A lignin active site precise regulation method based on multi-parameter synergistic oxidation and application thereof

Abstract

The application discloses a kind of based on multi-parameter collaborative oxidation lignin active site precision regulation method and its application.The regulation method includes the following steps: (1) acid solution or alkali solution is added to lignin, and is fully stirred to remove ash and residual carbohydrate in lignin, to obtain pretreated lignin;(2) the pretreated lignin is dispersed in solvent, then oxidizing agent (hydrogen peroxide, ammonium persulfate and sodium hypochlorite) and auxiliary oxidizing agent (zinc borate and sodium alginate) are added to reaction, to realize the collaborative regulation of phenolic hydroxyl and quinone group density.The oxidized modified lignin obtained by the method can improve the antibacterial efficiency, and can be widely applied in antibacterial drugs, antibacterial materials and other fields.

Description

Technical Field

[0001] This invention relates to lignin functionalization modification and antibacterial material technology, and particularly to a method for precise regulation of lignin active sites based on multi-parameter synergistic oxidation and its application. Background Technology

[0002] Lignin is the second most abundant natural polymer after cellulose, and a major byproduct of pulp and paper making and biorefining industries. Global annual production exceeds 50 million tons, but its utilization rate is currently less than 20%, with most being directly burned or discarded. This not only wastes resources but also causes environmental problems. The lignin molecule is rich in active groups such as phenolic hydroxyl, methoxy, and carboxyl groups. Among these, the phenolic hydroxyl and quinone groups are the core sites determining its antibacterial activity—phenolic hydroxyl groups can inhibit metabolism by disrupting bacterial cell membrane integrity, while quinone groups can damage bacterial DNA by generating oxidative stress. Therefore, modified lignin has a natural advantage in the field of antibacterial applications.

[0003] However, existing lignin oxidation modification technologies have three major bottlenecks that severely restrict their high-value applications: 1) Low precision in regulating active sites: Traditional oxidation methods (such as alkali-catalyzed oxygen oxidation and ozone oxidation) mostly use a single oxidant and coarse parameters (such as fixed temperature and excess oxidant), which cannot achieve independent or synergistic regulation of the density of phenolic hydroxyl groups and quinone groups. For example, although ozone oxidation can increase the quinone content, it is easy to cause excessive oxidation and degradation of phenolic hydroxyl groups, resulting in uneven distribution of active sites and density fluctuations of more than 30%; 2) Uncontrollable antibacterial properties: Due to the lack of quantitative correlation between active sites and antibacterial properties, the antibacterial effects of existing modified lignin (such as inhibition rate and inhibition spectrum) rely entirely on empirical processes, making it difficult to customize according to target scenarios (such as food packaging requiring an inhibition rate of ≥90%, and soil remediation requiring an inhibition rate of 50%-70%), resulting in poor product applicability; 3) Contradiction between environmental protection and cost: In order to make up for its own insufficient antibacterial properties, existing technologies often need to add exogenous antibacterial agents such as silver ions and quaternary ammonium salts after oxidation, which not only increases costs but also poses a risk of antibacterial agent migration and leakage (such as excessive migration of silver ions in food), and some oxidants (such as potassium dichromate) are prone to producing toxic byproducts, which do not meet the requirements of green processes.

[0004] Furthermore, existing research has not elucidated the antibacterial synergistic mechanism of lignin active sites. Most studies only evaluate performance based on macroscopic indicators such as the diameter of the inhibition zone, failing to establish a structure-activity relationship model of "oxidation parameters → active sites → antibacterial performance," leading to difficulties in process scale-up. Therefore, developing a precise, controllable, and environmentally friendly lignin oxidation method to achieve quantitative regulation of active sites and on-demand design of antibacterial performance is key to overcoming the bottleneck of high-value utilization of lignin. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for precise regulation of lignin active sites based on multi-parameter synergistic oxidation.

[0006] Another objective of this invention is to provide an application of the aforementioned method for precise regulation of lignin active sites based on multi-parameter synergistic oxidation.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A method for precise regulation of lignin active sites based on multi-parameter synergistic oxidation includes the following steps:

[0009] (1) Lignin pretreatment: Add acid or alkaline solution to lignin, stir thoroughly to remove ash (such as calcium and magnesium ions) and residual carbohydrates from lignin, then centrifuge to collect the precipitate, wash to pH 6-7, and finally freeze dry, crush and sieve to obtain pretreated lignin.

[0010] (2) Oxidation reaction: The pretreated lignin is dispersed in a solvent, and then an oxidant and a co-oxidant are added. The pH value is adjusted to 3-9, and the reaction is stirred at 30-80℃ for 0.5-4 h. After the reaction is completed, the lignin is centrifuged, washed, and freeze-dried to obtain oxidized modified lignin. The oxidant is hydrogen peroxide (H2O2), ammonium persulfate ((NH4)2S2O8) and sodium hypochlorite (NaClO); the co-oxidant is zinc borate and sodium alginate.

[0011] The lignin mentioned in step (1) is industrial lignin; preferably at least one of kraft lignin, enzymatically hydrolyzed lignin and alkali lignin; more preferably kraft lignin, with an initial phenolic hydroxyl density of 0.4 to 0.6 mmol / g and ash content ≤2%; more preferably kraft lignin, with an initial phenolic hydroxyl density of 0.5 mmol / g, ash content of 1.8% and moisture content ≤5%.

[0012] The acid solution mentioned in step (1) is a dilute acid solution; preferably an HCl solution; more preferably an HCl solution with a concentration of 0.05 to 0.2 mol / L; more preferably an HCl solution with a concentration of 0.1 mol / L.

[0013] The alkaline solution mentioned in step (1) is a dilute alkaline solution; preferably a NaOH solution; more preferably a NaOH solution with a concentration of 0.05 to 0.1 mol / L.

[0014] The solid-liquid ratio of lignin to acid solution (or alkaline solution) in step (1) is 1 g: (5-10) mL; preferably 1 g: 8 mL.

[0015] The stirring conditions described in step (1) are: 50-70℃, 300-500 rpm for 0.5-1.5 h; preferably: 60℃, 400 rpm for 1 h.

[0016] The centrifugation conditions described in step (1) are: 8000-12000 rpm for 10-15 min; preferably: 10000 rpm for 12 min.

[0017] The washing described in step (1) is performed using deionized water; preferably, it is performed using deionized water until pH=7.

[0018] The freeze-drying conditions described in step (1) are: -40 to -30°C, 0.01 to 0.03 mbar, freeze-drying for 24 to 36 h; preferably: -35°C, 0.02 mbar, freeze-drying for 28 h.

[0019] The sieving mentioned in step (1) is sieve 100 to 200 mesh; preferably sieve 150 mesh.

[0020] The pretreated lignin in step (1) has a particle size ≤150 μm and the reaction surface area is increased by 2 to 3 times.

[0021] The solvent mentioned in step (2) is at least one of deionized water and aqueous ethanol solution; preferably at least one of deionized water and aqueous ethanol solution with a volume percentage ≤30% (ethanol volume fraction ≤30% improves lignin dispersibility).

[0022] The solid-liquid ratio of the pretreated lignin to the solvent in step (2) is 1g:(10-20)mL; preferably 1g:15mL.

[0023] The molar ratio of hydrogen peroxide (H2O2), ammonium persulfate ((NH4)2S2O8) and sodium hypochlorite (NaClO) in step (2) is (3-6):(1-4):(1-4); preferably 6:2:2.

[0024] In step (2), hydrogen peroxide is preferably added as a hydrogen peroxide solution with a concentration of 30% by volume.

[0025] The amount of oxidant used in step (2) is calculated based on a final concentration of 0.1 to 1 mol / L (preferably 1 mol / L) in the reaction system and a molar ratio of lignin structural units to oxidant of 1:1 to 5, to avoid over-oxidation; wherein, the amount of hydrogen peroxide (H2O2) is calculated based on a final concentration of 0.3 to 0.6 mol / L (preferably 0.6 mol / L) in the reaction system; the amount of ammonium persulfate ((NH4)2S2O8) is calculated based on a final concentration of 0.1 to 0.4 mol / L (preferably 0.4 mol / L) in the reaction system; and the amount of sodium hypochlorite (NaClO) is calculated based on a final concentration of 0.1 to 0.4 mol / L (preferably 0.4 mol / L) in the reaction system.

[0026] The amount of oxidant added in step (2) is calculated based on its final concentration in the reaction system being 8% to 18% by mass; preferably, it is calculated based on its final concentration in the reaction system being 14% by mass.

[0027] The mass ratio of zinc borate to sodium alginate in step (2) is (1-5):(1-6); preferably 3:4.

[0028] The amount of zinc borate added in step (2) is calculated based on a final concentration of 2% to 10% by mass in the reaction system; preferably, it is calculated based on a final concentration of 6% by mass in the reaction system.

[0029] The amount of sodium alginate added in step (2) is calculated based on a final concentration of 2% to 12% by mass in the reaction system; preferably, it is calculated based on a final concentration of 8% by mass in the reaction system.

[0030] The reaction temperature described in step (2) is 30-80°C, wherein low temperature (30-50°C) is conducive to the retention of phenolic hydroxyl groups, and high temperature (60-80°C) is conducive to the formation of quinone groups; preferably 50-70°C; more preferably 55°C.

[0031] The pH value of the reaction described in step (2) is 3 to 9, wherein acidity (pH 3 to 5) promotes the formation of phenolic hydroxyl groups, neutrality (pH 6 to 7) is conducive to the stability of quinone groups, and alkalinity (pH 8 to 9) accelerates the oxidation rate; preferably 4.

[0032] The stirring rate in step (2) is 300-600 rpm to ensure that the oxidant and lignin are in full contact and to avoid excessive local concentration; preferably 500 rpm.

[0033] The reaction time described in step (2) is 0.5 to 4 h, wherein the phenolic hydroxyl group is dominant in the short time (0.5 to 1 h) and the proportion of quinone group increases in the long time (2 to 4 h); preferably 1.5 to 3 h; more preferably 2 h.

[0034] In step (2), the reaction is carried out under constant temperature and stirring under the set parameters. During the reaction, the changes in active sites can be monitored by sampling to ensure the precision of regulation.

[0035] The centrifugation conditions described in step (2) are: 8000-12000 rpm, centrifugation for 15-20 min; preferably: 10000 rpm, centrifugation for 15 min.

[0036] The washing described in step (2) is performed using deionized water or ethanol; preferably, it is washed 3 to 5 times with deionized water or ethanol to remove residual oxidants and small molecule oxidation products (such as vanillin and eugenol).

[0037] The freeze-drying conditions described in step (2) are: -50 to -30°C, 0.01 to 0.02 mbar, freeze-drying for 24 to 48 h, to avoid degradation of active sites caused by high-temperature drying; preferably: -41°C, 0.02 mbar, freeze-drying for 36 h.

[0038] An oxidatively modified lignin was prepared by the above-mentioned method for precise regulation of lignin active sites based on multi-parameter synergistic oxidation.

[0039] The phenolic hydroxyl group density of the oxidized modified lignin is 3-5 mmol / g (preferably 3.12-4.99 mmol / g), and the quinone group density is 0.2-0.85 mmol / g.

[0040] The application of the oxidized modified lignin in the preparation of antibacterial products.

[0041] The antibacterial products include at least one of antibacterial drugs, antibacterial materials, antibacterial coatings, food packaging materials, and agricultural soil antibacterial remediation agents.

[0042] The bacteria include at least one of Escherichia coli and Staphylococcus aureus; preferably at least one of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538.

[0043] The present invention has the following advantages and effects compared with the prior art:

[0044] 1. This invention provides an oxidation method that can precisely control the density of lignin active sites (phenolic hydroxyl groups, quinone groups) and antibacterial properties. Through multi-parameter synergistic oxidation, the density of phenolic hydroxyl groups (3.12-4.99 mmol / g) and quinone groups (0.2-0.85 mmol / g) can be independently or synergistically controlled with a control error of ≤5%, significantly improving the control precision.

[0045] 2. This invention establishes a quantitative law model of "oxidation parameters-active site density-antibacterial performance", which can increase the diameter of the inhibition zone from 10.2 mm to 19.5 mm (high antibacterial), with an inhibition rate covering 90.5%-98.6% and a minimum MIC of 0.28 mg / mL, meeting the needs of different scenarios (such as food packaging requiring an inhibition rate of ≥90%).

[0046] 3. The antibacterial mechanism of this invention is clear (the first time that "phenolic hydroxyl-quinone synergistic antibacterial" has been confirmed): the phenolic hydroxyl group disrupts the integrity of the bacterial cell membrane phospholipid bilayer by interacting with it; the quinone group damages bacterial DNA by promoting the generation of reactive oxygen species (ROS). The synergistic effect of the two increases the antibacterial efficiency by 1.5-2 times.

[0047] 4. The method of this invention is green and low-cost. The raw material is lignin, an industrial by-product. No exogenous antibacterial agents are required. An environmentally friendly oxidant is used. The process has no secondary pollution. The cost is reduced by more than 30% compared with the synthetic antibacterial agent (based on the comprehensive accounting of raw materials, reagents and energy consumption per unit mass of antibacterial product). The method uses easily degradable oxidants such as H2O2 and (NH4)2S2O8. There are no toxic by-products. The solvent is recyclable (recovery rate ≥80%). The carbon emissions of the process are reduced by more than 25% (based on the life cycle assessment comparison of the entire process (including solvent recovery and oxidant degradation) with the conventional chemical synthesis antibacterial agent process).

[0048] 5. The method of this invention has high industrial adaptability: the process steps are simple (pretreatment-oxidation-posttreatment), the equipment is conventional centrifuge and freeze-drying equipment, the parameters are easy to monitor, and continuous production can be achieved (batch processing capacity ≥10 kg).

[0049] 6. The modified lignin obtained by oxidation in this invention can be widely used in antibacterial coatings, food packaging materials, agricultural soil antibacterial remediation agents and other fields, providing a reliable path for the high-value utilization of industrial by-products and the preparation of green antibacterial materials. Attached Figure Description

[0050] Figure 1 This is a graph showing the relationship between total active site density and Escherichia coli inhibition rate;

[0051] Figure 2 This is a graph showing the relationship between total active site density and the inhibition zone of Escherichia coli;

[0052] Figure 3 This is a graph showing the relationship between total active site density and minimum inhibitory concentration (MIC).

[0053] Figure 4 This is a comparison chart of the density of hydroxyl / quinone groups in lignin and their antibacterial rate. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0055] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0056] 1. Industrial lignin: Kraft lignin, purchased from Shandong Longli Biotechnology Co., Ltd., with the following parameters: initial phenolic hydroxyl density 0.5 mmol / g, ash content 1.8%, and moisture content ≤5%.

[0057] 2. Oxidizing agents and co-oxidizing agents: H2O2 (30%, analytical grade), (NH4)2S2O8 (analytical grade), NaClO (10%, analytical grade), zinc borate (analytical grade), and sodium alginate (analytical grade) were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0058] 3. Testing equipment: UV-Vis spectrophotometer (UV-2600, Shimadzu), FTIR (Nicolet iS50, Thermo Fisher Scientific), XPS (ESCALAB 250Xi, Thermo Fisher Scientific), biosafety cabinet (BSC-1300IIA2, Haier).

[0059] 4. Detection method:

[0060] (1) Determination of active site density:

[0061] ① Phenolic hydroxyl group: Ultraviolet-visible spectrophotometry (UV-Vis) was used, with 280 nm as the characteristic absorption peak. Gallic acid was used as the standard curve to calculate the density (mmol / g); the average value of three determinations was taken.

[0062] ② Quinone group: Spectroscopy was performed using UV-Vis method (450 nm characteristic peak) or Fourier transform infrared spectroscopy (FTIR, 1650 cm⁻¹). -1 Characteristic peaks), p-benzoquinone was used as a standard curve, and the density (mmol / g) was calculated; the average value of three determinations was taken.

[0063] ③ Auxiliary characterization: X-ray photoelectron spectroscopy (XPS) was used to fit the O1s peak (phenolic hydroxyl oxygen: 532.6 eV, quinone oxygen: 531.8 eV) to verify the uniformity of the site distribution.

[0064] (2) Evaluation of antibacterial properties:

[0065] ① Test strains: Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538, both purchased from Beijing Beina Chuanglian Biotechnology Research Institute;

[0066] ②Inhibition zone diameter: Referring to GB / T 20944.3-2008 "Evaluation of antibacterial properties of textiles - Part 3: Shaking method", oxidized lignin (10 mg / mL) was added to a bacterial plate, and the diameter of the inhibition zone (mm) was measured after 24 h of incubation.

[0067] ③ Inhibition rate: The plate count method was used to calculate the ratio of the number of surviving bacteria to the initial number after 24 hours. The average value of three measurements was taken. Formula: Inhibition rate (%) = (1 - number of surviving bacteria / initial number) × 100;

[0068] ④Minimum inhibitory concentration (MIC): The lowest lignin concentration that inhibits bacterial growth is determined by serial dilution method to evaluate antibacterial potency. The average value of three determinations is taken.

[0069] Example 1: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 6% zinc borate + 8% sodium alginate)

[0070] (1) Pretreatment: Take 10 g of Kraft lignin, add 80 mL of 0.1 mol / L HCl solution, stir at 60℃ and 400 rpm for 1 h to remove ash (such as calcium and magnesium ions) and residual carbohydrates in the lignin, then centrifuge (10000 rpm, 12 min), collect the precipitate, wash with deionized water to pH 7, freeze dry (-35℃, 0.02 mbar, 28 h), pulverize and pass through a 150 mesh sieve to obtain pretreated lignin; after pretreatment, the lignin particle size is ≤150 μm (measured by laser particle size analyzer, D90=126 μm), and the specific surface area increases from 2.1 m² / g before pretreatment to 5.8 m² / g (measured by BET method), and the reaction specific surface area increases by about 2.8 times.

[0071] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.2 mol / L, respectively; then add zinc borate and sodium alginate to make their final concentrations in the reaction system 6% (w / v) and 8% (w / v), respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 2 h.

[0072] (3) Post-treatment: After the reaction was completed, the reaction solution was transferred to a centrifuge tube and centrifuged at 10,000 rpm for 15 min to collect the precipitate. Approximately 80 mL of deionized water was added to the precipitate, and after stirring to disperse, it was centrifuged again (10,000 rpm, 15 min). The washing was repeated three times to remove residual oxidizing agents, auxiliaries, and small molecule oxidation products. The washed precipitate was placed in a freeze dryer and freeze-dried at -41℃ and 0.02 mbar for 36 h to obtain oxidized lignin 1#.

[0073] (4) Detection: The density of phenolic hydroxyl groups and quinone groups, the inhibition zone and minimum inhibition zone concentration (MIC) of Escherichia coli and Staphylococcus aureus were detected respectively, and the inhibition rate was calculated.

[0074] Example 2: (0.3 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 6% zinc borate + 8% sodium alginate)

[0075] (1) Pretreatment: Same as in Example 1;

[0076] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.3 mol / L, 0.2 mol / L and 0.2 mol / L, respectively; then add zinc borate and sodium alginate to make their final concentrations in the reaction system 6% (w / v) and 8% (w / v), respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 2 h.

[0077] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 2#;

[0078] (4) Detection: The density of phenolic hydroxyl groups and quinone groups, the inhibition zone and minimum inhibition zone concentration (MIC) of Escherichia coli and Staphylococcus aureus were detected respectively, and the inhibition rate was calculated.

[0079] Example 3: (0.6 mol / L H2O2 + 0.1 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 6% zinc borate + 8% sodium alginate)

[0080] (1) Pretreatment: Same as in Example 1;

[0081] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.1 mol / L and 0.2 mol / L, respectively; then add zinc borate and sodium alginate to make their final concentrations in the reaction system 6% (w / v) and 8% (w / v), respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 2 h.

[0082] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 3#;

[0083] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0084] Example 4: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.1 mol / L NaClO + 6% zinc borate + 8% sodium alginate)

[0085] (1) Pretreatment: Same as in Example 1;

[0086] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.1 mol / L, respectively; then add zinc borate and sodium alginate to make their final concentrations in the reaction system 6% (w / v) and 8% (w / v), respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 2 h.

[0087] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 4#;

[0088] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0089] Example 5: (0.8 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 6% zinc borate + 8% sodium alginate)

[0090] (1) Pretreatment: Same as in Example 1;

[0091] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.8 mol / L, 0.2 mol / L and 0.2 mol / L, respectively; then add zinc borate and sodium alginate to make their final concentrations in the reaction system 6% (w / v) and 8% (w / v), respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 2 h.

[0092] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 5#;

[0093] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0094] Example 6: (0.6 mol / L H2O2 + 0.4 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 6% zinc borate + 8% sodium alginate)

[0095] (1) Pretreatment: Same as in Example 1;

[0096] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.4 mol / L and 0.2 mol / L, respectively; then add zinc borate and sodium alginate to make their final concentrations in the reaction system 6% (w / v) and 8% (w / v), respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 2 h.

[0097] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 6#;

[0098] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0099] Example 7: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.4 mol / L NaClO + 6% zinc borate + 8% sodium alginate)

[0100] (1) Pretreatment: Same as in Example 1;

[0101] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.4 mol / L, respectively; then add zinc borate and sodium alginate to make their final concentrations in the reaction system 6% (w / v) and 8% (w / v), respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 2 h.

[0102] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 7#;

[0103] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0104] Example 8: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 2% zinc borate + 8% sodium alginate)

[0105] (1) Pretreatment: Same as in Example 1;

[0106] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.2 mol / L, respectively; then add zinc borate and sodium alginate to make their final concentrations in the reaction system 2% (w / v) and 8% (w / v), respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 2 h.

[0107] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 8#;

[0108] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0109] Example 9: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 6% zinc borate + 2% sodium alginate)

[0110] (1) Pretreatment: Same as in Example 1;

[0111] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.2 mol / L, respectively; then add zinc borate and sodium alginate to make their final concentrations in the reaction system 6% (w / v) and 2% (w / v), respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 2 h.

[0112] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 9#;

[0113] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0114] Example 10: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 10% zinc borate + 8% sodium alginate)

[0115] (1) Pretreatment: Same as in Example 1;

[0116] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.2 mol / L, respectively; then add zinc borate and sodium alginate to make their final concentrations in the reaction system 10% (w / v) and 8% (w / v), respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 2 h.

[0117] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 10#;

[0118] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0119] Example 11: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 6% zinc borate + 12% sodium alginate)

[0120] (1) Pretreatment: Same as in Example 1;

[0121] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.2 mol / L, respectively; then add zinc borate and sodium alginate to make their final concentrations in the reaction system 6% (w / v) and 12% (w / v), respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 2 h.

[0122] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 11#;

[0123] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0124] Example 12: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 6% zinc borate + 8% sodium alginate)

[0125] (1) Pretreatment: Same as in Example 1;

[0126] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.2 mol / L, respectively; then add zinc borate and sodium alginate to make their final concentrations in the reaction system 6% (w / v) and 8% (w / v), respectively. Adjust the pH to 4, stir at 50℃ and 500 rpm for 2 h.

[0127] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 12#;

[0128] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0129] Example 13: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 6% zinc borate + 8% sodium alginate)

[0130] (1) Pretreatment: Same as in Example 1;

[0131] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.2 mol / L, respectively; then add zinc borate and sodium alginate to make their final concentrations in the reaction system 6% (w / v) and 8% (w / v), respectively. Adjust the pH to 4, stir at 70℃ and 500 rpm for 2 h.

[0132] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 13#;

[0133] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0134] Example 14: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 6% zinc borate + 8% sodium alginate)

[0135] (1) Pretreatment: Same as in Example 1;

[0136] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.2 mol / L, respectively; then add zinc borate and sodium alginate to make their final concentrations in the reaction system 6% (w / v) and 8% (w / v), respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 1.5 h.

[0137] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 14#;

[0138] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0139] Example 15: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 6% zinc borate + 8% sodium alginate)

[0140] (1) Pretreatment: Same as in Example 1;

[0141] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.2 mol / L, respectively; then add zinc borate and sodium alginate to make their final concentrations in the reaction system 6% (w / v) and 8% (w / v), respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 3 h.

[0142] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 15#;

[0143] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0144] Comparative Example 1: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8)

[0145] (1) Pretreatment: Same as in Example 1;

[0146] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2 and (NH4)2S2O8 to make their final concentrations in the reaction system 0.6 mol / L and 0.2 mol / L, respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 2 h.

[0147] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 16#;

[0148] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0149] Comparative Example 2: (0.6 mol / L H2O2 + 0.2 mol / L NaClO)

[0150] (1) Pretreatment: Same as in Example 1;

[0151] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2 and NaClO to make their final concentrations in the reaction system 0.6 mol / L and 0.2 mol / L, respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 2 h.

[0152] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 17#;

[0153] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0154] Comparative Example 3: (0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO)

[0155] (1) Pretreatment: Same as in Example 1;

[0156] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.2 mol / L and 0.2 mol / L, respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 2 h.

[0157] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 18#;

[0158] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0159] Comparative Example 4: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 8% sodium alginate)

[0160] (1) Pretreatment: Same as in Example 1;

[0161] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.2 mol / L, respectively; then add sodium alginate to make its final concentration in the reaction system 8% (w / v), adjust the pH to 4, and stir at 55℃ and 500 rpm for 2 h.

[0162] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 19#;

[0163] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0164] Comparative Example 5: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 6% zinc borate)

[0165] (1) Pretreatment: Same as in Example 1;

[0166] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.2 mol / L, respectively; then add zinc borate to make its final concentration in the reaction system 6% (w / v), adjust the pH to 4, and stir at 55℃ and 500 rpm for 2 h;

[0167] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 20#;

[0168] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0169] Comparative Example 6: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO)

[0170] (1) Pretreatment: Same as in Example 1;

[0171] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.2 mol / L, respectively. Adjust the pH to 4, stir at 55℃ and 500 rpm for 2 h;

[0172] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 21#;

[0173] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0174] Comparative Example 7: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 14% sodium alginate)

[0175] (1) Pretreatment: Same as in Example 1;

[0176] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.2 mol / L, respectively; then add sodium alginate to make its final concentration in the reaction system 14% (w / v), adjust the pH to 4, and stir at 55℃ and 500 rpm for 2 h.

[0177] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 22#;

[0178] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0179] Comparative Example 8: (0.6 mol / L H2O2 + 0.2 mol / L (NH4)2S2O8 + 0.2 mol / L NaClO + 14% zinc borate)

[0180] (1) Pretreatment: Same as in Example 1;

[0181] (2) Oxidation reaction: Take 5 g of pretreated lignin, disperse it in 100 mL of deionized water, and then add H2O2, (NH4)2S2O8 and NaClO to make their final concentrations in the reaction system 0.6 mol / L, 0.2 mol / L and 0.2 mol / L, respectively; then add zinc borate to make its final concentration in the reaction system 14% (w / v), adjust the pH to 4, and stir at 55℃ and 500 rpm for 2 h.

[0182] (3) Post-treatment: Same as in Example 1, to obtain oxidized lignin 23#;

[0183] (4) Detection: The density of phenolic hydroxyl / quinone groups, the inhibition zone of Escherichia coli / Staphylococcus aureus, and the minimum inhibition zone concentration (MIC) were detected respectively, and the inhibition rate was calculated.

[0184] Example of effect:

[0185] 1. Regulatory mechanisms of active sites

[0186] The results of the determination of phenolic hydroxyl and quinone group densities of the oxidized lignin prepared in Examples 1-15 and Comparative Examples 1-8 of this invention are shown in Table 1.

[0187] Table 1: Comparison of oxidation parameters and active site density (including concentration gradient) between the examples and comparative examples

[0188]

[0189] As shown in Table 1, this invention achieves precise control of phenolic hydroxyl group density within the range of 3.12–4.99 mmol / g and quinone group density within the range of 0.25–0.85 mmol / g through multi-parameter synergistic oxidation, with a control accuracy of ≤5% (taking the phenolic hydroxyl group density of Examples 1 and 5 as examples, the deviation between the target control value and the measured value is within 5%). Detailed analysis follows:

[0190] (1) Synergistic effect of oxidants: Comparative Examples 1-3 lacked one of NaClO, ammonium persulfate or H2O2, and their phenolic hydroxyl density (0.88-1.65 mmol / g) and quinone density (0.18-0.20 mmol / g) were significantly lower than those of Example 1 (4.99 mmol / g and 0.71 mmol / g), indicating that the synergistic use of the three oxidants is the key to achieving high-density regulation of active sites.

[0191] (2) Synergistic effect of adjuvants: Comparative Examples 4-6 lacked sodium alginate, zinc borate, or both, respectively. Their phenolic hydroxyl group density (1.54-2.61 mmol / g) and quinone group density (0.20-0.30 mmol / g) were lower than those of Example 1, indicating that the combined use of zinc borate and sodium alginate can effectively improve the density of active sites. The phenolic hydroxyl group density (2.78 mmol / g, 2.55 mmol / g) of Comparative Example 7 (14% sodium alginate) and Comparative Example 8 (14% zinc borate) was higher than that of Comparative Example 6, but still significantly lower than that of Example 1. This indicates that there is an optimal range for the dosage of adjuvants (2%-10% zinc borate, 2%-12% sodium alginate), and the effect of exceeding this range on improving the density of active sites is limited.

[0192] (3) Single-parameter regulation law:

[0193] ①H2O2 concentration (Examples 1, 2, 5): When the H2O2 concentration increased from 0.3 mol / L to 0.6 mol / L, the density of phenolic hydroxyl groups increased from 3.12 mmol / L to 4.99 mmol / L, and the density of quinone groups increased from 0.51 mmol / L to 0.71 mmol / L; when it was further increased to 0.8 mol / L, the density of phenolic hydroxyl groups decreased slightly to 4.42 mmol / L, indicating that the appropriate concentration can balance the oxidation depth and the retention of active sites.

[0194] ② Ammonium persulfate concentration (Examples 1, 3, 6): When the ammonium persulfate concentration increased from 0.1 mol / L to 0.4 mol / L, the quinone group density increased from 0.39 mmol / L to 0.85 mmol / L. The phenolic hydroxyl group density first increased and then stabilized, indicating that ammonium persulfate has a significant promoting effect on quinone group formation.

[0195] ③NaClO concentration (Examples 1, 4, 7): When the NaClO concentration increased from 0.1 mol / L to 0.4 mol / L, the density of phenolic hydroxyl groups increased from 4.10 mmol / L to 4.98 mmol / L, while the density of quinone groups first increased and then decreased, indicating that NaClO has a promoting effect on the retention of phenolic hydroxyl groups, but excessive amount may inhibit the formation of quinone groups.

[0196] ④ Reaction temperature (Examples 1, 12, 13): When the temperature increased from 50℃ to 70℃, the density of phenolic hydroxyl groups increased from 4.13 mmol / L to 4.76 mmol / L. The density of quinone groups was higher at 50℃ (0.53 mmol / L) and decreased to 0.49 mmol / L at 70℃. This indicates that low temperature (50-55℃) is conducive to the formation of quinone groups, while high temperature (70℃) is more conducive to the retention of phenolic hydroxyl groups.

[0197] ⑤ Reaction time (Examples 1, 14, 15): When the reaction time was extended from 1.5 h to 3 h, the density of phenolic hydroxyl groups increased from 4.22 mmol / L to 4.99 mmol / L and then slightly decreased to 4.58 mmol / L, while the density of quinone groups first increased and then decreased from 0.59 mmol / L. This indicates that 2 h is the optimal reaction time, which can achieve balanced regulation of phenolic hydroxyl and quinone groups.

[0198] The above results demonstrate that the present invention can achieve independent or synergistic regulation of the density of phenolic hydroxyl groups and quinone groups through multi-parameter synergistic oxidation, providing technical support for the targeted design of active sites in different antibacterial scenarios.

[0199] 2. Antibacterial properties

[0200] The inhibition zones, minimum inhibition zone concentrations (MICs), and inhibition rates of the oxidized lignin prepared in Examples 1-15 and Comparative Examples 1-8 against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538 are shown in Table 2.

[0201] Table 2: Comparison of antibacterial properties between the examples and comparative examples

[0202]

[0203] As shown in Table 2, the oxidized lignin prepared in this invention exhibits excellent antibacterial activity against both *Escherichia coli* and *Staphylococcus aureus*, with inhibition zone diameters ranging from 10.2 to 19.5 mm (high antibacterial activity), inhibition rates covering 90.5% to 98.6%, and a minimum MIC of 0.28 mg / mL. This meets the requirements of various applications (such as food packaging requiring an inhibition rate ≥90%). Detailed analysis follows:

[0204] (1) Synergistic effect: The antibacterial properties of Comparative Examples 1-6 (inhibition zone 10.2-14.4 mm, MIC 0.69-1.10 mg / mL) were significantly lower than those of Example 1 (inhibition zone 19.5 / 19.1 mm, MIC 0.28 mg / mL), indicating that the synergistic use of the oxidant combination and the adjuvant combination is the key to obtaining high antibacterial properties. Although the antibacterial properties of Comparative Example 7 (sodium alginate only) and Comparative Example 8 (zinc borate only) were better than those of Comparative Example 6, they were still significantly lower than those of Example 1, further verifying the synergistic effect of zinc borate and sodium alginate.

[0205] (2) Correlation between active sites and antibacterial performance: Comparing Table 1 and Table 2, it can be seen that the samples with both high phenolic hydroxyl density and high quinone density (such as Example 1 and Example 7) showed the best antibacterial performance, while the samples with high single active site density (such as Example 6 with high quinone density but slightly low phenolic hydroxyl density, and Example 5 with high phenolic hydroxyl density but slightly low quinone density) had lower antibacterial performance than Example 1, indicating the effectiveness of the "phenolic hydroxyl-quinone synergistic antibacterial" mechanism: phenolic hydroxyl disrupts the integrity of bacterial cell membrane, and quinone promotes the generation of reactive oxygen species that damage bacterial DNA. The synergy of the two significantly improves the antibacterial efficiency.

[0206] (3) Quantitative relationship between MIC and active site density: MIC is negatively correlated with phenolic hydroxyl group density and quinone group density. Example 1 (phenolic hydroxyl group 4.99 mmol / g, quinone group 0.71 mmol / g) had the lowest MIC (0.28 mg / mL); Example 2 (phenolic hydroxyl group 3.12 mmol / g, quinone group 0.51 mmol / g) had a MIC that increased to 0.55 mg / mL; and Comparative Example 2 (phenolic hydroxyl group 0.88 mmol / g, quinone group 0.20 mmol / g) had the highest MIC (1.10 mg / mL). This quantitative relationship provides data support for the establishment of the quantitative law model of "oxidation parameter-active site density-antibacterial performance" in this invention.

[0207] (4) Consistency of antibacterial spectrum: The antibacterial properties of all samples against Escherichia coli and Staphylococcus aureus showed a consistent trend, indicating that the oxidized modified lignin prepared in this invention has broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria.

[0208] 3. Quantitative Model Validation

[0209] like Figure 1-4 As shown:

[0210] ① Total active site density (phenolic hydroxyl groups + quinone groups) (x) vs. Escherichia coli inhibition rate (y):

[0211] y = 3.59x + 78.25, R 2 =0.9931;

[0212] ② Total active site density (x) vs. Escherichia coli inhibition zone (y):

[0213] y = 1.87x + 9.52, R 2 =0.9931;

[0214] ③ Total active site density (x) vs. minimum inhibitory concentration (MIC) (y):

[0215] y=2.15e −0.38x R 2 =0.9863;

[0216] ④ Comparison of phenolic hydroxyl / quinone group density (x) vs. antibacterial rate (y):

[0217] Phenolic hydroxyl group: y = 3.82x + 77.56, R 2 = 0.9863;

[0218] Quinone group: y = 12.58x + 75.82, R 2 = 0.4661.

[0219] This invention achieves precise regulation of lignin active sites and antibacterial properties through multi-parameter synergistic oxidation, solving the problems of low regulation precision and uncontrollable antibacterial properties in traditional methods. It provides a new technical path for the high-value utilization of industrial lignin and the preparation of green antibacterial materials, and has significant economic and environmental benefits.

[0220] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for precise regulation of lignin active sites based on multi-parameter synergistic oxidation, characterized in that, Includes the following steps: (1) Lignin pretreatment: Add acid or alkaline solution to lignin, stir thoroughly to remove ash and residual carbohydrates from lignin, then centrifuge to collect the precipitate, wash to pH 6-7, and finally freeze dry, crush and sieve to obtain pretreated lignin. (2) Oxidation reaction: The pretreated lignin is dispersed in a solvent, and then an oxidant and a co-oxidant are added. The pH value is adjusted to 3-9, and the reaction is stirred at 30-80℃ for 0.5-4 h. After the reaction is completed, the lignin is centrifuged, washed, and freeze-dried to obtain oxidized modified lignin. The oxidant is hydrogen peroxide, ammonium persulfate and sodium hypochlorite; the co-oxidant is zinc borate and sodium alginate.

2. The method according to claim 1, characterized in that: The amount of oxidant used in step (2) is calculated based on a final concentration of 0.1 to 1 mol / L in the reaction system; The molar ratio of hydrogen peroxide, ammonium persulfate and sodium hypochlorite in step (2) is 3-6:1-4:1-4; The amount of the oxidant added in step (2) is calculated based on its final concentration in the reaction system being 8% to 18% by mass. The mass ratio of zinc borate to sodium alginate in step (2) is 1-5:1-6.

3. The method according to claim 2, characterized in that: In step (2), the amount of hydrogen peroxide used is calculated based on a final concentration of 0.3–0.6 mol / L in the reaction system; the amount of ammonium persulfate used is calculated based on a final concentration of 0.1–0.4 mol / L in the reaction system; and the amount of sodium hypochlorite used is calculated based on a final concentration of 0.1–0.4 mol / L in the reaction system. The amount of zinc borate added in step (2) is calculated based on its final concentration in the reaction system being 2% to 10% by mass. The amount of sodium alginate added in step (2) is calculated based on its final concentration in the reaction system being 2% to 12% by mass.

4. The method according to claim 1, characterized in that: The reaction temperature described in step (2) is 50–70 °C; The stirring rate described in step (2) is 300–600 rpm; The reaction time described in step (2) is 1.5 to 3 hours.

5. The method according to claim 4, characterized in that: The reaction temperature described in step (2) is 55°C; The pH value of the reaction described in step (2) is 4; The stirring rate described in step (2) is 500 rpm; The reaction time described in step (2) is 2 h.

6. The method according to claim 1, characterized in that: The lignin mentioned in step (1) is industrial lignin; further, it is at least one of Kraft lignin, enzymatically hydrolyzed lignin, and alkali lignin. The acid solution mentioned in step (1) is an HCl solution; The alkaline solution mentioned in step (1) is a NaOH solution; In step (1), the solid-liquid ratio of lignin to acid solution is 1 g: 5-10 mL; the solid-liquid ratio of lignin to alkaline solution is 1 g: 5-10 mL. The solvent mentioned in step (2) is at least one of deionized water and aqueous ethanol solution; The solid-liquid ratio of the pretreated lignin to the solvent in step (2) is 1g:10-20mL.

7. The method according to claim 1, characterized in that: The stirring conditions described in step (1) are: 50-70℃, 300-500 rpm for 0.5-1.5 h; The centrifugation conditions described in step (1) are: 8000-12000 rpm, centrifugation for 10-15 min; The washing described in step (1) is performed using deionized water; The freeze-drying conditions described in step (1) are: -40 to -30°C, 0.01 to 0.03 mbar, freeze-drying for 24 to 36 hours; The sieving mentioned in step (1) refers to sieving through a 100-200 mesh sieve; The centrifugation conditions described in step (2) are: 8000-12000 rpm, centrifugation for 15-20 min; The washing described in step (2) is performed using deionized water or ethanol; The freeze-drying conditions described in step (2) are: -50 to -30°C, 0.01 to 0.02 mbar, freeze-drying for 24 to 48 hours.

8. An oxidized modified lignin, characterized in that: It was prepared by the precise regulation method of lignin active sites based on multi-parameter synergistic oxidation as described in any one of claims 1 to 7.

9. The application of the oxidized modified lignin according to claim 8 in the preparation of antibacterial products.

10. The application according to claim 9, characterized in that: The antibacterial products include at least one of antibacterial drugs, antibacterial materials, antibacterial coatings, food packaging materials, and agricultural soil antibacterial remediation agents; The bacteria mentioned include at least one of Escherichia coli and Staphylococcus aureus.

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