A method for preparing tubular bacterial cellulose hydrogel using a superhydrophobic mold

Abstract

The present invention relates to a method for preparing a tubular bacterial cellulose hydrogel using a super-hydrophobic mold, and belongs to the field of preparation of bacterial cellulose hydrogel. The present invention immerses the super-hydrophobic mold in a bacterial culture medium, so that an air film is formed between the surface of the super-hydrophobic mold and the bacterial culture medium, bacteria gather around the air film and secrete bacterial cellulose, and a layer of bacterial cellulose hydrogel is generated on the surface of the super-hydrophobic mold. The tubular bacterial cellulose hydrogel prepared by the present invention is integrally formed, does not need to be processed multiple times, and avoids the generation of cracks.

Description

Technical Field

[0001] The invention relates to the field of preparation of bacterial cellulose hydrogel, and more specifically, to a method for preparing tubular bacterial cellulose hydrogel by using a super-hydrophobic mold. Background Art

[0002] As a natural polymer material, bacterial cellulose hydrogel has good biocompatibility and degradability, and is widely used in biomedical devices, cosmetics, tissue engineering scaffolds, wound dressings and other fields. In the field of tissue engineering, bacterial cellulose hydrogel is designed to be tubular, which can be well used in vascular stents and artificial blood vessels. The production of traditional bacterial cellulose hydrogel is carried out by large-scale fermentation culture of Acetobacter xylinum. The culture medium containing Acetobacter xylinum has a high oxygen content at the liquid-gas junction, which induces Acetobacter xylinum to secrete a layer of bacterial cellulose hydrogel film at the gas-liquid junction. Therefore, if you want to prepare tubular bacterial cellulose hydrogel, you can only curl and bond the thin film of bacterial cellulose hydrogel. The preparation process is relatively complicated, the conditions are difficult to control, and the obtained tubular bacterial cellulose hydrogel is easy to tear at the bonding point, affecting the use effect, so we need to develop a new bacterial cellulose preparation method to obtain the required tubular bacterial cellulose hydrogel. Summary of the invention

[0003] The present invention solves the problem that the prior art cannot effectively produce tubular bacterial cellulose hydrogels. The present invention immerses a super-hydrophobic mold in a bacterial culture medium, so that an air film is formed between the surface of the super-hydrophobic mold and the bacterial culture medium. Bacteria gather around the air film and secrete bacterial cellulose, and a layer of bacterial cellulose hydrogel is generated on the surface of the super-hydrophobic mold. The tubular bacterial cellulose hydrogel prepared by the present invention is integrally formed, does not require multiple processing, and avoids the generation of cracks.

[0004] According to the purpose of the present invention, there is provided a method for preparing a tubular bacterial cellulose hydrogel using a superhydrophobic mold, comprising the following steps:

[0005] (1) immersing a straight super-hydrophobic mold into a culture medium, so that an air film is formed between the surface of the super-hydrophobic mold and the culture medium; the culture medium is used to cultivate microorganisms that can produce bacterial cellulose;

[0006] (2) After sterilizing the culture medium and the super-hydrophobic mold, microorganisms capable of producing bacterial cellulose are inoculated into the culture medium; the microorganisms gather around the air film in step (1) and secrete bacterial cellulose, so that a layer of bacterial cellulose hydrogel is generated on the surface of the super-hydrophobic mold.

[0007] Preferably, the super-hydrophobic mold is in the shape of a sheet, a cylinder or a prism.

[0008] Preferably, the prism shape is a triangular prism shape or a quadrangular prism shape.

[0009] Preferably, the upper end of the super-hydrophobic mold is fixed to the side wall of the container.

[0010] Preferably, the superhydrophobic mold is suspended in a culture medium.

[0011] Preferably, the super hydrophobic mold is obtained by coating a mold material with a nano-silicon dioxide dispersion and drying the resulting mold material.

[0012] Preferably, the superhydrophobic mold contact angle is greater than 150°.

[0013] Preferably, the microorganism is Acetobacter xylinum, Agrobacterium, Rhizobium or Sarcina.

[0014] Preferably, after step (2), the bacterial cellulose hydrogel generated on the surface of the superhydrophobic mold is demolded, and then soaked in alcohol and boiled in NaOH solution until white or transparent, and finally the tubular bacterial cellulose hydrogel is treated to be neutral using ultrapure water.

[0015] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[0016] The present invention proposes a method for preparing a tubular bacterial cellulose hydrogel using a super-hydrophobic mold. The microorganism (preferably Acetobacter xylinum) that can produce bacterial cellulose has an aerobic tendency to secrete bacterial cellulose, and the super-hydrophobic material has a very strong hydrophobic ability. The surface stable contact angle of the super-hydrophobic material is greater than 150°, and the rolling contact angle is less than 10°. Immersing the super-hydrophobic material in water can form an air film. The present invention immerses the super-hydrophobic mold in bacteria (preferably Acetobacter xylinum) culture medium, forms a layer of air film between the surface of the super-hydrophobic membrane tool and the bacteria (preferably Acetobacter xylinum) culture medium, sterilizes the bacteria (preferably Acetobacter xylinum) culture medium and the super-hydrophobic mold together and inoculates Acetobacter xylinum, and is placed under suitable conditions for cultivation, bacteria (preferably Acetobacter xylinum) gather around the air film and secrete bacterial cellulose, after sufficient culture time, the super-hydrophobic mold surface will generate a layer of bacterial cellulose hydrogel, the bacterial cellulose hydrogel generated on the super-hydrophobic mold surface is demoulded, and then is respectively soaked in alcohol and boiled in NaOH solution to white or transparent, and finally ultrapure water is used to process the tubular bacterial cellulose hydrogel to neutrality to obtain the tubular bacterial cellulose hydrogel prepared by the present invention. The method for preparing tubular bacterial cellulose hydrogel using super-hydrophobic molds involved in the present invention is simple to operate, easy to realize large-scale production, and the prepared tubular bacterial cellulose hydrogel is integrally formed, does not need to be processed multiple times, avoids the generation of cracks, and can prepare tubular bacterial cellulose hydrogels of specific inner diameters by controlling the size of the super-hydrophobic mold. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of a method for preparing tubular bacterial cellulose hydrogel using a superhydrophobic mold according to the present invention.

[0018] Figure 2 are photos of a superhydrophobic mold and a non-superhydrophobic mold immersed in a culture medium of Acetobacter xylinum.

[0019] Figure 3 This is a schematic diagram of the preparation of tubular bacterial cellulose hydrogel using a superhydrophobic mold.

[0020] Figure 4 The invention discloses a specific step for preparing a tubular bacterial cellulose hydrogel by using a super-hydrophobic mold.

[0021] Figure 5 This is a photo of tubular bacterial cellulose prepared using a superhydrophobic mold.

[0022] Figure 6 is a photograph of a tubular bacterial cellulose hydrogel cultured according to the present invention.

[0023] In all the drawings, the same reference numerals are used to denote the same elements or structures, wherein: 1-superhydrophobic mold, 2-culture container, 3-Acetobacter xylinum culture medium, 4-air membrane, 5-liquid surface, 6-Acetobacter xylinum, 7-tubular bacterial cellulose hydrogel. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0025] According to the present invention, the microorganism (preferably Acetobacter xylinum) that secretes bacterial cellulose has aerotaxis and can secrete bacterial cellulose at the liquid-gas interface where there is sufficient oxygen. The secreted bacterial cellulose exists in the form of a gel in the culture medium.

[0026] According to the present invention, a method for preparing a tubular bacterial cellulose hydrogel using a superhydrophobic mold is provided, and the specific steps include:

[0027] Step 1: Prepare a super-hydrophobic mold of a required size, prepare a culture container filled with Acetobacter xylinum culture medium, fix the super-hydrophobic mold in the culture container in a vertical posture, and immerse it in the Acetobacter xylinum culture medium for a period of time.

[0028] Step 2: Prepare a sterile environment.

[0029] Step 3: Inoculate Acetobacter xylinum seeds in a sterile environment.

[0030] Step 4: Place the culture container inoculated with Acetobacter xylinum seeds in an incubator for continuous cultivation for 4 days.

[0031] Step 5: Take out the super-hydrophobic mold and demold the bacterial cellulose hydrogel generated on the surface of the super-hydrophobic mold. Soak the demolded bacterial cellulose hydrogel in alcohol and boil it in NaOH solution until it turns white or transparent. Use ultrapure water to treat the tubular bacterial cellulose hydrogel to be neutral.

[0032] In some embodiments, the super hydrophobic mold is obtained by coating a mold material with a nano-silicon dioxide dispersion and drying the mold material; the mold material is paper, fiber material or rough wood.

[0033] According to the present invention, the super-hydrophobic mold required in step 1 has super-hydrophobic properties, and its contact angle is greater than 150°.

[0034] According to the present invention, the required super-hydrophobic mold can form an air film in the Acetobacter xylinum culture medium.

[0035] According to the present invention, the Acetobacter xylinum culture medium required in step 1 is prepared by containing 6.8 g / L disodium hydrogen phosphate dodecahydrate, 5 g / L yeast extract, 5 g / L peptone, 1.5 g / L citric acid monohydrate, and 20 g / L glucose. The Acetobacter xylinum culture medium can provide sufficient nutrition for the growth of Acetobacter xylinum and the secretion of bacterial cellulose.

[0036] According to the present invention, the sterile environment prepared in step 2 is a clean bench environment in the laboratory.

[0037] According to the present invention, the inoculation process of the Acetobacter xylinum seeds involved in step 3 is completed in a sterile environment, and is completed in a clean bench in a laboratory. The sterile environment avoids contamination by environmental bacteria and ensures the normal growth of Acetobacter xylinum and the normal secretion of bacterial cellulose after inoculation.

[0038] According to the present invention, the optimum culture temperature of Acetobacter xylinum is 30°C, and the incubator environment in step 4 is generally 30°C.

[0039] According to the present invention, the continuous culture for 4 days in step 4 can be adjusted according to the specific growth conditions of Acetobacter xylinum and the secretion conditions of bacterial cellulose.

[0040] According to the present invention, the super-hydrophobic mold in step 5 has bacterial cellulose hydrogel wrapped on its surface after the cultivation is completed, and the wrapped bacterial cellulose hydrogel can be removed by demoulding.

[0041] According to the present invention, the tubular bacterial cellulose hydrogel after demoulding in step 5 needs to be treated with alcohol, NaOH solution, pure water, etc. The purpose of alcohol soaking is to kill the xylinum Acetobacter present in the hydrogel. The purpose of using NaOH for cooking is to remove the residual culture medium and the endotoxins produced during the growth of xylinum Acetobacter, and the NaOH used for cooking is generally 1 mol / L. The ultrapure water treatment to neutrality is to treat the tubular bacterial cellulose hydrogel to neutrality for subsequent use.

[0042] According to the present invention, the process of forming the tubular bacterial cellulose hydrogel is completed in a culture container.

[0043] According to the present invention, the method for preparing tubular bacterial cellulose hydrogel using a superhydrophobic mold is easy to operate and has a simple process. Large-scale production can be achieved by placing multiple superhydrophobic molds in a culture container and increasing the number of culture containers.

[0044] According to the method for preparing tubular bacterial cellulose hydrogel using a superhydrophobic mold, the inner diameter of the prepared tubular bacterial cellulose hydrogel is the same as the outer diameter of the superhydrophobic mold, and the size of the air film can be changed by changing the size of the superhydrophobic mold, thereby adaptively producing tubular bacterial cellulose hydrogels of different diameters.

[0045] According to the present invention, the length of the tubular bacterial cellulose hydrogel prepared by the method for preparing a tubular bacterial cellulose hydrogel using a superhydrophobic mold is the same as the length of the superhydrophobic mold immersed in the Acetobacter xylinum culture medium, and the length of the generated air film can be changed by changing the length of the superhydrophobic mold immersed in the Acetobacter xylinum culture medium, thereby adaptively producing tubular bacterial cellulose hydrogels of different lengths.

[0046] According to the present invention, the thickness of the tubular bacterial cellulose hydrogel prepared by the method for preparing a tubular bacterial cellulose hydrogel using a superhydrophobic mold is related to the culture time. Within a certain time range, tubular bacterial cellulose hydrogels of different thicknesses can be adaptively produced by changing the culture time.

[0047] The present invention solves the problem that the prior art cannot effectively produce tubular bacterial cellulose hydrogels, and proposes a method for preparing tubular bacterial cellulose hydrogels using a superhydrophobic mold. The present invention immerses the superhydrophobic mold 1 in a xylinum acetobacter culture medium 3 through a liquid surface 5, so that a layer of air film 4 is formed between the surface of the superhydrophobic membrane 1 and the xylinum acetobacter culture medium 3, and the xylinum acetobacter culture medium 3 and the superhydrophobic mold 1 are sterilized together and inoculated with xylinum acetobacter 6, and cultured under suitable conditions, and the xylinum acetobacter 6 gathers around the air film 4 and secretes bacterial cellulose. After a sufficient culture time, a layer of tubular bacterial cellulose hydrogel 7 will be wrapped on the surface of the superhydrophobic mold 1, and the tubular bacterial cellulose hydrogel 7 generated on the surface of the superhydrophobic membrane 1 is demoulded, and then soaked in alcohol and boiled with a NaOH solution until white or transparent, and finally the tubular bacterial cellulose hydrogel 7 is treated with ultrapure water to be neutral to obtain the tubular bacterial cellulose hydrogel 7 prepared by the present invention. The method for preparing a tubular bacterial cellulose hydrogel using a superhydrophobic mold involved in the present invention is simple to operate and easy to realize large-scale production. The prepared tubular bacterial cellulose hydrogel is integrally formed and does not require multiple processing to avoid the generation of cracks. By controlling the size of the superhydrophobic mold, a tubular bacterial cellulose hydrogel with a specific inner diameter can be prepared.

[0048] Example 1

[0049] In order to elaborate on the method for preparing tubular bacterial cellulose hydrogel using superhydrophobic molds involved in the present invention, this embodiment will be combined with Figure 1 In this embodiment, the super-hydrophobic mold 1 is immersed in the Acetobacter xylinum culture medium 3, so that an air film 4 is formed between the surface of the super-hydrophobic mold 1 and the Acetobacter xylinum culture medium 3, and the Acetobacter xylinum 6 gathers around the air film 4 and secretes bacterial cellulose, and a tubular bacterial cellulose hydrogel 7 is generated on the surface of the super-hydrophobic mold 1.

[0050] Example 2

[0051] In order to explain the method of immersing the super-hydrophobic mold 1 in the Acetobacter xylinum culture medium 3 in Example 1 so that an air film 4 is formed between the surface of the super-hydrophobic film 1 and the Acetobacter xylinum culture medium 3, this example will be combined with Figure 2 In this embodiment, the super-hydrophobic mold 1 is immersed in the Acetobacter xylinum culture medium 3, and an air layer 4 is formed, and the air layer 4 presents a silvery gas state. In this embodiment, the non-super-hydrophobic mold is immersed in the Acetobacter xylinum culture medium 3, and no silvery air film appears.

[0052] Example 3

[0053] In order to explain the specific principle of Acetobacter xylinum 6 gathering around the air film 4 and secreting bacterial cellulose in Example 1, this example will be combined with Figure 3In this embodiment, Acetobacter xylinum 6 has aerotaxis and moves to places with high oxygen content, and secretes bacterial cellulose using oxygen as raw material, and the super-hydrophobic mold 1 is immersed in the Acetobacter xylinum culture medium 3 to form a layer of air film 4, which is connected to the atmosphere and contains oxygen components, so that Acetobacter xylinum can move around the air film 4 and secrete bacterial cellulose. In the process of secreting bacterial cellulose, Acetobacter xylinum gradually consumes the oxygen in the air film 4 and makes the secreted bacterial cellulose tightly wrapped on the surface of the super-hydrophobic mold, forming a layer of tubular bacterial cellulose hydrogel 7.

[0054] Example 4

[0055] In order to explain the process of preparing tubular bacterial cellulose hydrogel using superhydrophobic mold described in Example 1, this example will be combined with Figure 4 The specific preparation process of the tubular bacterial cellulose hydrogel 7 prepared by using a superhydrophobic mold designed in the present invention is described.

[0056] Step 1: Prepare a super-hydrophobic mold of a required size, prepare a culture container 2 filled with Acetobacter xylinum culture medium 3, fix the super-hydrophobic mold 1 in the culture container in a vertical posture, and immerse it in the Acetobacter xylinum culture medium 3 for a period of time.

[0057] Step 2: Prepare a sterile environment.

[0058] Step 3: Inoculate Acetobacter xylinum seeds in a sterile environment.

[0059] Step 4: The culture container 2 inoculated with the Acetobacter xylinum 6 seeds is placed in an incubator for continuous culture for 4 days.

[0060] Step 5: Take out the superhydrophobic mold 1 and demold the bacterial cellulose hydrogel generated on the surface of the superhydrophobic mold 1. The demolded bacterial cellulose hydrogel is soaked in alcohol and boiled in NaOH solution until it turns white or transparent. Ultrapure water is used to treat the tubular bacterial cellulose hydrogel 7 to be neutral.

[0061] Example 5

[0062] The method for preparing tubular bacterial cellulose hydrogel using a superhydrophobic mold involved in the present invention is simple and can be mass-produced. In this embodiment, the method described in the present invention is used to expand the culture under laboratory conditions, such as Figure 5 As shown, a plurality of superhydrophobic molds 1 are placed in a culture container 2, and the total number of culture containers 2 is increased, so that a larger number of tubular bacterial cellulose hydrogels 7 can be prepared at one time.

[0063] Example 6

[0064] The method for preparing a tubular bacterial cellulose hydrogel using a superhydrophobic mold according to the present invention can prepare a tubular bacterial cellulose hydrogel 7 with a specific inner diameter by changing the size of the superhydrophobic mold 1 .

[0065] Example 7

[0066] The method for preparing tubular bacterial cellulose hydrogel using a superhydrophobic mold according to the present invention can cultivate tubular bacterial cellulose hydrogel 7. Figure 6 a and b in the figure show the tubular bacterial cellulose hydrogel 7 after culture and treatment, which is in the shape of an elongated tube, white in color and has high light transmittance; Figure 6 The tubular bacterial cellulose hydrogel 7 shown in c in the figure is freeze-dried, showing the white color of dry bacterial cellulose, with good shape preservation and can be re-moistened after being soaked in water.

[0067] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a tubular bacterial cellulose hydrogel using a superhydrophobic mold, characterized in that: The following steps are involved: (1) vertically fixing a straight super-hydrophobic mold in a culture container and immersing it in a culture medium, so that an air film is formed between the surface of the super-hydrophobic mold and the culture medium; the culture medium is used to culture Acetobacter xylinum; The super hydrophobic mold is obtained by coating the mold material with a nano-silicon dioxide dispersion and drying it. The surface stable contact angle of the super hydrophobic mold is greater than 150° and the rolling contact angle is less than 10°. (2) After sterilizing the culture medium and the super-hydrophobic mold, inoculating Acetobacter xylinum into the culture medium; the Acetobacter xylinum aggregates around the air film in step (1) and secretes bacterial cellulose, so that bacterial cellulose hydrogel is generated on the surface of the super-hydrophobic mold.

2. The method for preparing tubular bacterial cellulose hydrogel using a superhydrophobic mold according to claim 1, characterized in that: The super hydrophobic mold is in the shape of a sheet, a cylinder or a prism.

3. The method for preparing tubular bacterial cellulose hydrogel using a superhydrophobic mold according to claim 2, characterized in that: The prism shape is a triangular prism shape or a quadrangular prism shape.

4. The method for preparing tubular bacterial cellulose hydrogel using a superhydrophobic mold according to claim 1, characterized in that: The upper end of the super hydrophobic mold is fixed on the side wall of the container.

5. The method for preparing tubular bacterial cellulose hydrogel using a superhydrophobic mold according to claim 1 or 4, characterized in that: The superhydrophobic mold is suspended in culture medium.

6. The method for preparing tubular bacterial cellulose hydrogel using a superhydrophobic mold according to claim 1, characterized in that: After step (2), the bacterial cellulose hydrogel generated on the surface of the super-hydrophobic mold is demolded, and then soaked in alcohol and boiled in a NaOH solution until it becomes white or transparent, and finally the tubular bacterial cellulose hydrogel is treated with ultrapure water to be neutral.

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