do not possess antibacterial activity, thereby affecting the antibacterial activity of the components. Taking all factors into consideration, in order to obtain a plant-derived antibacterial agent that exhibits good antibacterial activity against all four tested bacteria, Fr.2-6 and Fr.2-7 were selected for the next step of isolation.

[0065] Components 2-6 were separated by silica gel column chromatography and their antibacterial activity was detected.

[0066] Fr.2-6 was eluted with a mixture of petroleum ether:ethyl acetate (V / V) ratios of 1:0, 10:1, 8:1, 6:1, 4:1, 3:1, 2.5:1, 2:1, 1:1, 1:2, 1:4, 1:10, and 0:1, along with methanol. The mixture was eluted using a 200-300 mesh silica gel column, yielding 28 fractions. These 28 fractions were then spotted onto a thin-layer chromatography silica gel plate, developed in a thin-layer developing tank, and finally visualized using a thin-layer chromatography imaging system. The results are shown below. Figure 3 As shown. Fractions with similar specific migration values ​​under the same developing solvent conditions were combined to obtain 15 components.

[0067] As shown in Table 2, the 15 obtained components were numbered, their separation yields were calculated, and their antibacterial activity was determined. The results showed that, compared with other components, components Fr.2-6-9, Fr.2-6-10, Fr.2-6-11, and Fr.2-6-15 all exhibited antibacterial activity against *Escherichia coli*, *Salmonella typhimurium*, *Bacillus cereus*, and *Staphylococcus aureus*, indicating that the main antibacterial components were concentrated in these components. Therefore, to comprehensively analyze their main antibacterial active components, the above four components were combined and analyzed by UPLC-MS.

[0068] Table 2. Antibacterial rates of Fr.2-6 column chromatography components

[0069]

[0070] Components 2-7 were separated by silica gel column chromatography and their antibacterial activity was detected.

[0071] Fr.2-7 used a mixture of petroleum ether:ethyl acetate (V / V) ratios of 5:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:7, 1:10, 1:15, 1:20, 1:50 and ethyl acetate:methanol (V / V) ratios of 50:1, 10:1, 3:1, 1:1, 1:3, 1:10 as eluents. The mixture was eluted by silica gel column chromatography (200-300 mesh), yielding 51 fractions. These 51 fractions were then spotted onto a silica gel plate for thin-layer chromatography (TLC), developed in a TLC developing tank, and finally visualized using a TLC imaging system. The results are shown below. Figure 4 As shown in the figure. Fractions with similar specific migration values ​​under the same developing solvent conditions were combined to obtain 22 components.

[0072] As shown in Table 3, the 22 obtained components were numbered, their separation yields were calculated, and their antibacterial activity was determined. The results showed that, compared with other components, Fr.2-7-5, Fr.2-7-6, and Fr.2-7-7 all exhibited antibacterial activity against *Escherichia coli*, *Salmonella typhimurium*, *Bacillus cereus*, and *Staphylococcus aureus*, indicating that the main antibacterial components were concentrated in these components. Therefore, to comprehensively analyze their main antibacterial active components, the above three components were combined and analyzed by UPLC-MS.

[0073] Table 3 Antibacterial rates of Fr.2-7 column chromatography components

[0074]

[0075] Example 3

[0076] Ultra-high performance liquid chromatography-mass spectrometry analysis

[0077] Chromatographic conditions

[0078] Chromatographic separation was performed using an EXION LC system (SCIEX) ultra-high performance liquid chromatograph via a UPLC Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm). Mobile phase A was 0.01% aqueous acetic acid, and mobile phase B was 50% acetonitrile / isopropanol solution. The column temperature was set to 25 °C, the autosampler temperature to 4 °C, the injection volume to 2 μL, and the flow rate to 0.3 mL / min.

[0079] Mass spectrometry conditions

[0080] A SCIEX 6500 QTRAP+ triple quadrupole mass spectrometer equipped with an IonDrive Turbo V ESI ion source was used, employing electrospray ionization (ESI) with switching between positive and negative ion scanning modes. Data acquisition was performed in multiple reaction monitoring (MRM) mode. Specific parameters were as follows: ion spray voltage of 5500 V (positive ion) or -4500 V (negative ion), curtain gas pressure of 35 psi, ion source temperature of 400 °C, and pressures of both nebulizer gas and auxiliary gas of 50 psi.

[0081] Calculation of relative content of compounds

[0082] The relative contents of each compound were calculated using the peak area normalization method according to formula (4):

[0083] Relative content (%) = ×100% (4)

[0084] Experimental results:

[0085] Table 4 UPLC-MS Analysis Results

[0086]

[0087] The collected optimal antibacterial components were analyzed by UPLC-MS, and the total ion chromatogram is shown below. Figure 5 The relative contents of compounds obtained from mass spectrometry qualitative analysis were calculated using the peak area normalization method. Compounds with relative contents greater than 1% in Fr.2-6 and Fr.2-7 include 2-methylcitric acid, coniferaldehyde, 2,6-dimethoxybenzoquinone, vanillin, benzoic acid, 9-hydroxy-10,12-dienoic acid, and 4-hydroxybenzaldehyde. Considering that compounds with relative contents less than 1% may contribute limitedly to the overall antibacterial activity, these compounds were not included in this analysis to improve research efficiency and highlight key research areas.

[0088] Overall, these compounds encompass various types, including organic acids, aldehydes, phenols, and amides. Among them, eugenol exerts its antibacterial effect by inhibiting the bacterial secretion system, while sinigral inhibits cytoplasmic membrane ATPases and disrupts bacterial cell wall and cell membrane structures. Vanillin, salicylic acid, and benzoic acid primarily inhibit bacterial growth by damaging cell membrane integrity and interfering with energy metabolism pathways. Furthermore, 4-hydroxybenzaldehyde and sennaol have been shown to effectively inhibit bacterial biofilm formation. Thistlein reduces intracellular ATP levels by inhibiting ATP synthases.

[0089] Based on the above understanding of the antibacterial mechanisms of various compounds, in order to further study the main antibacterial active ingredients of the chloroform extract of Pyrus pyrifolia root and promote the development of novel plant-derived antibacterial agents, this invention selects the monomeric compounds in Table 4 that are stably obtainable and whose mechanisms of action are not yet clear, determines their minimum inhibitory concentration, and studies their antibacterial mechanisms.

[0090] Example 4

[0091] Determination of the minimum inhibitory concentration (MIC) of a monomeric compound.

[0092] The minimum inhibitory concentration (MIC) of the compound was determined using the microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI). The compound was dissolved in DMSO to a concentration of 100 mg / mL. In sterile 96-well plates, 100 μL of the compound solution diluted with LB liquid medium (final concentration gradients of 4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, and 0.03125 mg / mL) and 100 μL of a solution with a final concentration of 1×10⁻⁶ mg / mL were added, respectively. 5 CFU / mL bacterial suspension. Three replicates and control wells (compound-free, sterile, cinnamaldehyde-free) were included for each batch. The 96-well plates were incubated at 37 °C and 200 r / min for 24 h with shaking. OD values ​​before and after incubation were measured using a UV-Vis spectrophotometer. 600 Changes, of which no significant changes (△OD) 600 The lowest concentration (<0.05) is the MIC value of the compound for the test strain.

[0093] Table 5 shows the MICs of the obtained, stably obtainable monomeric compounds with unclear antibacterial mechanisms against the four tested bacteria. Cinnamaldehyde, a known plant-derived preservative explicitly permitted for use under GB 2760, was also determined under the same conditions for comparative analysis. Under the experimental conditions, the MICs of cinnamaldehyde against *Escherichia coli*, *Salmonella typhimurium*, *Bacillus cereus*, and *Staphylococcus aureus* were 0.5 mg / mL, 0.5 mg / mL, 0.5 mg / mL, and 1 mg / mL, respectively. Among the 13 monomeric compounds tested, 2,6-dimethoxybenzoquinone exhibited the strongest antibacterial effect, with MICs of 0.125 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, and 0.0625 mg / mL against the above four bacteria, all lower than that of cinnamaldehyde. Although 2-methylcitric acid had the highest relative content in Fr.2-6, reaching 9.21%, its MIC was not detected in this experiment. The MICs of coniferaldehyde, 4-hydroxy-3-(3-methylbut-2-enyl)benzoic acid, and ethyl 3,4-dihydroxybenzoate against two Gram-positive bacteria were all 2 mg / mL; cinnamamide showed an MIC of 4 mg / mL against only two Gram-negative bacteria; hesperidin had an MIC of 1 mg / mL against two Gram-negative bacteria and Bacillus cereus; 3,4-dihydrocoumarin had an MIC of 4 mg / mL against four bacteria; and the remaining compounds did not show a detectable MIC.

[0094] Notably, 2,6-dimethoxybenzoquinone not only exhibited significant antibacterial activity against all four tested bacteria, but also showed relatively high concentrations in Fr.2-6 and Fr.2-7. Based on this, it is speculated to be a key antibacterial active ingredient in the chloroform extract of *Pyrus pyrifolia* root, and has value for further research as a potential plant-derived antibacterial agent.

[0095] Table 5 Minimum inhibitory concentrations of monomeric compounds

[0096]

[0097] Example 5

[0098] Identification of characteristic antibacterial components in pear root chloroform extract

[0099] 2,6-Dimethoxybenzoquinone standard and *Pyrus pyrifolia* root chloroform extract were dissolved in methanol to prepare solutions with concentrations of 0.5 mg / mL and 10 mg / mL, respectively. After filtration through a 0.22 μm organic filter membrane, the solutions were analyzed by high-performance liquid chromatography (HPLC). Chromatographic conditions: An Elite SinoChrom ODS-BP column (4.6 nm × 250 mm, 5.0 μm) was used; the mobile phase was methanol-5% acetic acid aqueous solution (24: 76, v / v); the detection wavelength was 289 nm; the column temperature was 25 °C; the flow rate was 1.0 mL / min; and the injection volume was 20 μL.

[0100] Identification of characteristic antibacterial components in the chloroform extract of Pyrus pyrifolia root:

[0101] The component analysis of the above-mentioned *Pyrus pyrifolia* root chloroform extract revealed that 2,6-dimethoxybenzoquinone is the key antibacterial active ingredient in the extract. For further comparison and identification, 2,6-dimethoxybenzoquinone standard and *Pyrus pyrifolia* root chloroform extract were subjected to high-performance liquid chromatography (HPLC) detection. Figure 6 ).

[0102] Chromatographic results showed that the retention time of the 2,6-dimethoxybenzoquinone standard was 8.45 min. Comparison revealed that the chloroform extract of *Pyrus pyrifolia* root exhibited a characteristic peak matching that of the standard at the same retention time, preliminarily identifying them as the same substance. Previous studies have shown that 2,6-dimethoxybenzoquinone exists in wheat germ, red wine, and *Ficus lyrata*. Current research on this compound mainly focuses on its antioxidant and anticancer activities, while research on its antibacterial mechanism remains lacking.

[0103] This invention employs an activity-tracking method to repeatedly perform column chromatography on the chloroform extract of *Pyrus pyrifolia* root to separate its antibacterial active components, and then uses ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) to identify the main substances. Subsequently, the minimum inhibitory concentration (MIC) of monomeric compounds with high content, stable availability, and unclear antibacterial mechanisms was determined to identify the main antibacterial active components of the *Pyrus pyrifolia* root chloroform extract, providing a theoretical basis for the development of novel plant-derived antibacterial agents. Specific research results are as follows:

[0104] (1) Separation of antibacterial active substances by silica gel column chromatography and detection of antibacterial activity: In Fr.2-6, the four components Fr.2-6-9, Fr.2-6-10, Fr.2-6-11 and Fr.2-6-15 showed major antibacterial effects against Escherichia coli, Salmonella typhimurium, Bacillus cereus and Staphylococcus aureus. Therefore, these four components were combined for UPLC-MS analysis. In Fr.2-7, the three components Fr.2-7-5, Fr.2-7-6 and Fr.2-7-7 showed major antibacterial effects against the above four bacteria. Therefore, these three components were combined for UPLC-MS analysis.

[0105] (2) Ultra-high performance liquid chromatography-mass spectrometry analysis: Through analysis of the above combined components, a total of 27 compounds with relative contents higher than 1% were identified. Among them, 2-methylcitric acid, coniferaldehyde, 2,6-dimethoxybenzoquinone, cinnamamide, 5,7-dihydroxychromone, 4-hydroxy-3-(3-methylbut-2-enyl)benzoic acid, ryegrass lactone, vanillic acid, ethyl 3,4-dihydroxybenzoate, hesperidin, indole-3-carboxaldehyde, naringenin chalcone, and 3,4-dihydrocoumarin were not only stable to obtain, but their antibacterial mechanisms were not yet clear. Therefore, these monomeric compounds were selected for the determination of minimum inhibitory concentration.

[0106] (3) Determination of the minimum inhibitory concentration (MIC) of the monomeric compound: 2,6-dimethoxybenzoquinone showed the most significant inhibitory effects against *Escherichia coli*, *Salmonella typhimurium*, *Bacillus cereus*, and *Staphylococcus aureus*, with MIC values ​​of 0.125, 0.125, 0.0625, and 0.0625 mg / mL, respectively. Meanwhile, the relative content of 2,6-dimethoxybenzoquinone in the extract was relatively high. Therefore, it is inferred that 2,6-dimethoxybenzoquinone is a key antibacterial active ingredient in the chloroform extract of *Pyrus pyrifolia* root and has the potential to be further studied as a potential antibacterial agent.

[0107] (4) Identification of characteristic antibacterial components in the chloroform extract of *Pyrus pyrifolia* root: Further comparative identification was performed using 2,6-dimethoxybenzoquinone standard. The comparison revealed that the chloroform extract of *Pyrus pyrifolia* root exhibited characteristic peaks matching those of the standard at the same retention time, confirming them as the same substance. Currently, research on the antibacterial mechanism of 2,6-dimethoxybenzoquinone remains incomplete.

[0108] Example 6

[0109] Experimental strains: Staphylococcus aureus (ATCC 6538) was purchased from Beijing Baocang Biotechnology Co., Ltd., and Escherichia coli (ATCC 25922) was purchased from Beijing Baocang Biotechnology Co., Ltd.

[0110] Using an inoculation loop, streak the bacteria onto LB solid medium and incubate at 37 °C for 12 h. Pick a single colony and inoculate it into LB liquid medium, then incubate at 37 °C and 200 rpm for 10–14 h until OD (dose retardation) is reached. 600 The concentration should be around 0.8, and diluted as needed for the experiment.

[0111] Effect on bacterial growth curves:

[0112] The inhibitory effect of 2,6-dimethoxybenzoquinone on the tested strains was evaluated using the growth curve method. A 1 L Erlenmeyer flask was used, and 200 mL of LB liquid medium diluted to a concentration of 1×10⁻⁶ was added. 5 CFU / mL bacterial suspensions were added with 2,6-dimethoxybenzoquinone to final concentrations of 1 / 2 MIC, 1 MIC, and 2 MIC, respectively, with a control containing no compound. The suspensions were treated with shaking at 37 °C and 200 r / min. 2 mL samples were taken every 2 h, and the absorbance (OD) was measured at 600 nm. 600 Plot the time-growth curve.

[0113] At a wavelength of 600 nm, the absorbance of a bacterial suspension is directly proportional to its concentration; therefore, by measuring the OD... 600 To determine the effect of 2,6-dimethoxybenzoquinone on the growth of Staphylococcus aureus and Escherichia coli. Figure 7 As shown, the control group of Staphylococcus aureus ( Figure 7 A) and Escherichia coli ( Figure 7 B) Both bacteria exhibited typical S-shaped growth curves. *Staphylococcus aureus* entered the logarithmic growth phase 6 hours after inoculation and reached the stationary phase after 18 hours. *Escherichia coli* grew even faster, entering the logarithmic growth phase at 4 hours and reaching the stationary phase after 12 hours. The inhibitory effect of 2,6-dimethoxybenzoquinone on both tested bacteria was mainly manifested in delaying their logarithmic growth phase. *Staphylococcus aureus* in the 1 / 2 MIC treatment group showed growth inhibition until 22 hours before entering the logarithmic growth phase; *Escherichia coli* in the 1 / 2 MIC treatment group was inhibited in the first 18 hours of culture, then rapidly increased, with a higher proliferation rate in the later stages than *Staphylococcus aureus*. When the concentration of 2,6-dimethoxybenzoquinone was 1 MIC and 2 MIC, the OD values ​​of the bacterial suspensions for both bacteria were... 600The levels remained consistently low, with almost no growth, indicating that 2,6-dimethoxybenzoquinone at this treatment concentration had a strong inhibitory effect on the growth of both bacteria, effectively preventing bacterial increase and essentially suppressing bacterial growth and reproduction. Overall, the higher the concentration of 2,6-dimethoxybenzoquinone, the more pronounced the inhibitory effect on the two bacteria. Particularly under treatment conditions of 2 MIC and 1 MIC, this compound effectively inhibited the growth and reproduction of the two tested bacteria, demonstrating good potential for antibacterial applications.

[0114] Example 7

[0115] Effects on bacterial cell membrane permeability:

[0116] Take the bacterial suspension described above and centrifuge at 4 °C and 5000 r / min for 10 min, discarding the supernatant. Wash the bacterial cells repeatedly with sterile physiological saline until the conductivity remains almost constant. Then, resuspend the bacterial cells in 20 mL of sterile physiological saline and adjust the bacterial concentration to 1 × 10⁻⁶. 8 CFU / mL was added, and 2,6-dimethoxybenzoquinone was added to make the final concentrations 1 / 2 MIC, 1 MIC and 2 MIC, respectively. Each group had three replicates and a control without the compound. The suspension was shaken at 37 °C and 200 r / min, and the suspensions were taken out at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h and 7 h, respectively. The conductivity of the bacterial cells in the suspension was measured using a conductivity meter. Finally, the bacterial suspension was boiled, cooled and the final conductivity was measured. The relative conductivity was calculated according to formula (5):

[0117] Relative conductivity (%) = ×100% (5)

[0118] In the formula, L0 is the initial conductivity, L1 is the conductivity measured at each time point, and L2 is the final conductivity after boiling.

[0119] Effects on bacterial cell membrane integrity:

[0120] Take the bacterial suspension described above, centrifuge at 4 °C and 5000 r / min for 10 min, discard the supernatant, and wash the bacterial cells three times with sterile PBS. Then, resuspend the bacterial cells in 50 mL of sterile PBS and adjust the bacterial concentration to 1 × 10⁻⁶. 8CFU / mL was added, and 2,6-dimethoxybenzoquinone was added to achieve final concentrations of 1 / 2 MIC, 1 MIC, and 2 MIC, respectively. Each group had three replicates and a control without the compound. The mixture was treated with shaking at 37 °C and 200 r / min. At 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, and 7 h, 1 mL of bacterial culture was collected and centrifuged at 4 °C and 5000 r / min for 10 min. The supernatant was collected, and the absorbance (OD) was measured at 260 nm. 260 ).

[0121] When bacteria are exposed to antimicrobial substances, their cell membrane structure is damaged, resulting in a loss of their original protective capabilities. This damage causes intracellular charged ions and other small molecules to leak out, leading to an increase in the conductivity of the bacterial suspension. Therefore, by measuring changes in the relative conductivity of the bacterial suspension, the effect of antimicrobial substances on bacterial cell membrane permeability can be indirectly assessed. Figure 8 This shows the change in the relative conductivity of Staphylococcus aureus and Escherichia coli after treatment with 2,6-dimethoxybenzoquinone compared to the control group. In the control group, Staphylococcus aureus (… Figure 8 The relative conductivity of A) increased continuously in the first 4 hours, and remained relatively stable from 4 to 7 hours; Escherichia coli ( Figure 8 B) The relative conductivity of the control group increased continuously in the first 2 hours, then stabilized between 2 and 7 hours. This may be due to normal cell lysis and death, leading to an increase in relative conductivity. For the experimental group, after treatment with 1 / 2 MIC, 1 MIC, and 2 MIC concentrations of 2,6-dimethoxybenzoquinone, the relative conductivity of the two tested bacteria overlapped over time, with no significant difference for most of the time. Even so, when the concentrations of 2,6-dimethoxybenzoquinone were 1 / 2 MIC, 1 MIC, and 2 MIC, the relative conductivity of both tested bacteria was higher than that of the control group with increasing time. Although the relative conductivity decreased at some time points, it still showed an overall upward trend. Previous studies on the antibacterial mechanism of limonene against Pseudomonas aeruginosa and the antibacterial mechanism of gingerol against Bacillus cereus have also shown similar results. The above results indicate that 2,6-dimethoxybenzoquinone can increase the cell membrane permeability of the two tested bacteria, leading to leakage of intracellular components and thus exerting an antibacterial effect.

[0122] The integrity and permeability of the cell membrane are fundamental to bacterial life processes. When antibacterial substances act on bacteria, the bacterial cell membrane may be damaged, causing large molecules (such as nucleic acids) to leak into the bacterial suspension. Since nucleic acids have absorbance at 260 nm, they can be used as an indicator of bacterial cell membrane integrity. Figure 9As shown, for Staphylococcus aureus ( Figure 9 A) The extracellular nucleic acid leakage was highest in the 2 MIC treatment group at all time points, followed by the 1 MIC treatment group, and relatively lower in the 1 / 2 MIC treatment group, but the extracellular nucleic acid content in all three treatment groups was higher than that in the control group. This indicates that the higher the concentration of 2,6-dimethoxybenzoquinone, the more intracellular nucleic acid of Staphylococcus aureus leaks to the extracellular space. With increasing concentration, the damaging effect of 2,6-dimethoxybenzoquinone on bacterial cells is enhanced, disrupting the integrity of the cell membrane and thus causing more nucleic acid leakage. During the treatment period of 2-4 h, the extracellular nucleic acid content in each treatment group increased rapidly, and then the upward trend slowed down. This indicates that in the early stage of treatment, 2,6-dimethoxybenzoquinone can act on the cell membrane of Staphylococcus aureus relatively quickly, promoting nucleic acid leakage, and the leakage rate slows down with time. For Escherichia coli ( Figure 9 B) The concentration of 2,6-dimethoxybenzoquinone was also positively correlated with the amount of extracellular nucleic acid leakage; that is, the higher the concentration, the stronger the destructive effect on the *E. coli* cell membrane and the greater the amount of nucleic acid leakage. After 2 hours of treatment, the amount of nucleic acid leakage increased significantly, and continued to increase with the extension of treatment time and the increase of treatment concentration, and was significantly higher than that of the control group. Overall, 2,6-dimethoxybenzoquinone had a destructive effect on the cell membranes of both tested bacteria, leading to nucleic acid leakage. Moreover, the higher the concentration and the longer the treatment time, the more significant the destructive effect. This corresponds to the results of the previous study on the effect of this substance on bacterial cell membrane permeability, further proving that 2,6-dimethoxybenzoquinone can damage bacterial cell membranes and cause intracellular substances to leak out.

[0123] Example 8

[0124] Effects on bacterial cell walls

[0125] Take the above bacterial suspension, centrifuge at 4 °C and 5000 r / min for 10 min, discard the supernatant, and resuspend the bacterial cells in 3 mL of sterile physiological saline. Then, add the bacterial suspension to 50 mL of LB liquid medium to adjust the bacterial concentration to 1 × 10⁻⁶. 8CFU / mL was added, and 2,6-dimethoxybenzoquinone was added to achieve final concentrations of 1 / 2 MIC, 1 MIC, and 2 MIC, respectively. Three replicates and a control without the compound were included for each group. The mixture was shaken at 37 °C and 200 r / min, and 1 mL samples were taken at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, and 7 h. The samples were centrifuged at 4 °C and 5000 r / min for 10 min, and the supernatant was collected as the test solution. In each well of a 96-well plate, 100 μL of the test solution, 50 μL of 2 mmol / L pNPP solution, and 50 μL of DEA buffer were added, mixed well, and incubated at 37 °C for 35 min. After the reaction, 100 μL of the reaction solution was transferred from each well to a new 96-well plate, and an equal volume of 6 mol / L NaOH solution was added to terminate the reaction. The absorbance was then measured at 405 nm. The pNP concentration in the test solution was calculated based on the pNP standard curve. The enzyme activity of generating 1 μmol of pNP within 1 min is defined as 1 U. The AKP activity is calculated according to formula (6):

[0126] AKP activity (U) = (6)

[0127] In the formula, C pNP The concentration of p-nitrophenol (pNP) calculated for the standard curve. The total volume of the reaction system (50 mL) T represents the sample volume (0.1 mL) and T represents the reaction time (35 min).

[0128] Establishment of the pNP standard curve: Weigh 25 mg of pNP, dissolve it in 80 mL of distilled water by sonication, and finally bring the volume to 100 mL to prepare a pNP stock solution with a concentration of 0.25 mg / mL. Pipette 0 μL, 3.75 μL, 7.5 μL, 15 μL, 30 μL, 60 μL, and 120 μL of pNP stock solution into 10 mL stoppered centrifuge tubes, respectively, and add distilled water to a final volume of 2 mL. Add 100 μL of solution from each centrifuge tube to a 96-well plate, then add an equal volume of 6 mol / L NaOH solution, mix well, and measure the absorbance at 405 nm. The standard curve is established as: y = 2.0786x + 0.0443, R0 2 =0.9981, where x is the pNP concentration (mg / mL) and y is the absorbance.

[0129] Alkaline phosphatase (AKP) is a non-specific phospholipase primarily found in the periplasmic space between the bacterial cell wall and cell membrane. Under normal circumstances, it does not permeate into the extracellular space. However, when the cell wall structure is damaged, large amounts of AKP leak out. Therefore, it can serve as an indicator of cell wall integrity. Figure 10 As shown, the extracellular AKP activity of the control groups of the two tested bacteria only increased slightly and remained essentially unchanged. Compared with the cell membrane damage observed in the control group in the previous study, this may be because when bacteria are in LB liquid medium, they can obtain the nutrients needed to repair the cell wall, such as specific enzymes and peptidoglycan precursors, thereby repairing the cell wall. In the treatment groups, the AKP activity in the 2 MIC treatment group was generally the highest at all time points, followed by the 1 MIC treatment group, and the 1 / 2 MIC treatment group was relatively lower, but all were higher than the control group. This indicates that the higher the concentration of 2,6-dimethoxybenzoquinone, the greater the amount of intracellular AKP leaked extracellularly into the two tested bacteria. For Staphylococcus aureus (… Figure 10 A), after 2 hours of treatment at each concentration, AKP leakage began to increase significantly; for Escherichia coli ( Figure 10 (B) At treatment concentrations of 1 / 2 MIC and 1 MIC, AKP leakage began to increase significantly after 2 hours, while at a 2 MIC concentration, AKP leakage began to increase significantly after 1 hour. Combined with previous studies, this further demonstrates that 2,6-dimethoxybenzoquinone can disrupt the integrity of bacterial cell walls. Cell wall formation is a crucial step in maintaining cell morphology during bacterial growth. It provides rigid support for the cell, protecting bacteria from external damage. Furthermore, studies have found that the lipopolysaccharide component unique to the cell walls of Gram-negative bacteria may be related to their pathogenicity. Therefore, cell wall biosynthesis is an important physiological process for bacterial survival and a significant target for antibacterial substances.

[0130] Example 9

[0131] Effects on bacterial biofilms

[0132] Determination of the ability to inhibit biofilm formation

[0133] Biomembrane quantification was performed using crystal violet staining. 200 μL of a 1×10⁻⁶ solution was added to each well of a 96-well plate. 5CFU / mL bacterial suspension was treated with 2,6-dimethoxybenzoquinone to final concentrations of 0 (control group), 1 / 8 MIC, 1 / 4 MIC, 1 / 2 MIC, 1 MIC, 2 MIC, and 4 MIC, with three replicates per group. After treatment with shaking at 37 °C and 200 r / min for 24 h, the culture medium was removed, and the biofilm was gently washed three times with PBS. Subsequently, the biofilm was fixed with 99% methanol solution, and after 15 min, the methanol was removed and the residual liquid was air-dried. 200 μL of 0.1% crystal violet solution was added to each well for staining for 15 min. After removing excess dye, the biofilm was slowly washed three times with PBS to remove residual crystal violet. After drying, 200 μL of 33% acetic acid solution was added to each well to dissolve the dye, and the resulting solution was transferred to a new 96-well plate. The absorbance was measured at 590 nm, and the biofilm formation inhibition rate was calculated according to formula (7):

[0134] Biofilm formation inhibition rate (%) = (7)

[0135] Determination of biofilm scavenging capacity

[0136] To induce biofilm formation, 200 μL of a 1×10⁻⁶ solution was added to each well of a 96-well plate. 5 CFU / mL bacterial suspension. Cultured with shaking at 37 °C and 200 r / min for 24 h. After biofilm formation, remove the culture medium and gently wash the biofilm three times with PBS, then allow to air dry at room temperature. Add 200 μL of 2,6-dimethoxybenzoquinone diluted with LB liquid medium to each well, resulting in final concentrations of 0 (control group), 1 / 8 MIC, 1 / 4 MIC, 1 / 2 MIC, 1 MIC, 2 MIC, and 4 MIC, with three replicates per group. The following steps are the same as those described in the biofilm formation inhibition assay. Calculate the biofilm clearance rate according to formula (8):

[0137] Biofilm removal rate (%) = (8)

[0138] Determination of the ability to inhibit biofilm formation

[0139] Bacterial biofilm formation is a dynamic and continuous process, primarily influenced by key factors such as the biofilm's three-dimensional structure, physiological function, and chemical composition. This study employed crystal violet staining for quantitative analysis of biofilms. This staining agent can stain biomolecules such as nucleic acids, proteins, and lipids within the biofilm. After washing away the crystal violet solution, the bacterial culture was dissolved in a solution of acetic acid of a specific concentration, and the absorbance value was used to determine the strength of the bacterial biofilm-forming ability. The experimental results are as follows: Figure 8As shown, 2,6-dimethoxybenzoquinone inhibited biofilm formation in both tested bacteria, and the inhibitory effect was positively correlated with concentration. For Staphylococcus aureus (… Figure 11 A) When the treatment concentration was below 1 MIC, the inhibition rate increased significantly with increasing concentration; when the treatment concentration was 1 MIC, the inhibition rate was 84.8 ± 0.6%, almost reaching the maximum inhibition rate, and thereafter, even with increasing concentration, the inhibition rate did not show a significant difference. 2,6-Dimethoxybenzoquinone against *Escherichia coli* (… Figure 11 B) The inhibitory effect on biofilm formation also exhibited the same characteristics, with an inhibition rate of 58.28 ± 0.92% at a treatment concentration of 1 MIC. At concentrations of 1 / 2 MIC and above, 2,6-dimethoxybenzoquinone showed stronger inhibitory activity against Staphylococcus aureus biofilms than against Escherichia coli. Overall, the inhibitory effect of 2,6-dimethoxybenzoquinone on biofilm formation may mainly be achieved through multiple pathways, including inhibiting the synthesis of extracellular matrix components, interfering with interbacterial signal transduction, and regulating the expression of biofilm-related genes. Furthermore, as discussed earlier, 2,6-dimethoxybenzoquinone can damage bacterial cell walls by causing the leakage of AKP enzymes. This process may release signaling molecules that regulate the expression of biofilm-related genes, ultimately leading to a reduction in biofilm formation.

[0140] Determination of biofilm scavenging capacity

[0141] The results of the scavenging effects of different concentrations of 2,6-dimethoxybenzoquinone on mature biofilms of Staphylococcus aureus and Escherichia coli are as follows: Figure 12 As shown in the figure. The results showed that the ability of 2,6-dimethoxybenzoquinone to remove mature biofilms from the two tested bacteria exhibited a significant concentration-dependent effect; the higher the concentration, the better the removal effect. Similar to its ability to inhibit biofilm formation, overall, 2,6-dimethoxybenzoquinone showed a significant effect on Staphylococcus aureus (Staphylococcus aureus). Figure 12 A) Biofilm removal ability is stronger than that of Escherichia coli ( Figure 12(B) Under high concentration conditions, the clearance rate of 2,6-dimethoxybenzoquinone against Staphylococcus aureus biofilm increased significantly with increasing concentration. At concentrations of 1 MIC, 2 MIC, and 4 MIC, the biofilm clearance rates were 35.34±1.85%, 39.19±4.36%, and 46.00±0.87%, respectively. For Escherichia coli, the increase in biofilm clearance rate of 2,6-dimethoxybenzoquinone gradually leveled off at high concentrations, with clearance rates of 31.87±0.77%, 35.88±1.79%, and 37.16±0.5% at concentrations of 1 MIC, 2 MIC, and 4 MIC, respectively. The biofilm clearance effect of 2,6-dimethoxybenzoquinone is mainly achieved through multiple pathways, including disrupting the spatial structure of the biofilm and reducing its metabolic activity. However, compared to its ability to inhibit biofilm formation, 2,6-dimethoxybenzoquinone has a weaker ability to clear mature biofilms. This may be because the matrix components of mature biofilms provide mechanical stability and cohesion for the connections between bacterial cells, giving the biofilm a certain structural stability, thus making it difficult to eradicate multicellular aggregates of biofilms that colonize the body surface or tissues.

[0142] Example 10

[0143] Effects on bacterial genomic DNA

[0144] The binding ability of 2,6-dimethoxybenzoquinone to bacterial genomic DNA was observed using agarose gel electrophoresis.

[0145] (1) Bacterial genomic DNA was extracted according to the operating procedure of the bacterial genomic DNA extraction kit, and its concentration was detected using an ultra-micro UV spectrophotometer. The DNA was then diluted to 100 μg / mL with dd H2O. Subsequently, 10 μL of 2,6-dimethoxybenzoquinone diluted with dd H2O was added to 10 μL of genomic DNA and mixed thoroughly to achieve final concentrations of 0 (control group), 1 / 8 MIC, 1 / 4 MIC, 1 / 2 MIC, 1 MIC, 2 MIC, and 4 MIC, respectively. The mixtures were then incubated at 4 °C for 2 h.

[0146] (2) Nucleic acid electrophoresis detection of bacterial genomic DNA binding: Take 25 mL of TAE working solution and place it in an Erlenmeyer flask. Add 0.25 g of agarose, mix thoroughly, and heat in a microwave oven until completely dissolved. After the solution temperature drops to approximately 60 °C, add 1.25 μL of nucleic acid dye and gently shake to mix. Slowly pour the mixture into the gel casting plate, insert the comb, and let it stand for about 30 min. After the gel has formed, remove the comb and place the gel along with the tray into the electrophoresis tank. Pour in an appropriate amount of TAE working solution to completely submerge the gel surface. Use a pipette to add the DNA solution prepared above into the gel wells, set the voltage to 120 V, and run for 25 min. After electrophoresis, remove the gel and observe the DNA bands in a gel imaging system.

[0147] DNA plays a crucial role in the physiological processes of bacterial growth, development, and genetic regulation. Certain plant-derived antibacterial components can directly bind to DNA through covalent or non-covalent interactions, thereby altering its spatial conformation and causing structural damage, such as nucleic acid chain breaks. The effects of 2,6-dimethoxybenzoquinone on the genomic DNA of Staphylococcus aureus and Escherichia coli were analyzed by agarose gel electrophoresis. Figure 13 The results showed that, compared with the control group, there were almost no significant changes in the genomic DNA bands and band brightness of the two tested bacteria treated with different concentrations of 2,6-dimethoxybenzoquinone. This indicates that the antibacterial effect of 2,6-dimethoxybenzoquinone on Staphylococcus aureus and Escherichia coli is not achieved by directly binding to bacterial genomic DNA and affecting its conformation and structure.

[0148] Example 11

[0149] Effects on bacterial metabolism

[0150] Bacterial metabolic activity assay

[0151] Take the aforementioned bacterial suspension and adjust the bacterial concentration to 1×10⁻⁶ using LB liquid medium. 8 CFU / mL was added to 2,6-dimethoxybenzoquinone to achieve final concentrations of 0 (control group), 1 / 8 MIC, 1 / 4 MIC, 1 / 2 MIC, 1 MIC, 2 MIC, and 4 MIC, with three replicates for each group. After treatment with shaking at 37 °C and 200 r / min for 6 h, the bacterial suspension from each treatment group was collected, centrifuged at 5000 g for 10 min at 4 °C, the supernatant was discarded, and the bacteria were washed three times with sterile PBS buffer. The bacterial concentration was then adjusted to OD0.05. 600 =0.5. Add 250 mg / mL iodonitrosotetrazole violet (INT) solution to the bacterial suspension to a final concentration of 1 mmol / L, incubate at 37 °C for 30 min, and measure the absorbance (OD) at 630 nm. 630 ).

[0152] Bacterial oxidative damage assay

[0153] Centrifuge the aforementioned bacterial suspension at 4 °C and 5000 r / min for 10 min, discarding the supernatant. Wash the bacterial cells three times with sterile PBS buffer and resuspend them. Add 100 mg / mL DCFH-DA solution to the bacterial suspension to a final concentration of 2 × 10⁻⁶. -5 Incubate at 37 °C and 200 r / min for 1 h using a mol / L solution. Wash the cells three times with sterile PBS and adjust the bacterial concentration to 1 × 10⁻⁶. 8 CFU / mL of 2,6-dimethoxybenzoquinone was added to achieve final concentrations of 0 (control group), 1 / 8 MIC, 1 / 4 MIC, 1 / 2 MIC, 1 MIC, 2 MIC, and 4 MIC, with three replicates for each group. After treatment at 37 °C and 200 r / min for 6 h with shaking, the fluorescence intensity of each group was measured. The excitation wavelength was set to 485 nm, the emission wavelength to 524 nm, the voltage to 700 V, and the excitation and emission slits to 10 nm.

[0154] Succinate dehydrogenase (SDH) activity assay

[0155] SDH activity was determined using a succinate dehydrogenase assay kit. The aforementioned bacterial suspension was adjusted to a concentration of 1 × 10⁻⁶ using LB liquid medium. 8 CFU / mL, add 2,6-dimethoxybenzoquinone to make the final concentrations 0 (control group), 1 / 8 MIC, 1 / 4 MIC, 1 / 2 MIC, 1 MIC, 2 MIC and 4 MIC respectively, with three replicates for each group. After shaking treatment at 37 °C and 200 r / min for 6 h, take the bacterial suspension of each group, centrifuge at 4 °C and 5000 r / min for 10 min, discard the supernatant, and measure SDH activity. Calculate the protein content in the test solution according to the Bradford method standard curve. Define 1 nmol of 2,6-dichlorophenolindophenol consumed per minute in the reaction system per mg of tissue protein as one enzyme activity unit. Calculate SDH activity according to formulas (9) and (10):

[0156] (9)

[0157] (10)

[0158] In the formula, 5 and 0 represents the absorbance of the test solution at 5 min and 0 min, respectively. The total volume of the reaction system (1 mL) The volume of sample added is 0.05 mL; T is the reaction time (5 min); ε is the molar extinction coefficient of 2,6-dichloroindophenol (2.1 × 10⁻⁶). 4 L / mol / cm), d is the optical path length of the cuvette (1 cm), C pr The sample protein concentration (mg / mL) calculated for the standard curve.

[0159] Establishment of the Bradford method standard curve: 0, 0.03, 0.06, 0.12, 0.24, 0.48, 0.72, 0.84, and 0.96 mL of bovine serum albumin solution (0.1 mg / mL) were pipetted into 10 mL stoppered centrifuge tubes. The volume was brought to 1 mL with distilled water. 5 mL of Coomassie Brilliant Blue (obtained by diluting 5× Coomassie Brilliant Blue G-250 5-fold) was added to each tube. After mixing and standing for 2 min, the absorbance was measured at 595 nm. The standard curve was established as: y = 0.5472x + 0.4015, R0 2 =0.9972, where x is the soluble protein content (mg / mL) and y is the absorbance.

[0160] Effects on bacterial metabolic activity

[0161] Iodonitrobetrazolium violet (INT) is a reagent used to detect cellular metabolic activity. It contains tetrazolium, which reacts directly with activated hydrogen ions produced by living cells of the test bacteria outside the cell to form a stable, purple-red, insoluble formazan. The absorbance of this product at 630 nm is directly proportional to the metabolic activity of the test bacteria, thus it can be used as an indicator of cell viability. Figure 14 As shown, when the concentration of 2,6-dimethoxybenzoquinone gradually increased from 1 / 8 MIC to 1 MIC, the metabolic activity of the two tested bacteria decreased relatively slowly; however, when the concentration reached 2 MIC and 4 MIC, the decrease in metabolic activity of the two tested bacteria intensified, and then hardly changed. Specifically, for Staphylococcus aureus (… Figure 14 A) As the concentration of 2,6-dimethoxybenzoquinone gradually increases from 1 / 8 MIC to 1 MIC, its OD... 630 The value decreased from 0.8±0.07 to 0.66±0.03, showing a significant change at a concentration of 1 MIC; for Escherichia coli (… Figure 14 B) As the concentration of 2,6-dimethoxybenzoquinone gradually increased from 1 / 8 MIC to 1 MIC, OD 630The concentration decreased from 0.69±0.02 to approximately 0.6±0.04, and the cellular metabolic activity was significantly lower than the control group at all concentrations of 2,6-dimethoxybenzoquinone. In summary, the results indicate that 2,6-dimethoxybenzoquinone has the ability to reduce bacterial cell metabolic activity. This may be because 2,6-dimethoxybenzoquinone disrupts the bacterial cell membrane, leading to increased cell membrane permeability, leakage of nucleic acids and charged ions, and consequently, cellular metabolic disorders, ultimately inhibiting bacterial growth.

[0162] Effects on bacterial oxidative damage

[0163] 2',7'-Dichlorofluorescein diacetate (DCFH-DA) is a label-free, oxidation-sensitive fluorescent probe. It is non-fluorescent and can freely cross the cell membrane. Once inside the cell, it is hydrolyzed by intracellular esterases into DCFH, which cannot penetrate the cell membrane. In the cell, reactive oxygen species (ROS) oxidize the non-fluorescent DCFH into fluorescent dichlorofluorescein (DCF). Based on this principle, the level of intracellular ROS can be determined by detecting the fluorescence intensity of DCF. Figure 15 As shown, both tested bacteria exhibited significant oxidative damage at the minimum treatment concentration, and the degree of damage increased with increasing 2,6-dimethoxybenzoquinone concentration. Overall, 2,6-dimethoxybenzoquinone showed a stronger oxidative damaging effect on Staphylococcus aureus. For Staphylococcus aureus (… Figure 15 A) As the concentration of 2,6-dimethoxybenzoquinone increases, the fluorescence intensity shows a significant upward trend; for Escherichia coli ( Figure 15 B) As the concentration of 2,6-dimethoxybenzoquinone increased, the fluorescence intensity gradually increased, but when the concentration exceeded 1 / 2 MIC, the fluorescence intensity no longer changed significantly. This indicates that 2,6-dimethoxybenzoquinone can induce oxidative damage in *E. coli*. In the low concentration range, the degree of damage increases with increasing concentration; after reaching 1 / 2 MIC, even with further increases in concentration, the degree of oxidative damage no longer significantly increases. ROS is closely related to cell growth and apoptosis, playing an important role in maintaining intracellular redox balance. When ROS levels are abnormal, intracellular oxidative stress changes, leading to cellular metabolic disorders. The results show that 2,6-dimethoxybenzoquinone can increase the total amount of ROS in bacterial cells, thereby inducing damage and death in *Staphylococcus aureus* and *Escherichia coli* cells.

[0164] Effects on bacterial succinate dehydrogenase activity

[0165] The tricarboxylic acid cycle (TCA) is a typical pathway of cellular respiratory metabolism, providing energy for cellular metabolic processes. Succinate dehydrogenase (SDH), an intracellular enzyme involved in the TCA cycle, catalyzes the interconversion of succinate and fumarate in the TCA cycle; therefore, intracellular SDH activity can reflect cellular metabolic status. Figure 16 The effects of different concentrations of 2,6-dimethoxybenzoquinone on the SDH activity of Staphylococcus aureus and Escherichia coli were demonstrated. The results showed that the effect of 2,6-dimethoxybenzoquinone on the SDH activity of the tested bacteria was concentration-dependent. For Staphylococcus aureus (… Figure 16 A) As the concentration of 2,6-dimethoxybenzoquinone gradually increased from 1 / 8 MIC to 1 / 2 MIC, the SDH activity decreased from 2.93±0.54 U / mg to 2.56±0.49 U / mg, a relatively small decrease; at concentrations of 1 MIC and higher, the SDH activity decreased significantly, indicating that low concentrations of 2,6-dimethoxybenzoquinone had little effect on the SDH activity of Staphylococcus aureus, while high concentrations showed a significant inhibitory effect. For Escherichia coli (… Figure 16 B) When the concentration of 2,6-dimethoxybenzoquinone was 1 / 4 MIC, it significantly inhibited SDH activity, and the inhibitory effect was more pronounced at concentrations of 1 MIC and above. Experimental results indicate that 2,6-dimethoxybenzoquinone treatment has a damaging effect on Staphylococcus aureus and Escherichia coli. It inhibits SDH activity, interferes with the TCA cycle of the two tested bacteria, and thus hinders normal cellular energy metabolism. This inhibitory effect may be due to 2,6-dimethoxybenzoquinone interfering with the biosynthesis of SDH-related proteins. Furthermore, the accumulation of intracellular metabolites malonic acid and oxaloacetic acid also inhibits SDH activity. Simultaneously, the bacterial respiratory chain is located within the cell membrane, which plays a crucial role in maintaining normal bacterial physiological functions. Therefore, 2,6-dimethoxybenzoquinone may disrupt bacterial energy metabolism by damaging the respiratory chain enzyme system and disrupting membrane integrity. This hypothesis is consistent with previous findings that 2,6-dimethoxybenzoquinone can disrupt the normal cell membrane structure of Staphylococcus aureus and Escherichia coli.

[0166] This invention focuses on 2,6-dimethoxybenzoquinone, the main antibacterial component of the chloroform extract of *Pyrus pyrifolia* root. *Staphylococcus aureus* and *Escherichia coli* were selected as test strains to explore its antibacterial mechanism from multiple perspectives. The main research results are as follows:

[0167] (1) Effect on bacterial growth curve: The effect of 2,6-dimethoxybenzoquinone on bacterial growth was evaluated by plotting time-growth curves. Treatment with this substance inhibited bacterial growth, resulting in significant changes in the growth curve, and the inhibitory effect was dose-dependent. Treatment with 1 / 2 MIC concentration of 2,6-dimethoxybenzoquinone delayed the logarithmic growth phase of the bacteria; while treatments with 1 MIC and 2 MIC concentrations almost halted bacterial growth.

[0168] (2) Effects on bacterial cell membranes: After treatment with 2,6-dimethoxybenzoquinone, the relative extracellular conductivity of bacteria increased, and the amount of nucleic acid leakage increased. This indicates that the substance can disrupt the bacterial cell membrane, affecting its permeability and integrity, thereby exerting an antibacterial effect.

[0169] (3) Effect on bacterial cell wall: After treatment with 2,6-dimethoxybenzoquinone, the extracellular AKP content increased, indicating that 2,6-dimethoxybenzoquinone can destroy the cell wall of bacteria and eventually lead to cell death.

[0170] (4) Effects on bacterial biofilm: The inhibitory and scavenging abilities of 2,6-dimethoxybenzoquinone on bacterial biofilm were evaluated using crystal violet staining. The results showed that, at the tested concentrations, both high and low concentrations of 2,6-dimethoxybenzoquinone significantly inhibited biofilm formation and scavenged biofilm, reducing the bacteria's ability to resist adverse external environments. Both its inhibitory and scavenging abilities on bacterial biofilm exhibited a dose-dependent effect.

[0171] (5) Effect on bacterial genomic DNA: Agarose gel electrophoresis results showed that there were almost no significant changes in the bacterial genomic DNA bands and band brightness in the treatment group, indicating that the antibacterial effect of 2,6-dimethoxybenzoquinone on Staphylococcus aureus and Escherichia coli was not achieved by directly binding to bacterial genomic DNA and affecting its conformation and structure.

[0172] (6) Effects on bacterial metabolism: 2,6-Dimethoxybenzoquinone reduces the energy metabolism of bacterial cells, decreases cell metabolic activity in a dose-dependent manner, causes bacterial oxidative damage, and inhibits SDH activity.

Claims

1. The application of pear extract in food storage and preservation, characterized in that, The pear extract is obtained by chloroform extraction of ethanol extract from pear root and concentration under reduced pressure using a rotary evaporator.

2. The application according to claim 1, characterized in that, The preparation method of the pear extract includes the following steps: The root of *Pyrus pyrifolia* was pulverized and sieved to obtain root powder. The root powder was mixed evenly with ethanol solution and extracted with ultrasonic assistance. The ultrasonic extract was filtered under reduced pressure, the filtrate was collected, and concentrated under reduced pressure using a rotary evaporator to obtain a concentrate. The concentrate was extracted with chloroform, the chloroform extract was collected, and the extract was evaporated to dryness using a rotary evaporator to obtain *Pyrus pyrifolia* extract.

3. The application according to claim 1, characterized in that, The pear extract includes organic acids, aldehydes, phenols, ketones, esters, and amides.

4. The application according to claim 1, characterized in that, The pear extract includes 2-methylcitric acid, coniferaldehyde, 2,6-dimethoxybenzoquinone, eugenol, cinnamamide, 5,7-dihydroxychromone, 4-hydroxy-3-(3-methylbut-2-enyl)benzoic acid, ryegrass lactone, vanillin, vanillic acid, ethyl 3,4-dihydroxybenzoate, benzoic acid, hesperidin, indole-3-carboxaldehyde, sagerol, naringenin chalcone, and 3,4-dihydrocoumarin.

5. The application according to claim 1, characterized in that, The pear extract includes 2,6-dimethoxybenzoquinone.

6. The application according to claim 1, characterized in that, The pear extract is used for antibacterial purposes.

7. The application according to claim 6, characterized in that, The bacteria include Escherichia coli, Salmonella typhimurium, Bacillus cereus, and Staphylococcus aureus.

8. The application according to claim 1, characterized in that, The pear extract is used to delay the logarithmic growth phase of bacteria, increase bacterial cell membrane permeability, and induce leakage of intracellular components.

9. The application according to claim 1, characterized in that, The pear extract is used to disrupt the integrity of bacterial cell walls, inhibit biofilm formation, promote the clearance of mature bacterial biofilms, induce oxidative damage in bacteria, reduce bacterial cell metabolic activity, inhibit bacterial SDH activity, and interfere with the bacterial TCA cycle.

10. A biological preservative, characterized in that, The biological preservative includes pear extract, which is obtained by chloroform extraction of ethanol extract of pear root and concentration under reduced pressure using a rotary evaporator.

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