Method and application of preparing lysozyme dimer by combined application of thermal catalysis and chemical modification

Abstract

The present invention provides a method and application for preparing lysozyme dimers by combining thermal catalysis with chemical modification, and belongs to the field of material preparation technology. The preparation method of the lysozyme dimer provided by the present invention comprises the following steps: lysozyme is mixed with an aqueous solution containing an oxidant H2O2, the pH is adjusted with a citric acid / disodium hydrogen phosphate solution, stirring at room temperature after complete mixing, then stirring and heating in a water bath, cooling and adding catalase to remove residual H2O2, then adding glutaraldehyde for cross-linking reaction, and then terminating the reaction by adding Tris-HCl to obtain a lysozyme dimer. The preparation method of the present invention increases the content of the lysozyme dimer and also increases the lysozyme antibacterial activity. The content of the prepared lysozyme dimer is up to more than 50%. The preparation method of the present invention is simple to operate, and the good antibacterial activity of the lysozyme dimer has a prospect for drug development.

Description

Technical Field

[0001] The present invention relates to the technical field of material preparation, in particular to a method and application of preparing a lysozyme dimer by combining thermal catalysis and chemical modification. Background Art

[0002] Lysozyme, chemically known as N-acetylmuramylin hydrolase, is also known as muramidase. Lysozyme, found in egg white, for example, exhibits strong heat and acid resistance and good stability in saline solutions. Lysozyme, a natural immune factor, is widely present in various microorganisms, animals, and plants. It hydrolyzes the peptidoglycan scaffolding of the cell wall of Gram-positive bacteria, altering the osmotic pressure inside the cell and causing cell lysis and death. However, it has no significant effect on Gram-negative bacteria.

[0003] Lysozyme dimer is a non-specific immune factor formed by using egg white lysozyme through modern biotechnology to transform monomeric lysozyme and its molecules. Compared with ordinary lysozyme, the potency of lysozyme dimer can be increased by 20-30 times. It plays the role of building a health protection wall in the human and animal bodies.

[0004] Currently, the main method for preparing lysozyme dimers is to directly cross-link lysozyme monomers using the chemical cross-linking agent glutaraldehyde. The lysozyme dimer content prepared by this method can reach up to 25%. However, the reaction conditions are relatively harsh. Excessive glutaraldehyde content can easily lead to extensive denaturation of lysozyme, making it impossible to cross-link and obtain lysozyme dimers with higher content.

[0005] Research has shown that dimers formed by modifying lysozyme monomers have broad application prospects not only in the food industry but also in medicine, pharmacology, and veterinary medicine. However, during the preparation process, lysozyme dimers are often affected by factors such as reaction materials, reaction temperature and time, and pH, leading to a decrease in content, which can also affect their efficacy. Therefore, providing a method for preparing lysozyme dimers to increase their content, antibacterial activity, and broad-spectrum antibacterial properties is a technical problem that urgently needs to be solved. Summary of the Invention

[0006] The present invention aims to provide a method and application for preparing lysozyme dimers by combining thermal catalysis and chemical modification to address the problems of the prior art. The preparation method of the present invention can increase the content of lysozyme dimers and improve the antibacterial activity compared to lysozyme monomers.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a method for preparing a lysozyme dimer, comprising the following steps:

[0009] After lysozyme and an oxidant are mixed, pH adjustment treatment, stirring treatment, heating treatment, cooling treatment and cross-linking treatment are sequentially performed to obtain the lysozyme dimer.

[0010] Preferably, the oxidant is a hydrogen peroxide solution;

[0011] The volume percentage of hydrogen peroxide in the mixed solution obtained by mixing the lysozyme and the oxidant is 1%-10%.

[0012] When preparing lysozyme dimers in the present invention, the relative content of lysozyme dimers increases with increasing H2O2 concentration. However, if the H2O2 concentration is high, the strong interaction between H2O2 and lysozyme destroys the higher-order structure of the protein, which is not conducive to the formation of lysozyme dimers and reduces enzyme activity. Therefore, the final concentration of the H2O2 solution is preferably 1%-10%, which can relatively increase the lysozyme dimer content. The 1%-10% H2O2 solution is prepared by mixing a 30% H2O2 solution with pure water, then uniformly mixing with a lysozyme solution, and finally adding a small amount of pure water to a final concentration of 1%-10%. The mixing method is preferably stirring or shaking.

[0013] Preferably, the concentration of lysozyme in the mixed solution obtained after the lysozyme and the oxidant are mixed is 10 mg / mL-20 mg / mL.

[0014] Preferably, the pH value is 3-7.

[0015] In the present invention, an overly alkaline environment is not conducive to the formation of lysozyme dimers, will lead to the decomposition of the oxidant H2O2 and is not conducive to the aggregation of lysozyme monomers during the thermal reaction. It may also cause the denaturation of lysozyme due to excessive alkalinity. Therefore, in order to increase the content of the prepared lysozyme dimer, the pH value of the solution is preferably 3-7.

[0016] Preferably, the stirring process includes a first stirring process and a second stirring process;

[0017] The first stirring process is performed at a rotation speed of 100-150 rpm and a time of 10-25 min;

[0018] The second stirring treatment lasts for 24 hours.

[0019] In the present invention, the oxidation reaction time (second stirring treatment time) affects the amount of lysozyme dimer generated. Therefore, the present invention conducts an oxidation reaction between the lysozyme solution and the H2O2 aqueous solution at room temperature for 24 hours to increase the amount of lysozyme dimer generated.

[0020] Preferably, the heating treatment is carried out at a temperature of 70°C-80°C and for a time of 10-30 minutes.

[0021] In the present invention, since too low a temperature is not conducive to the thermal reaction aggregation of lysozyme, resulting in a decrease in the amount of lysozyme dimer generated, the present invention performs a thermal aggregation reaction at 70°C-80°C. The present invention increases the amount of lysozyme dimer generated by setting the thermal aggregation reaction temperature.

[0022] Preferably, the cross-linking agent used in the cross-linking treatment is glutaraldehyde solution;

[0023] The volume percentage of glutaraldehyde in the mixed solution obtained by the cross-linking treatment is 0.4%-0.5%.

[0024] In the present invention, the concentration of the cross-linking agent glutaraldehyde affects the amount of lysozyme dimer generated, so the final concentration of glutaraldehyde is preferably 0.4%-0.5%. The relative content of lysozyme dimer increases with increasing glutaraldehyde concentration. However, if the glutaraldehyde concentration is high, due to its high toxicity, it will cause a large amount of lysozyme denaturation.

[0025] The present invention utilizes H2O2 solution as an oxidant, then utilizes a heating reaction to promote lysozyme aggregation, and then adds a cross-linking agent, glutaraldehyde, for cross-linking, thereby increasing the lysozyme dimer content by up to 50% or more. While increasing the lysozyme content, the bactericidal biological function of lysozyme is also enhanced.

[0026] The present invention provides a lysozyme dimer prepared by the above preparation method.

[0027] The present invention provides the use of the lysozyme dimer in the preparation of antibacterial products.

[0028] More preferably, the bacteria include Staphylococcus aureus, Propionibacterium acnes, Escherichia coli, Pseudomonas aeruginosa and Candida albicans.

[0029] Preferably, the antibacterial product includes an antibacterial drug and a disinfectant.

[0030] The present invention discloses the following technical effects:

[0031] The present invention provides a method for preparing a lysozyme dimer and its application. The method utilizes H2O2 as an oxidant to react with lysozyme, aggregating the lysozyme by heating, and finally uses glutaraldehyde for a cross-linking reaction, thereby significantly increasing the content of the resulting lysozyme dimer. The oxidant concentration, cross-linker concentration, oxidation time, thermal reaction temperature, and raw material concentrations set during the preparation of the lysozyme dimer not only increase the content of the lysozyme dimer but also enhance the bactericidal biological function of the lysozyme. The content of the lysozyme dimer obtained by the preparation is as high as 50% or more. The preparation methods of the lysozyme dimer of the present invention are simple to operate, and the lysozyme dimer has excellent antibacterial and broad-spectrum properties, showing promising prospects for pharmaceutical development. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 The effect of different pH on the content of lysozyme dimer formation;

[0034] Figure 2 The effect of different heating temperatures on the content of lysozyme dimer formation;

[0035] Figure 3 The effect of different heating time on the content of lysozyme dimer formation;

[0036] Figure 4 The effect of different oxidation times on the content of lysozyme dimer formation;

[0037] Figure 5 The effect of different lysozyme concentrations on the content of lysozyme dimer formation;

[0038] Figure 6 The effect of different oxidant concentrations on the content of lysozyme dimer formation;

[0039] Figure 7 The effect of different cross-linking agent glutaraldehyde concentrations on the content of lysozyme dimer formation;

[0040] Figure 8 This is the chromatogram measured when the H2O2 concentration is 2.5%;

[0041] Figure 9 is the inhibition rate of lysozyme dimer and lysozyme monomer against Staphylococcus aureus;

[0042] Figure 10is the inhibition rate of lysozyme dimer and lysozyme monomer against Propionibacterium acnes;

[0043] Figure 11 is the inhibition rate of lysozyme dimer and lysozyme monomer against Escherichia coli;

[0044] Figure 12 is the inhibition rate of lysozyme dimer and lysozyme monomer against Pseudomonas aeruginosa;

[0045] Figure 13 is the inhibition rate of lysozyme dimer and lysozyme monomer against Candida albicans. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] In the following examples, lysozyme was purchased from Nanning Pangbo Bioengineering Co., Ltd.; 30% hydrogen peroxide solution was purchased from Guangdong Guanghua Science and Technology Co., Ltd.; disodium hydrogen phosphate and glutaraldehyde were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; and citric acid was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd.

[0052] Example 1

[0053] A method for preparing a lysozyme dimer comprises the following steps:

[0054] Accurately weigh 0.5 g of lysozyme sample and add it to 40 mL of H2O2 aqueous solution. Adjust the pH to 3 with citric acid / disodium hydrogen phosphate solution, then add pure water to 50 mL to make the final H2O2 concentration in the solution 10% (V / V). Stir at 120 rpm for 10 minutes to dissolve. After complete dissolution, stir at room temperature for 24 hours. Then, stir and heat in a 75°C water bath for 20 minutes. Cool and add catalase to remove residual H2O2. Then, add glutaraldehyde to a final concentration (V / V) of 0.5% to carry out cross-linking reaction. Finally, terminate the reaction by adding Tris-HCl to obtain lysozyme dimer.

[0055] Example 2

[0056] The only difference from Example 1 is that the pH of this example is 4.

[0057] Example 3

[0058] The only difference from Example 1 is that the pH of this example is 5.

[0059] Example 4

[0060] The only difference from Example 1 is that the pH of this example is 6.

[0061] Example 5

[0062] The only difference from Example 1 is that the pH of this example is 7.

[0063] Comparative Example 1

[0064] The only difference from Example 1 is that the pH of this comparative example is 1.

[0065] Comparative Example 2

[0066] The only difference from Example 1 is that the pH of this comparative example is 2.

[0067] Experimental Example 1 Effect of pH on the Content of Lysozyme Dimer

[0068] The lysozyme dimer prepared in Examples 1-4 and Comparative Examples 1-2 was tested by high performance liquid chromatography for content. A hydrophilic silica gel high-efficiency size exclusion chromatography column was used for the test. The hydrophilic silica gel column used was a TSKgel G2000 SWXL column from Tosoh Corporation, with a column diameter of 7.5 mm and a length of 60 cm. 30.3 g of urea, 6.76 g of potassium dihydrogen phosphate, and 39.642 g of ammonium sulfate were weighed and dissolved in 700 mL of water. 100 mL of acetonitrile was added, and the pH was adjusted to 3 with trifluoroacetic acid. Finally, the volume was fixed to 1 L to obtain the mobile phase. The detection wavelength was 280 nm. The flow rate was 0.35 mL per minute, the sample volume was 20 μL, and the chromatogram was recorded to obtain the lysozyme dimer content. The results are as follows: Figure 1 As shown. Figure 1 It can be seen that changes in environmental pH have a significant impact on the formation of lysozyme dimers. Between pH 1 and 5, an increase in buffer pH promotes the polymerization of lysozyme dimers, reaching a maximum lysozyme dimer content of 49.3% at pH 5. When the pH exceeds 5, the lysozyme dimer content shows a downward trend, which may be due to the fact that the acidic environment facilitates the oxidation of H2O2. Therefore, pH 3-7 was selected for the preparation of lysozyme dimers.

[0069] Example 6

[0070] A method for preparing a lysozyme dimer comprises the following steps:

[0071] Accurately weigh 0.5 g of lysozyme sample and add it to 40 mL of H2O2 aqueous solution. Adjust the pH to 5 with citric acid / disodium hydrogen phosphate solution, then add pure water to 50 mL to make the final H2O2 concentration in the solution 10% (V / V). Stir at 120 rpm for 10 minutes to dissolve. After complete dissolution, stir at room temperature for 24 hours. Then, stir and heat in a 70°C water bath for 20 minutes. Cool and add catalase to remove residual H2O2. Then, add glutaraldehyde to a final concentration (V / V) of 0.5% to carry out cross-linking reaction. Finally, terminate the reaction by adding Tris-HCl to obtain lysozyme dimer.

[0072] Example 7

[0073] The only difference from Example 6 is that the heating temperature in this example is 75°C.

[0074] Example 8

[0075] The only difference from Example 6 is that the heating temperature in this example is 80°C.

[0076] Comparative Example 3

[0077] The difference from Example 6 is that the heating temperature of this comparative example is 40°C.

[0078] Comparative Example 4

[0079] The difference from Example 6 is that the heating temperature of this comparative example is 50°C.

[0080] Comparative Example 5

[0081] The difference from Example 6 is that the heating temperature of this comparative example is 60°C.

[0082] Comparative Example 6

[0083] The difference from Example 6 is that the heating temperature of this comparative example is 90°C.

[0084] Experimental Example 2 Effect of Heating Temperature on the Content of Lysozyme Dimer

[0085] The lysozyme dimers prepared in Examples 6-8 and Comparative Examples 3-6 were tested for content using high performance liquid chromatography. The chromatographic conditions were the same as those in Experimental Example 1. The chromatograms were recorded to obtain the lysozyme dimer content. The results were as follows: Figure 2 As shown. Figure 2 It can be seen that when the temperature reaches 40℃-75℃, the lysozyme dimer content shows an upward trend, with a significant increase at 70℃. The lysozyme dimer content is the highest at 75℃. As the temperature increases, the lysozyme dimer content shows a downward trend, but the lysozyme dimer content at 80℃ is still higher than that at 40℃, 60℃, and 90℃. Excessively high temperature can lead to lysozyme inactivation, resulting in a decrease in the amount of lysozyme dimer generated. Therefore, lysozyme dimer can be generated in large quantities at 75℃.

[0086] Example 9

[0087] Accurately weigh 0.5 g of lysozyme sample and add it to 40 mL of H2O2 aqueous solution. Adjust the pH to 5 with citric acid / disodium hydrogen phosphate solution, then add pure water to 50 mL to make the final H2O2 concentration in the solution 10% (V / V). Stir at 120 rpm for 10 minutes to dissolve. After complete dissolution, stir at room temperature for 24 hours. Then, stir and heat in a 75°C water bath for 10 minutes. Cool and add catalase to remove residual H2O2. Then add glutaraldehyde to a final concentration (V / V) of 0.5% to carry out cross-linking reaction. Finally, terminate the reaction by adding Tris-HCl to obtain lysozyme dimer.

[0088] Example 10

[0089] The only difference from Example 9 is that the heating time in this example is 20 minutes.

[0090] Example 11

[0091] The only difference from Example 9 is that the heating time in this example is 30 minutes.

[0092] Comparative Example 7

[0093] The only difference from Example 9 is that the heating time of this comparative example is 40 min.

[0094] Comparative Example 8

[0095] The only difference from Example 9 is that the heating time of this comparative example is 50 min.

[0096] Comparative Example 9

[0097] The only difference from Example 9 is that the heating time of this comparative example is 60 min.

[0098] Experimental Example 3 Effect of Heating Time on the Content of Lysozyme Dimer

[0099] The lysozyme dimers prepared in Examples 9-11 and Comparative Examples 7-9 were tested for content by high performance liquid chromatography. The chromatographic conditions were the same as those in Experimental Example 1. The chromatograms were recorded to obtain the lysozyme dimer content. The results were as follows: Figure 3 As shown. Figure 3 As can be seen, the lysozyme dimer content increases between 10 and 20 minutes, reaching its highest level at 20 minutes. As the heating time continues to increase, the lysozyme dimer content shows a significant downward trend. Although the lysozyme dimer content at 30 minutes shows a downward trend, it is significantly higher than the lysozyme dimer content at 10, 40, 50, and 60 minutes. Prolonged, high-temperature reactions can easily inactivate and denature a large amount of lysozyme, leading to a sharp decrease in production. Therefore, a 20-minute heating time is ideal for preparing lysozyme dimer.

[0100] Example 12

[0101] Accurately weigh 0.5 g of lysozyme sample and add it to 40 mL of H2O2 aqueous solution. Adjust the pH to 5 with citric acid / disodium hydrogen phosphate solution, then add pure water to 50 mL to make the final H2O2 concentration in the solution 10% (V / V). Stir at 120 rpm for 10 minutes to dissolve. After complete dissolution, stir at room temperature for 24 hours. Then, stir and heat in a 75°C water bath for 20 minutes. Cool and add catalase to remove residual H2O2. Then, add glutaraldehyde to a final concentration (V / V) of 0.5% to carry out a cross-linking reaction. Finally, terminate the reaction by adding Tris-HCl to obtain a lysozyme dimer.

[0102] Comparative Example 10

[0103] The only difference from Example 12 is that this comparative example is stirred at room temperature for 1 hour.

[0104] Comparative Example 11

[0105] The only difference from Example 12 is that this comparative example is stirred at room temperature for 2 h.

[0106] Comparative Example 12

[0107] The only difference from Example 12 is that this comparative example is stirred at room temperature for 6 hours.

[0108] Comparative Example 13

[0109] The difference between this embodiment and embodiment 12 is that the stirring in this embodiment is carried out at room temperature for 48 hours.

[0110] Experimental Example 4 Effect of oxidation time on the content of prepared lysozyme dimer

[0111] The lysozyme dimer prepared in Example 12 and Comparative Examples 10-13 was tested for content by high performance liquid chromatography. The chromatographic conditions were the same as those in Experimental Example 1. The chromatogram was recorded to obtain the lysozyme dimer content. The results are as follows: Figure 4 As shown. Figure 4 It can be seen that when the stirring time (oxidation time) is between 1 h and 24 h, the lysozyme dimer content shows an upward trend, and the lysozyme dimer content is highest at 24 h. As the oxidation time continues to increase, the lysozyme dimer content shows a downward trend. Therefore, an oxidation time of 24 h is selected to prepare lysozyme dimer.

[0112] Example 13

[0113] Accurately weigh 0.5 g of lysozyme sample and add it to 40 mL of H2O2 aqueous solution. Adjust the pH to 5 with citric acid / disodium hydrogen phosphate solution, then add pure water to 50 mL to make the final H2O2 concentration in the solution 10% (V / V). Stir at 120 rpm for 10 minutes to dissolve. After complete dissolution, stir at room temperature for 24 hours. Then, stir and heat in a 75°C water bath for 20 minutes. Cool and add catalase to remove residual H2O2. Then, add glutaraldehyde to a final concentration (V / V) of 0.5% to carry out a cross-linking reaction. Finally, terminate the reaction by adding Tris-HCl to obtain a lysozyme dimer.

[0114] Example 14

[0115] The only difference from Example 13 is that in this example, 0.75 g is weighed into 40 mL of H2O2 aqueous solution, the pH is adjusted to 5 with citric acid / disodium hydrogen phosphate solution, and then pure water is added to make the volume to 50 mL, so that the final concentration of H2O2 in the solution is 10% (V / V) and the lysozyme concentration is 15 mg / mL.

[0116] Example 15

[0117] The only difference from Example 13 is that in this example, 1.0 g is weighed into 40 mL of H2O2 aqueous solution, the pH is adjusted to 5 with citric acid / disodium hydrogen phosphate solution, and then pure water is added to make the volume to 50 mL, so that the final concentration of H2O2 in the solution is 10% (V / V) and the lysozyme concentration is 20 mg / mL.

[0118] Comparative Example 14

[0119] The only difference from Example 13 is that in this comparative example, 0.05 g is weighed into 40 mL of H2O2 aqueous solution, the pH is adjusted to 5 with citric acid / disodium hydrogen phosphate solution, and then pure water is added to make the volume to 50 mL, so that the final concentration of H2O2 in the solution is 10% (V / V) and the lysozyme concentration is 1 mg / mL.

[0120] Comparative Example 15

[0121] The difference from Example 13 is that in this comparative example, 0.25 g is weighed into 40 mL of H2O2 aqueous solution, the pH is adjusted to 5 with citric acid / disodium hydrogen phosphate solution, and then pure water is added to make the volume to 50 mL, so that the final concentration of H2O2 in the solution is 10% (V / V) and the lysozyme concentration is 5 mg / mL.

[0122] Experimental Example 5 Effect of lysozyme concentration on the content of prepared lysozyme dimer

[0123] The lysozyme dimers prepared in Examples 13-15 and Comparative Examples 14-15 were tested for content using high performance liquid chromatography. The chromatographic conditions were the same as those in Experimental Example 1. The chromatograms were recorded to obtain the lysozyme dimer content. The results were as follows: Figure 5 As shown. Figure 5 It can be seen that when the lysozyme concentration is between 1 mg / mL and 15 mg / mL, the lysozyme dimer content shows an upward trend. At 10 mg / mL and 20 mg / mL, it is significantly increased. At 15 mg / mL, the lysozyme dimer content reaches a maximum of 54.6%. As the lysozyme content continues to increase, the lysozyme dimer content does not show an upward trend. Therefore, a lysozyme concentration of 15 mg / mL was selected for the preparation of lysozyme dimer.

[0124] Example 16

[0125] Accurately weigh 0.5 g of lysozyme sample and add it to 40 mL of H2O2 aqueous solution. Adjust the pH to 5 with citric acid / disodium hydrogen phosphate solution, then add pure water to 50 mL to make the final H2O2 concentration in the solution 1% (V / V). Stir at 120 rpm for 10 minutes to dissolve. After complete dissolution, stir at room temperature for 24 hours. Then, stir and heat in a 75°C water bath for 20 minutes. Cool and add catalase to remove residual H2O2. Then, add glutaraldehyde to a final concentration (V / V) of 0.5% to carry out a cross-linking reaction. Finally, terminate the reaction by adding Tris-HCl to obtain a lysozyme dimer.

[0126] Example 17

[0127] The only difference from Example 16 is that the final concentration of H2O2 in this example is 2.5% (V / V).

[0128] Example 18

[0129] The only difference from Example 16 is that the final concentration of H2O2 in this example is 5% (V / V).

[0130] Example 19

[0131] The only difference from Example 16 is that the final concentration of H2O2 in this example is 10% (V / V).

[0132] Comparative Example 16

[0133] The only difference from Example 16 is that the final concentration of H2O2 in this comparative example is 15% (V / V).

[0134] Comparative Example 17

[0135] The only difference from Example 16 is that the final concentration of H2O2 in this comparative example is 20% (V / V).

[0136] Experimental Example 6 Effect of Oxidant Concentration on the Content of Lysozyme Dimer

[0137] The lysozyme dimers prepared in Examples 16-19 and Comparative Examples 16-17 were tested for content using high performance liquid chromatography. The chromatographic conditions were the same as those in Experimental Example 1, and the chromatograms were recorded. The chromatogram of Example 17 was as follows: Figure 8 As shown, the lysozyme dimer content was obtained, and the results were as follows Figure 6 As shown. Figure 6It can be seen that when the H2O2 content is between 1% and 2.5% by volume, the lysozyme dimer content shows an upward trend. The highest lysozyme dimer content is achieved at 2.5% by volume, reaching a maximum of 58.7%. As the H2O2 content continues to increase, the lysozyme dimer content shows a significant downward trend, but it remains significantly higher than 15% to 20% when the H2O2 content is between 1% and 10%. Excessive oxidant concentrations can denature and inactivate proteins, leading to a decrease in lysozyme dimer content. Therefore, a 2.5% by volume H2O2 content is selected for preparing lysozyme dimer.

[0138] Example 20

[0139] Accurately weigh 0.5 g of lysozyme sample and add it to 40 mL of H2O2 aqueous solution. Adjust the pH to 5 with citric acid / disodium hydrogen phosphate solution, then add pure water to 50 mL to make the final H2O2 concentration in the solution 10% (V / V). Stir at 120 rpm for 10 minutes to dissolve. After complete dissolution, stir at room temperature for 24 hours. Then, stir and heat in a 75°C water bath for 20 minutes. Cool and add catalase to remove residual H2O2. Then, add glutaraldehyde to a final concentration (V / V) of 0.4% to carry out a cross-linking reaction. Finally, terminate the reaction by adding Tris-HCl to obtain a lysozyme dimer.

[0140] Example 21

[0141] The difference from Example 20 is that the glutaraldehyde concentration (V / V) in this example is 0.5%.

[0142] Comparative Example 18

[0143] The difference from Example 20 is that the glutaraldehyde concentration (V / V) in this comparative example is 0.1%.

[0144] Comparative Example 19

[0145] The difference from Example 20 is that the glutaraldehyde concentration (V / V) in this comparative example is 0.2%.

[0146] Comparative Example 20

[0147] The difference from Example 20 is that the glutaraldehyde concentration (V / V) in this comparative example is 0.6%.

[0148] Comparative Example 21

[0149] The difference from Example 20 is that the glutaraldehyde concentration (V / V) in this comparative example is 0.8%.

[0150] Experimental Example 7 Effect of the concentration of cross-linking agent glutaraldehyde on the content of prepared lysozyme dimer

[0151] The lysozyme dimers prepared in Examples 20-21 and Comparative Examples 18-21 were tested for content using high performance liquid chromatography. The chromatographic conditions were the same as those in Experimental Example 1. The chromatograms were recorded. The results are shown in FIG. Figure 7 .Depend on Figure 7 It can be seen that when the cross-linking agent glutaraldehyde concentration (V / V) is between 0.1% and 0.4%, the lysozyme dimer content shows an upward trend, and the highest lysozyme dimer content is generated at a cross-linking agent concentration (V / V) of 0.4%. As the glutaraldehyde content continues to increase, the lysozyme dimer content shows a significant downward trend. Although a downward trend appears at a cross-linking agent concentration (V / V) of 0.5%, it is still higher than at other concentrations. Excessively high glutaraldehyde concentrations can denature the protein, resulting in a flocculent lysozyme dimer that cannot be obtained. Therefore, a cross-linking agent glutaraldehyde concentration of 0.4% to 0.5% is selected to prepare lysozyme dimer.

[0152] Experimental Example 8 Antibacterial Effect of Lysozyme Dimer

[0153] 1. Strain source:

[0154] Pseudomonas aeruginosa #5336 (published in the "Catalogue of Chinese Medicinal Microorganisms" and promised to be released to the public for 20 years) used in the experiment was provided and subcultured by Guangxi Medical University; Escherichia coli (published in the "Catalogue of Chinese Microorganisms" and promised to be released to the public for 20 years) and Staphylococcus aureus (published in the "Catalogue of Chinese Microorganisms" and promised to be released to the public for 20 years) were provided and subcultured by Guangxi University of Chinese Medicine; Candida albicans (preservation number: GDMCC NO.2.178) and Propionibacterium acnes (preservation number: GDMCC NO.1.162) were purchased from Guangdong Provincial Microorganism Culture Collection and subcultured; and the above-mentioned Pseudomonas aeruginosa #5336, Escherichia coli, Staphylococcus aureus, Candida albicans, and Propionibacterium acnes were all used in the document "Synthesis, antibacterial activity, and 3D-QASR studies of matrine-indolederivatives as potential antibiotics".

[0155] 2. Reagents and instruments are shown in Table 1.

[0156] Table 1 Reagents and instruments

[0157] Instrument name model Manufacturer Autoclave YX-280B Guangzhou Kangmai Medical Equipment Co., Ltd. Constant temperature oven DKN612C Shanghai Junhe Instrument Co., Ltd. Large ultra-clean workbench SW-CJ-1FDA Beijing Huawei Zhongyi Technology Co., Ltd. Constant temperature incubator FYL-YS-430L Beijing Fuyin Electric Co., Ltd. Adjustable shaker THY-211D Shanghai Zhichu Instrument Co., Ltd. solid culture medium LB agar Beijing Luqiao Technology Co., Ltd. cell culture dish 60mm Wuhan Savier Biotechnology Co., Ltd. Ultrafiltration tube 15mL3KD Millipore

[0158] 3. Preparation of solid culture medium:

[0159] Dissolve 4g of LB agar in 100mL of deionized water and stir with a glass rod until dissolved. Cover the bottle with sealing film and secure with a rubber band to prevent contamination. Stir the culture medium in an autoclave at 121°C for 20 minutes. Remove the culture medium and add 5mL to each culture dish in a clean bench. Cool the mixture and prepare the solid culture medium.

[0160] 4. Antibacterial effect detection method:

[0161] Lysozyme dimer powder was obtained by freeze drying. Lysozyme dimer (lysozyme dimer prepared in Example 17) was weighed and dissolved in sterile distilled water to prepare a solution of the desired concentration. Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans, and Propionibacterium acnes were diluted with sterile saline to a concentration of 1×10 4 cfu / mL-9×10 4 cfu / mL. 5 mL of lysozyme dimer solution of different concentrations, 4 mL of sterile saline and 1 mL of the above-mentioned diluted bacterial suspension were added to each test tube. The final concentrations of lysozyme dimer in the obtained mixed solutions were 1 mg / mL, 2 mg / mL, 4 mg / mL, 8 mg / mL and 16 mg / mL, respectively. After mixing for 10 minutes, 200 μL of solution was added to the solid culture medium, spread evenly with a coating stick, and poured into a constant temperature incubator. After incubation for 24 hours, live bacteria colony technique was performed. At the same time, follow the above steps, use lysozyme monomer (manufacturer: Nanning Pangbo Bioengineering Co., Ltd., specification: 20,000u / mg) as the control group, and sterile saline as the blank control group; repeat the experiment 3 times and calculate the average value. The calculation formula is shown below:

[0162]

[0163] In the formula, α is the average colony count of the blank control group, β is the average colony count of the test sample or the control group, and the results are rounded to integers. The inhibition rates are shown in Tables 2-6 and Figure 9-13 shown.

[0164] Table 2 Staphylococcus aureus inhibition rate

[0165] Lysozyme monomer concentration (mg / mL) Inhibition rate (%) Lysozyme dimer concentration (mg / mL) Inhibition rate (%) 1 2.4 1 11.1 2 41.8 2 75.5 4 44.0 4 96.9 8 57.5 8 100 16 73.3 16 100

[0166] From Table 2 and Figure 9 It can be seen that the lysozyme dimer has a significantly higher inhibition rate against Staphylococcus aureus than the lysozyme monomer, and the antibacterial effect of the lysozyme dimer increases with increasing concentration. At 2 mg / mL-8 mg / mL, the inhibition rate against Staphylococcus aureus can reach over 70%. At a lysozyme dimer concentration of 8 mg / mL, the inhibition rate against Staphylococcus aureus reaches 100%. Therefore, the lysozyme dimer prepared by the present invention has a significantly improved antibacterial effect against Staphylococcus aureus (Gram-positive bacteria).

[0167] Table 3 Propionibacterium acnes inhibition rate

[0168]

[0169]

[0170] From Table 3 and Figure 10 It can be seen that lysozyme monomer has no significant antibacterial effect on Propionibacterium acnes, but lysozyme dimer has a significant antibacterial effect, which increases with increasing concentration. At 4 mg / mL-8 mg / mL, the inhibition rate against Propionibacterium acnes can reach over 70%. At a lysozyme dimer concentration of 8 mg / mL, the inhibition rate against Propionibacterium acnes reaches 100%. Therefore, the lysozyme dimer prepared by the present invention has a significantly improved antibacterial effect against Propionibacterium acnes (Gram-positive bacteria).

[0171] Table 4 Escherichia coli inhibition rate

[0172] Lysozyme monomer concentration (mg / mL) Inhibition rate (%) Lysozyme dimer concentration (mg / mL) Inhibition rate (%) 1 0.6 1 60.2 2 0.9 2 75.6 4 8.0 4 85.6 8 14.6 8 100 16 31.7 16 100

[0173] From Table 4 and Figure 11 It can be seen that the lysozyme dimer has a significantly higher inhibition rate against Escherichia coli than the lysozyme monomer, and the antibacterial effect of the lysozyme dimer increases with increasing concentration. At 1 mg / mL-8 mg / mL, the inhibition rate against Escherichia coli can reach more than 60%. At a lysozyme dimer concentration of 8 mg / mL, the inhibition rate against Escherichia coli reaches 100%. Therefore, the lysozyme dimer prepared by the present invention has a significantly improved antibacterial effect against Escherichia coli (Gram-negative bacteria).

[0174] Table 5 Pseudomonas aeruginosa inhibition rate

[0175] Lysozyme monomer concentration (mg / mL) Inhibition rate (%) Lysozyme dimer concentration (mg / mL) Inhibition rate (%) 1 0.4 1 68.9 2 0.9 2 82.4 4 1.2 4 95.9 8 6.9 8 100 16 16.6 16 100

[0176] From Table 4 and Figure 12 It can be seen that the lysozyme dimer has a significantly improved inhibition rate against Pseudomonas aeruginosa compared to the lysozyme monomer, and the antibacterial effect of the lysozyme dimer increases with increasing concentration. At 1 mg / mL-8 mg / mL, the inhibition rate against Pseudomonas aeruginosa can reach more than 65%. When the lysozyme dimer concentration is 8 mg / mL, the inhibition rate against Pseudomonas aeruginosa reaches 100%. Therefore, the lysozyme dimer prepared by the present invention has a significantly improved antibacterial effect on Pseudomonas aeruginosa (Gram-negative bacteria).

[0177] Table 6 Candida albicans inhibition rate

[0178] Lysozyme monomer concentration (mg / mL) Inhibition rate (%) Lysozyme dimer concentration (mg / mL) Inhibition rate (%) 1 0 1 14.4 2 0 2 16.4 4 0.3 4 58.8 8 0.5 8 91.4 16 0.6 16 100

[0179] From Table 6 and Figure 13As shown, lysozyme monomer had no antibacterial effect against Candida albicans, but lysozyme dimer exhibited a significant antibacterial effect, which increased with increasing concentration. At concentrations of 4 mg / mL to 8 mg / mL, the inhibition rate against Candida albicans reached over 55%. At a concentration of 16 mg / mL, the inhibition rate against Candida albicans reached 100%. Therefore, the lysozyme dimer prepared by the present invention exhibited an antibacterial effect against Candida albicans (fungus) compared to lysozyme monomer.

[0180] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a lysozyme dimer, characterized in that: The following steps are involved: After mixing lysozyme and an oxidant, sequentially performing a pH adjustment treatment, a stirring treatment, a heating treatment, a cooling treatment, and a cross-linking treatment to obtain the lysozyme dimer; The oxidant is a hydrogen peroxide solution; the volume percentage of hydrogen peroxide in the mixed solution obtained by mixing the lysozyme and the oxidant is 1%-10%; the concentration of lysozyme in the mixed solution obtained by mixing the lysozyme and the oxidant is 10mg / mL-20mg / mL; The pH value is 3-7; The stirring process includes a first stirring process and a second stirring process; the first stirring process has a rotation speed of 100-150 rpm and a time of 10-25 minutes; the second stirring process has a time of 24 hours; The heating temperature is 70-80°C and the time is 10-30 minutes; The cross-linking agent used in the cross-linking treatment is glutaraldehyde solution; the volume percentage of glutaraldehyde in the mixed solution obtained by the cross-linking treatment is 0.4%-0.5%.

2. A lysozyme dimer prepared by the preparation method according to claim 1.

3. Use of the lysozyme dimer according to claim 2 in the preparation of antibacterial products.

4. The use according to claim 3, characterized in that The antimicrobial products include antimicrobial drugs and disinfectants.

Images