Preparation method of bacterial cellulose membrane with traditional Chinese medicine antibacterial function

Abstract

The invention relates to a preparation method of a bacterial cellulose membrane with a traditional Chinese medicine antibacterial function, which is characterized in that a scutellaria baicalensis leach liquor is used as a substrate, and functional bacterial cellulose loaded with baicalin and baicalein is synthesized through microbial fermentation. Determining that the functional bacterial cellulose membrane contains characteristic compounds such as baicalin and baicalein through liquid phase detection; an electron microscope detects that a characteristic compound of scutellaria baicalensis exists in a three-dimensional network structure of the bacterial cellulose membrane, and antibacterial function detection shows that the bacterial cellulose membrane has an inhibition effect on staphylococcus aureus and escherichia coli. According to the method, the bacterial cellulose is synthesized in situ by adopting a traditional Chinese medicine substrate, the method is simple, convenient and green, the antibacterial concentration is explored, and the synthesized bacterial cellulose has a relatively good antibacterial function and has a wide application prospect in the field of biomedical materials.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine and biopharmaceutical materials, and specifically relates to a bacterial cellulose membrane containing the antibacterial function of traditional Chinese medicine prepared by using natural traditional Chinese medicine as a substrate and synthesized by microorganisms, and can be applied in the field of biological antibacterial. Background Art

[0002] Bacterial cellulose (BC) is a natural cellulose nanomaterial formed by enzymatic polymerization of glucose via β-1,4-glycosidic bonds using Gram-negative aerobic bacteria as a substrate. BC possesses advantages such as a fine network structure, high purity, good biocompatibility, non-toxicity, high mechanical strength, high swelling capacity, and controllable synthesis. Physical, chemical, and microbiological methods can be used to impart antibacterial and anti-inflammatory properties to BC, leading to its widespread application in food, biomedicine, and environmental fields.

[0003] Currently, methods for imparting antimicrobial properties to bacterial cellulose using physical impregnation and chemical modification are well established. Physical impregnation conditions are mild, but the interaction between the functional substances and bacterial cellulose is weak, resulting in a loose bond. Chemical modification, on the other hand, involves modifying the hydroxyl groups of bacterial cellulose with chemical substances, allowing the functional substances to bind to the bacterial cellulose through chemical or metallic bonds. This creates a stronger effect, but the chemicals are often toxic, impacting the environment and product safety.

[0004] To impart antimicrobial properties to bacterial cellulose, the main antimicrobial agents used include metal ions such as silver, zinc, and titanium; organic antimicrobials such as alcohols and quaternary ammonium salts; and natural extracts. However, the process of loading these metal ions or quaternary ammonium salts presents safety concerns. Selecting an excellent antimicrobial substance and enhancing the antimicrobial activity of bacterial cellulose membranes through safe and environmentally friendly synthesis methods are pressing technical challenges facing those skilled in the art. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing a bacterial cellulose film with the antibacterial function of traditional Chinese medicine.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for preparing a bacterial cellulose film having the antibacterial function of traditional Chinese medicine comprises the following steps:

[0008] (1) Using a characteristic culture medium as a solvent, the raw material of Scutellaria baicalensis was extracted at 80-85°C for 3 hours, and the sediment was filtered to remove the extract containing the effective antibacterial components of Scutellaria baicalensis; the mass ratio of Scutellaria baicalensis to the solvent was 1:10-50;

[0009] (2) The extract is sterilized by high pressure to obtain a synthetic culture medium of bacterial cellulose, which is then cooled for later use;

[0010] (3) Taking out the refrigerated bacterial cellulose synthesizing bacteria, inoculating them into LB solid medium under sterile conditions, and streaking the plates to activate the microorganisms;

[0011] (4) inoculating the activated microorganisms into the synthetic culture medium of step (2) by single colony picking, and culturing in a shaking incubator at 30° C. and 200 rpm for 24 h to obtain a seed solution;

[0012] (5) inoculating the seed solution into the synthetic culture medium of step (2) at an inoculum amount of 5%-10%, culturing in a shaking incubator at 30° C. and 200 rpm for 24 hours, and then culturing statically for 5 days to obtain a functional bacterial cellulose membrane;

[0013] (6) The functional bacterial cellulose membrane is rinsed with deionized water for 5-6 times to remove surface microorganisms and other impurities, thereby obtaining a functional bacterial cellulose membrane containing effective antibacterial components of Scutellaria baicalensis.

[0014] The characteristic culture medium composition of step (1) is: 1L water, 20g glucose, and 5g yeast powder.

[0015] In step (1), the raw material of Scutellaria baicalensis is crushed into 200-mesh granules with a water content of less than 10%;

[0016] The mass ratio of the scutellaria raw material to the solvent is 1:10.

[0017] The high-pressure sterilization conditions of the extract in step (2) are: high-pressure sterilization at 121° C. for 20 min.

[0018] The LB solid culture medium in step (3) was prepared by mixing 3 g of beef extract, 10 g of peptone, 5 g of NaCl, and 18 g of agar powder, adding distilled water to make up to 1 L, and sterilizing by autoclaving at 121° C. for 20 min.

[0019] Beneficial effects of the present invention:

[0020] 1. The present invention uses Scutellaria baicalensis as a substrate, prepares an appropriate ratio of Scutellaria baicalensis extract, and adopts microbial fermentation to obtain a bacterial cellulose membrane with antibacterial function.

[0021] 2. Liquid chromatography-mass spectrometry (LC-MS) analysis of the active ingredients of traditional Chinese medicine in the soaking solution confirmed the presence of the corresponding characteristic compounds, baicalin and baicalein, on the BC membrane. Electron microscopy confirmed the presence of these compounds within the 3D network structure of the BC membrane. Infrared spectroscopy confirmed the presence of characteristic peaks for baicalin on the BC membrane, demonstrating its successful binding to the membrane. Antibacterial testing confirmed the excellent antibacterial efficacy of the baicalin and baicalein compounds loaded into the BC membrane. Experiments confirmed that the BC membrane exhibited an inhibition zone diameter of 0.9 cm against Escherichia coli and 1.4 cm against Staphylococcus aureus.

[0022] 3. The present invention adopts traditional Chinese medicine substrates and utilizes bacterial cellulose synthesizing bacteria to directly synthesize bacterial cellulose. The method is simple and green, and the antibacterial concentration is clearly explored. The synthesized bacterial cellulose has excellent antibacterial function and has broad application prospects in the field of biomedical materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The culture process of bacterial cellulose membrane under different solid-liquid ratio conditions;

[0024] Figure 2 Liquid phase detection diagram of bacterial cellulose membrane with solid-liquid ratio of 1:10;

[0025] Among them, a is the liquid chromatogram of standard baicalin and baicalein, and b is the liquid chromatogram of bacterial cellulose membrane with a solid-liquid ratio of 1:10;

[0026] Figure 3 Scanning electron microscope images of cellulose membranes under different solid-liquid ratios;

[0027] Figure 4 IR spectra of bacterial cellulose membranes under different solid-liquid ratios;

[0028] Figure 5 Antibacterial results of bacterial cellulose membranes under different solid-liquid ratios. DETAILED DESCRIPTION

[0029] The specific embodiments of the present invention are further described in detail below with reference to the examples.

[0030] Example 1 Preparation method of bacterial cellulose membrane with traditional Chinese medicine antibacterial function

[0031] The scutellaria baicalensis root is crushed and extracted with a characteristic culture medium as a solvent at a certain ratio to obtain a culture medium. After microorganisms are inoculated into the culture medium, a bacterial cellulose membrane is synthesized. The specific steps are as follows:

[0032] (1) Prepare characteristic culture medium: 20 g glucose, 5 g yeast powder and 1 L water, adjust pH to 6.5, and sterilize.

[0033] (2) 100 g of Scutellaria baicalensis raw material was taken and crushed into 200-mesh Chinese medicine granules (with a water content of less than 10%). The culture medium of step (1) was used as a solvent and a mixed solution with a solid-liquid mass ratio of 1:10 was used for extraction. The extraction was carried out at 85° C. for 3 h. The residue was filtered and removed to obtain an extract containing the effective antibacterial components of Scutellaria baicalensis.

[0034] (3) The scutellaria baicalensis extract was sterilized by high pressure at 121°C for 20 min to obtain a synthetic culture medium of bacterial cellulose, which was then cooled in a refrigerator for later use.

[0035] (4) Prepare LB solid medium: 3 g beef extract, 10 g peptone, 5 g NaCl, and 18 g agar powder. Add distilled water to 1 L and sterilize by autoclaving at 121°C for 20 min.

[0036] (5) Take out the refrigerated laboratory-isolated Bacillus xylosus from the refrigerator, take 100 μL of it and inoculate it into LB solid culture medium under sterile conditions, and use a coating stick to streak the plate to activate the microorganisms.

[0037] (6) The activated microorganisms in step (5) were further inoculated into 50 mL of bacterial cellulose synthetic culture medium in step (3) by single colony picking, and cultured in a shaking incubator at 30° C. and 200 rpm for 24 h to obtain seed solution.

[0038] (7) The seed liquid obtained in step (6) was inoculated into 100 mL of the bacterial cellulose synthesis medium in step (3) at a 10% inoculum amount, and cultured on a shaking table at a temperature of 30°C and 200 rpm for 24 hours, and then cultured at 30°C for 5 days to obtain a bacterial cellulose film.

[0039] (8) The obtained bacterial cellulose membrane was washed 5 times with deionized water to remove surface microorganisms and other impurities, thereby obtaining a clean functional bacterial cellulose membrane containing the effective ingredients of Scutellaria baicalensis.

[0040] Example 2 Preparation method of bacterial cellulose membrane with traditional Chinese medicine antibacterial function

[0041] The process steps are basically the same as those in Example 1, except that different ratios of Scutellaria baicalensis and culture medium are used to prepare the bacterial cellulose membrane.

[0042] The extraction was performed using Scutellaria baicalensis and culture medium at a solid-liquid mass ratio of 1:20, 1:30, 1:40, and 1:50. The other steps were the same as in Example 1 to obtain bacterial cellulose films containing Scutellaria baicalensis medicinal ingredients under different solid-liquid ratios. The culture process of bacterial cellulose films with different solid-liquid ratios can be found in Figure 1 .

[0043] As the static culture time increases, Scutellaria baicalensis extract culture media with different solid-liquid ratios can synthesize bacterial cellulose films, and the films gradually thicken. The experiment also found that bacterial cellulose synthesized at a higher solid-liquid ratio is slower, indicating that an excessively high solid-liquid ratio will inhibit the synthesis of bacterial cellulose by microorganisms.

[0044] Example 3 Detection and characterization of the effective components of the functional bacterial cellulose membrane of the present invention

[0045] 3.1 Active ingredient detection

[0046] The obtained bacterial cellulose membrane was soaked in a PBS buffer solution with a pH of 6.5-7.5 for 24 hours, and the effective components in the soaking solution were detected by liquid phase analysis.

[0047] The bacterial cellulose membrane in Example 1 of the present invention was subjected to liquid phase detection. Figure 2 Figure a is the liquid chromatogram of standard baicalin and baicalein, and Figure b is the liquid chromatogram with a solid-liquid ratio of 1:10. It can be seen that the peak time of baicalin in the soaking solution is 5.093 min, and the peak time of baicalin of the standard is 5.069 min; the peak time of baicalein is 22.613 min, and the peak time of baicalein of the standard is 22.582 min, which are basically consistent.

[0048] The test results show that the bacterial cellulose membrane of the present invention still retains the effective antibacterial components of Scutellaria baicalensis after being washed with deionized water for many times, and the bacterial cellulose membrane contains baicalin as the main component.

[0049] 3.2 Structural characterization The bacterial cellulose membrane was freeze-dried and its surface structural characteristics were observed using a scanning electron microscope.

[0050] Figure 3 The microstructures of cellulose membranes grown under different solid-liquid ratios are shown in Figure 5. (a) 1:10 solid-liquid ratio; (b) 1:20 solid-liquid ratio; (c) 1:30 solid-liquid ratio; (d) 1:40 solid-liquid ratio; (e) 1:50 solid-liquid ratio; and (f) bacterial cellulose (BC) grown without Scutellaria baicalensis extract. The scale bar in the figure is 500 μm.

[0051] As can be seen from the figure, the bacterial cellulose membrane cultured without Scutellaria baicalensis extract has fine cellulose and many pores, while the cellulose at high solid-liquid ratios of 1:10 and 1:20 is tightly bound, has few pores, and has a good three-dimensional network structure. This structure can well encapsulate the effective ingredients of traditional Chinese medicine such as baicalin and baicalein; the low solid-liquid ratio of 1:30-1:50 has a less tight binding and more pores.

[0052] 3.3 Functional group characterization

[0053] The effective components present in the bacterial cellulose membrane of the present invention were characterized by infrared spectrometer.

[0054] Figure 4 BC in the middle is bacterial cellulose without Scutellaria baicalensis extract, 1:10-1:50 respectively represent bacterial cellulose membrane synthesized with Scutellaria baicalensis extract at a solid-liquid ratio of 1:10-1:50, and pure BC at 3345cm -1 , 2 916cm -1 The peaks at 1060 cm-1 correspond to the -OH and CH bending vibrations on cellulose. -1 The peaks near the basalin can be attributed to the stretching vibration peaks of the CO bond. The characteristic peaks of baicalin include 1659 (1660) cm -1 Nearby conjugated carbonyl stretching vibration, 1600 (1608) and 1500 (1449) cm -1 Vibration of benzene ring skeleton, single substitution of benzene ring 750 (740) cm -1 , CH bending vibration 1465-1340(1469)cm -1 , carboxylic acid C=O peak at 1760-1700 (1720) cm -1 , COH bending vibration 1298 cm -1 BC membrane at 3500-3000cm -1 There is a wide hydroxyl band at the center, while the Scutellaria baicalensis / BC composite materials with different proportions have a wider band, indicating that the number of hydroxyl groups increases. At the same time, the composite materials include the characteristic peaks of baicalin, conjugated carbon group, benzene ring skeleton vibration peak, etc., which shows the successful combination of baicalin.

[0055] Example 4 Antibacterial effect test of the functional bacterial cellulose film of the present invention

[0056] Detection method: Escherichia coli and Staphylococcus aureus were spread on LB plates for antibacterial detection of bacterial cellulose membranes.

[0057] The bacterial cellulose membrane was prepared into a 6 mm bacterial cellulose membrane sheet, which was aseptically attached to an LB solid plate coated with Escherichia coli or Staphylococcus aureus. The antibacterial effect of the membrane sheet was determined by the disc diffusion method.

[0058] The test results show that when the solid-liquid ratio of Scutellaria baicalensis and culture medium is 1:10, the diameter of the inhibition zone for Escherichia coli reaches 0.9 cm; the inhibition zone for Staphylococcus aureus is 1.4 cm. Other solid-liquid ratios have almost no antibacterial effect on Escherichia coli; the size of the inhibition zone for Staphylococcus aureus varies with the solid-liquid ratio. The inhibition zone is 1.2 cm when the solid-liquid ratio is 1:20, 1.1 cm when the solid-liquid ratio is 1:30, 1.1 cm when 1:40, and 0.9 cm when 1:50. This shows that the effect of Scutellaria baicalensis extract on Gram-positive bacteria is stronger than that on Gram-negative bacteria, and the microorganisms that synthesize bacterial cellulose are Gram-negative bacteria, which is conducive to their production of bacterial cellulose. At the same time, when the solid-liquid ratio of Scutellaria baicalensis extract is high, the inhibitory effect on the two bacteria is enhanced, resulting in a slow process of synthesizing bacterial cellulose (see Figure 5 ).

[0059] It can be seen that the bacterial cellulose membrane synthesized in the present invention has excellent antibacterial properties.

Claims

1. A method for preparing bacterial cellulose membrane with antibacterial function of traditional Chinese medicine, characterized in that: The following steps are involved: (1) Using a characteristic culture medium as a solvent, extracting the raw material of Scutellaria baicalensis at 80-85° C. for 3 hours, filtering to remove the sediment, and obtaining an extract containing effective antibacterial components of Scutellaria baicalensis; the mass ratio of Scutellaria baicalensis to the solvent is 1:10-50; (2) sterilizing the extract by high pressure to obtain a synthetic culture medium of bacterial cellulose, and cooling it for later use; (3) taking out the refrigerated bacterial cellulose-producing bacteria, inoculating them into LB solid medium under sterile conditions, and streaking the plates to activate the microorganisms; (4) inoculating the activated microorganisms into the synthetic culture medium of step (2) by single colony picking, and culturing in a shaking incubator at 30° C. and 200 rpm for 24 h to obtain a seed solution; (5) inoculating the seed solution into the synthetic culture medium of step (2) at an inoculation rate of 5% to 10%, culturing in a shaking incubator at 30° C. and 200 rpm for 24 h, and then culturing statically for 5 days to obtain a functional bacterial cellulose membrane; (6) Rinse the functional bacterial cellulose membrane with deionized water for 5-6 times to remove surface microorganisms and other impurities, thereby obtaining a functional bacterial cellulose membrane containing effective antibacterial components of Scutellaria baicalensis.

2. The preparation method according to claim 1, characterized in that: The characteristic culture medium composition of step (1) is: 1L water, 20g glucose, and 5g yeast powder.

3. The preparation method according to claim 1, characterized in that: In step (1), the raw material of Scutellaria baicalensis is crushed into 200-mesh granules with a water content of less than 10%.

4. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of the scutellaria raw material to the solvent is 1:

10.

5. The preparation method according to claim 1, characterized in that: The high-pressure sterilization conditions of the extract in step (2) are: high-pressure sterilization at 121° C. for 20 min.

6. The preparation method according to claim 1, characterized in that: Preparation method of the LB solid culture medium in step (3): Mix 3 g beef extract, 10 g peptone, 5 g NaCl and 18 g agar powder, add distilled water to make up to 1 L, and sterilize under high pressure at 121° C. for 20 min.

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