CN101914434B - Device and method for dynamically preparing heterocavity bacterium cellulose materials - Google Patents

Abstract

The invention relates to a device and method for dynamically preparing special-shaped cavity bacterial cellulose materials. The device includes a thermometer port, an acid liquid and alkali liquid addition port, a handle, a ventilation port, a pH meter port, a nutrient feeding port, a rotating shaft, and a fermenter , a motor, a water bath device, and a mould; its preparation includes: after the bacterial cellulose production strain is inserted into the liquid medium for expansion, it is transferred to a fermentation device equipped with a mold of a specific shape for dynamic perturbation culture, and the empty space can be harvested after cultivation. Lumen profiled cellulose material. The special-shaped cavity BC material prepared by the invention is not only controllable in size and shape, but also the mold of the fermentation device can be disassembled and reused, and the preparation method is simple and easy, the cost is low, the production efficiency is high, and it is suitable for industrial production; Special-shaped cavity BC materials can be widely used as substitutes for hollow organs such as artificial blood vessels and nerve fiber catheters, and can also be used as food wrapping materials such as meat casings and jelly casings.

Description

Device and method for dynamically preparing special-shaped cavity bacterial cellulose material

technical field

The invention belongs to the field of preparation of bacterial cellulose materials, in particular to a device and method for dynamically preparing special-shaped cavity bacterial cellulose materials.

Background technique

Bacterial Cellulose (BC) is a kind of pure cellulose produced by microorganisms. Bacterial cellulose, like plant fiber, is a straight chain formed by β-D-glucose through β-1,4-glucosidic bonds. The straight chains are parallel to each other, not in a helical conformation, and without branching structure, also known as β - 1,4-glucan. Bacterial cellulose has unique bioaffinity, biocompatibility and no allergic reaction, as well as high water holding capacity, degree of polymerization, crystallinity, good three-dimensional nanofiber network structure, high tension and strength, especially good Therefore, it has a wide range of uses in artificial blood vessels, tissue engineering scaffolds, artificial skin, and treatment of skin injuries. It is one of the hotspots in international biomedical materials research.

At present, the preparation of bacterial cellulose materials generally adopts the conventional static culture method, and a piece of cellulose film is obtained. Using conventional liquid deep paddle vertical stirring technology will cause the variation and degeneration of Acetobacter xylinum, causing it to no longer synthesize cellulose, while the dynamic fermentation technology (in the air-lift fermenter) commonly used at present that causes less strain degradation can only Granular bacterial cellulose materials can be prepared. Because cellulose is soluble in certain chemical reagents, it has long been hoped that the bacterial cellulose solution will be poured into a mold to "cast" a shaped cavity biomaterial. However, the effect is not ideal, and the obtained cellulose product does not have the excellent characteristics of the original bacterial cellulose, which may be due to its physical structure being changed and resulting in performance degradation. Since the synthesis of cellulose by Acetobacter xylinum is an oxygen-consuming secondary metabolic process, cellulose is easily formed at the junction of air and culture solution, so oxygen is an important factor for the formation of cellulose. Existing studies have shown that as long as a mold with good surface oxygen permeability and a certain shape is provided in the static fermentation broth, a cellulose film of this shape can be formed on the surface of the mold, so that it is easy to prepare tangible biomedical materials by using bacteria on-line fermentation. become possible.

The first successful preparation of a shaped bacterial cellulose product using this principle was a glove-shaped artificial skin (UKPatent 12,169,543; White and Brown, 1989). The technology uses oxygen-permeable hand-shaped micro-woven fabrics (microwaven textiles) as a mold and cultivates them under static conditions. In 1990 and 1991, according to the same principle, the Japanese Yamanaka used an oxygen-permeable hollow tube, such as a mold made of cellophane, Teflon, silica gel, ceramics, etc., to inject a culture solution containing live bacteria under static fermentation conditions. Preparation of artificial blood vessels has been successful (EP Patent 0,396,344; JP Patent 3,272,772). He implanted the prepared BC tube material into the aorta and jugular vein with adult mongrel dogs as the experimental subjects, and found that although there was slight thrombus adsorption at the suture of the blood vessel and the inner wall of the BC tube, the BC tube always maintained good patency Spend. In 2001 and 2003, German Klemm et al. immersed a mold consisting of a glass cylinder inside and a glass tube outside into bacterial culture solution, and successfully prepared small-diameter artificial blood vessels (1-3mm inner diameter) through static fermentation (trade name: BASYC). , and proved that the antithrombotic effect of the artificial blood vessel is very good through mouse experiments (WO Patent 0,161,026, US Patent 2,003,013,163). Rats were not treated with any anticoagulant drugs, and it was observed that the carotid artery-BC tube complex was wrapped by connective tissue, covered with small blood vessels similar to vasa vasorum, and the BC tube was completely wrapped by living tissue without any rejection reaction. All implanted BC tubes maintained a 100% patency rate after surgery, and there was no phenomenon of thrombus clotting or tissue proliferation. It can be seen that the hollow bacterial cellulose tube is a good material for preparing artificial blood vessels <6mm. In addition, the hollow fiber tube can also be used as a trachea, ureter, cartilage scaffold, a sheath covering nerve fibers, and a substitute for certain hollow organs, etc. (WO Patent 0,161,026; US Patent 2,003,013,163). In 2005, Svensson et al. from the United States and the Swedish International Cooperation Group reported the experimental results of using bacterial cellulose as a cartilage tissue engineering scaffold, and pointed out that the effect was better. In 2006, Backdahl et al pointed out that the material could be used for tissue engineering blood vessels in the future by studying the mechanical properties of bacterial cellulose and the interaction with human smooth muscle cells. The above studies have proved that the gel-like tubular BC material is used as a vascular tissue substitute in microsurgery because of its high mechanical strength, large water holding capacity, very regular inner surface and excellent biocompatibility. It has great application prospect.

In addition to being used in medical materials such as artificial blood vessels, BC can also be used in traumatic dressings such as artificial skin. The unique three-dimensional network structure of microbial cellulose has many "pores" inside, has good air permeability and water permeability, and can absorb 60 to 700 times its dry weight of water, which exists in the form of free water. Using BC as a wound dressing can quickly absorb wound blood and tissue fluid, prevent wound infection and suppuration, and provide a moist environment for tissue regeneration near chronic wounds to promote wound healing and reduce pain. At the same time, the cellulose will not adhere to the wound, will not cause secondary damage, and will not remain when peeled off. For the first time, the glove-shaped artificial skin successfully prepared by using oxygen-permeable hand-shaped micro-woven fabrics for mold static culture has good effects in the treatment of large-area burn/scald hand skin. Since 1987, Brazil has continuously reported more than 400 examples of good therapeutic effects of bacterial cellulose membranes on burns, scalds, bedsores, frostbite, skin grafts and chronic skin ulcers. Commercial dressings such as artificial skin made from it (trade name BioFill), gauze, bandages and "band-aids" are now on the market. Another BC wound dressing called Xcell has also been used to promote healing of chronic wounds and has also shown good therapeutic results. Studies have shown that this BC dressing can promote wound healing more effectively than dressings made of other materials in the application of chronic wounds. Compared with other artificial skin and traumatic dressings, the main features of this film are high mechanical strength under wet conditions, good permeability to liquid, gas and electrolyte, good compatibility with skin, and non-irritating. It can effectively relieve pain, prevent bacterial infection and absorb the fluid exuded from the wound, promote the rapid healing of the wound, and is conducive to the growth of skin tissue. The film is used as a carrier for slow-release drugs to carry various drugs, which is beneficial to drug delivery on the skin surface, and promotes the healing and rehabilitation of wounds. Therefore, the cellulose has broad market application prospects as a biological material with great application potential.

However, the disadvantages of the prior art methods are that specific oxygen-permeable molds and static fermentation preparation are required, the production cycle is long, the production cost is high, and the production efficiency is low, so industrial production and application cannot be carried out.

Contents of the invention

The technical problem to be solved by the present invention is to provide a device and method for dynamically preparing a special-shaped cavity bacterial cellulose material. Not only the size and shape of the special-shaped cavity BC material prepared by the method are controllable, but also the mold of the fermentation device can be controlled. It is disassembled and reused, and the preparation method is simple and easy, with low cost and high production efficiency, and is suitable for industrial production; the special-shaped cavity BC material can be widely used as a substitute for hollow organs such as artificial blood vessels and nerve fiber catheters, and can also be used as a Food packaging materials such as meat casings, jelly casings, etc.

A device for dynamically preparing special-shaped cavity bacterial cellulose materials according to the present invention includes a thermometer port, an acid liquid alkali liquid addition port, a handle, a vent port, a pH meter port, a nutrient feeding port, a rotating shaft, a fermenter, a motor, and a water bath The device, the mold, is characterized in that: one end of the rotating shaft is connected to a fixed motor output shaft, and the other end of the rotating shaft extends into the fermentation tank from the center of one end surface of the fermentation tank; a mold is fixed on the rotation shaft in the fermentation tank.

The rotating shaft is installed and fixed with a turntable in the inner cavity of the fermenter, and more than two molds are symmetrically fixed on the rotating shaft through the peripheral edge of the turntable.

The mold is stick-shaped or glove-shaped.

The mold is a glove-shaped mold, wherein more than one glove-shaped mold is sequentially inserted on the rotating shaft.

The cross-sectional shape of the mold is circular, square, oval, triangular, heart-shaped or five-pointed star-shaped.

The mold is a solid or hollow structure.

The thermometer port, the acid and alkali liquid addition port, the pH meter port, the nutrient feeding port, the ventilation port and the handle are all located on the outer surface of the fermenter.

The fermenter has any shape.

The material of the mold is glass, pottery, purple sand, metal, silica gel, wood, cellulose, rubber, polyester, nylon (Nylon), Orlon (Orlon), polyvinyl alcohol (PVA), polyvinyl alcohol (Ivalon) , polyester (Dacron), Teflon (Teflon), expanded polytetrafluoroethylene (ePTFE) and silk and other polymer materials, as well as one or more combinations of inorganic organic materials such as plastics.

A kind of method for dynamically preparing special-shaped cavity bacterial cellulose material of the present invention comprises:

(1) Bacteria culture

Insert the bacterial cellulose production strain into the liquid medium for expansion, and cultivate it on a shaking table under the conditions of 20-30°C and 100-250r/min or static culture for 12-48 hours before use;

(2) Fermentation preparation of heterosexual cavity BC material

Transfer the liquid culture medium containing the production strain prepared in step (1) to the above-mentioned fermentation device, then rotate the mold at a speed of 3-20rpm to carry out disturbed culture, and after dynamic culture at 20-32°C for 4-20 days, you can harvest the empty lumen profiled cellulose material;

(3) Material handling

Remove the prepared hollow special-shaped bacterial cellulose material from the mold, soak it in 0.5-2wt% NaOH solution, and treat it in a water bath at 70-100°C for 30-120 minutes, so that the bacterial cellulose material is white and translucent; Then wash until neutral to obtain the special-shaped bacterial cellulose product.

The BC production strains in the step (1) are Acetobacter sp., Gluconobacter sp., Gluconacetobacter sp., Rhizobium sp. ), Sarcina sp., Pseudomounas sp., Achromobacter sp., Alcaligenes sp., Aerobacter sp. , Azotobacter sp., Agrobacterium sp., Seudomonas cepacia, Campylobacter jejuni or kombucha; wherein the preferred strain is Acetobacter xylinum (Acetobacterxylinum) or kombucha;

Among them, the strains other than kombucha are inserted into the liquid seed medium according to the inoculation amount of 2 to 3 inoculation loops; Culture medium;

Or prepare the liquid seeds of the production bacteria first, and then transfer them to the liquid fermentation medium; the strains other than kombucha are inserted into the liquid seed medium to prepare the seed liquid according to the inoculation amount of 2 to 3 inoculation loops, and then press 3vol% to 20vol % of the inoculum is transferred to the liquid fermentation medium; kombucha is inserted into the liquid seed medium according to the inoculum of 1-10 discs with a diameter of 0.5cm containing bacteria BC film, and 1-10 discs with a diameter of 0.5-1cm The inoculum of the disc containing bacteria BC membrane was transferred to the liquid fermentation medium.

The components of the liquid seed culture medium and liquid fermentation medium other than kombucha are: per 1L of water, 20-200g of mannitol, glucose, maltose, sucrose or fructose, 3g of peptone or tryptone, yeast extract 5g, pH3.0-7.5, sterilized at 121°C for 20min; or mannitol, glucose, sucrose or fructose 20-200g, yeast extract 5g, peptone or tryptone 5g, citric acid 115g, Na ₂ HPO ₄ 2.7g, water 1L, pH3.0-7.5, sterilized at 121°C for 20min;

Kombucha liquid seed medium and liquid fermentation medium are composed of: (1) per 1L of water, 1-10g of green tea or black tea (5g of tea leaves is optimal), 10-200g of glucose, sucrose or fructose, peptone or tryptone 3g of peptone, 5g of yeast extract, pH3.0-7.5, pasteurized for 30min; (2) Glucose, sucrose or fructose, green tea or black tea, and water are made into a medium, wherein the mass ratio of sugar, tea, and water 5:0.1-0.4:100-200, pH3.0-7.5, pasteurized for 30 minutes; or (3) per 1L of water, mannitol, glucose, sucrose or fructose 20-200g, peptone or tryptone 3g, yeast Extract 5g, pH 3.0-7.5, sterilized at 121°C for 20 minutes; among them, the optimal medium is the formula of (1).

The liquid medium containing the production strain in the step (2) is the liquid seed medium containing the production strain prepared in step (1) or the liquid fermentation medium containing the production strain.

In addition, in order to accelerate the formation of cellulose film on the mould, air with an oxygen content of 1-100 vol% can be injected into the rotating shaft or the inner cavity of the mould. When the oxygen content is 100 vol%, pure oxygen is introduced.

The existing technology for preparing BC hollow special-shaped materials generally adopts a single-layer oxygen-permeable mold, which is low in efficiency and has rough outer or inner surfaces. In the container, the mold rotates around the axis of the rotating shaft on the turntable, so that the mold is slowly and cyclically submerged in the liquid culture medium, and the bacterial cellulose in the fermentation broth is entangled and adsorbed to form a certain shape on the surface of the mold. Hollow bacterial cellulose material.

Because it is a substatic culture (the rotation speed of the rotating shaft is very slow, <20rpm, and the bacteria grow in a substatic environment), the mold can be continuously alternated in the liquid medium and the oxygen-containing medium, which not only provides enough for the liquid medium The dissolved oxygen in the liquid promotes the rapid reproduction of bacteria in the liquid and the production of cellulose, which is beneficial to the winding of the mold; it also helps the bacteria in the cellulose film fixed on the mold to have access to nutrients, and at the same time allows the bacteria to fully contact Oxygen, thereby promoting the proliferation of bacterial cells and secreting cellulose, and efficiently preparing bacterial cellulose materials. A variety of factors contributed to the dramatic increase in production efficiency.

According to the size and shape of the BC material to be prepared, select the appropriate mold material to prepare special-shaped BC materials with cross-sectional shapes such as circles, squares, ellipses, triangles, hearts, and five-pointed stars.

The special-shaped hollow bacterial cellulose material prepared by the present invention can be used as a new type of nano-biological material not only for artificial blood vessels and sheaths covering nerve fibers, but also for artificial trachea, ureter, tissue engineering scaffolds, artificial skin, meninges, and It can be used as a substitute for certain hollow organs; or it can be used as an edible food wrapping material (such as meat casings, jelly casings, etc.), and has a very broad application range and bright application prospects.

Beneficial effect

(1) Compared with other physical and chemical methods, the special-shaped cavity BC material prepared by the present invention retains its unique three-dimensional network nanostructure, high chemical purity, high crystallinity, high degree of polymerization, high strength and good biological properties. Compatibility and other advantages; and the preparation method is simple and easy, low cost, high production efficiency, suitable for large-scale industrial production;

(2) The length, inner diameter, thickness and other sizes and shapes of the special-shaped cavity BC material prepared by the present invention are not limited, can be manually regulated, and the inside and outside of the material have smooth and even surfaces;

(3) The mold of the fermentation device can be disassembled and reused, and the prepared various special-shaped BC materials can be peeled off without any damage on the surface; in order to further improve the preparation efficiency of the special-shaped BC materials, the horizontal bioreactor can Equipped with multiple molds to increase production.

Description of drawings

Figure 1a is a schematic diagram 1 of a fermentation device for preparing tubular bacterial cellulose materials, wherein the mold is a stick or a pipe, and the cavity of the mold can be filled with oxygen or air;

Figure 1b is a schematic diagram 2 of a fermentation device for preparing hand-shaped bacterial cellulose materials, wherein the mold is hand-shaped, and the cavity of the mold can be filled with oxygen or air;

Fig. 2 is the design model of the turntable structure for preparing tubular bacterial cellulose material;

Figure 3 shows the results of bacterial cellulose prepared from different rod-shaped mold materials, the top is a silica gel rod, the middle is a wooden rod, and the bottom is a frosted glass rod;

Fig. 4 is the real photo of the fermentation device equipped with frosted glass rod molds for preparing tubular bacterial cellulose materials;

Fig. 5 is the actual picture of the tubular bacterial cellulose material prepared by fermentation for 5-7 days;

Figure 6 is an electron microscope image of the tube-shaped bacterial cellulose material prepared by fermentation for 5 days; wherein A is the outer surface, B is the inner surface, C is a section (cross section) perpendicular to the direction of the central axis of the tube, and D is the direction along the central axis of the tube Section (longitudinal section);

Fig. 8 is a graph showing the absolute tensile force (axial tensile force) of bacterial cellulose tubes tested along the direction of the central axis of the tube as a function of time for 1 to 8 days of fermentation;

Fig. 9 is a graph showing the absolute tensile force (radial tensile force) and the relative dry weight tensile force over time of bacterial cellulose tubes prepared by fermentation for 1-8 days along the direction perpendicular to the central axis of the tube.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Taking the preparation of tubular BC materials as an example

Example 1

1. Design and assembly of fermentation equipment

1.1 Design thinking

Since Acetobacterxylinum belongs to aerobic bacteria, it needs a lot of oxygen in the long-term fermentation and metabolism process. The growth of bacterial cellulose belongs to adsorption growth and must be supported by the medium. Therefore, the bacterial cellulose must be grown in a certain shape. , bacterial cellulose must be grown aerobically on a mold material of a specific shape. If the bacterial cellulose material to be prepared is a tubular material, stick-shaped, rod-shaped or tubular molds should be considered, not only to allow the bacterial cellulose to grow on these molds, but also to provide sufficient oxygen. For this reason, we have designed a A horizontal bioreactor equipped with a stick-shaped mold, which allows the mold to be slowly circulated and repeatedly submerged in the liquid medium for disturbed culture, so that the bacterial cellulose produced in the medium can be wound and adsorbed on the mold, and sometimes submerged in the medium The cycle of supplementing nutrients in the medium and exposing to air or oxygen is repeated until the desired tubular bacterial cellulose material is formed in the culture. In order to speed up the growth and synthesis and secretion of cellulose fixed on the mold, it is very helpful to fill the mold with 1-100% oxygen or air.

1.2 Structural characteristics of horizontal disturbance bioreactor

The reactor is composed of upper and lower half cylinders or glass containers of other shapes. The volume of the bioreactor is 1000mL, the effective volume is 500mL, the turntable is driven by a motor, and the liquid volume of the reactor is 300-350mL. The schematic diagram of the equipment is shown in the figure As shown in 1a-b: there is an opening with a diameter of 1cm on the top, and the temperature, ventilation and feeding operations are performed respectively. The fermented liquid in the reactor is immersed in about 2/5 of the turntable to ensure that when a stick-shaped mold rotates to the lowest end, the entire mold can be completely submerged in the fermented liquid.

1.3 Turntable structure design of bioreactor

The mold is rotated at a certain speed, and the bacterial cellulose is wound on the mold. Therefore, the most important part of this experiment is on the rod-shaped mold fixed on the turntable.

Two different types of turntables are used, one is that the mold is clamped between two turntables, the other is that the turntable is at one end of the mold, and the other end leaves a certain length of mold (see Figure 2), the required tubular bacterial fiber The element mainly grows and winds on the mold, and the two turntables mainly support the mold.

1.4 Screening of reactor turntable and rod mold materials

Considering that the tube-shaped bacterial cellulose material is prepared in this test, the turntable disc is composed of non-toxic polyethylene plastic sheet or other non-toxic materials, and the rotating shaft is composed of glass rods or stainless steel rods and other rigid materials. Due to the consideration of the toxicity of the mold material, the adsorption of bacterial cellulose, the mechanical strength and the ease of processing, it can be glass, ceramics, purple sand, metal, silica gel, wood, cellulose, rubber, polyester, nylon (Nylon), Orlon (Orlon), polyvinyl alcohol (PVA), polyvinyl alcohol (Ivalon), polyester (Dacron), Teflon (Teflon), expanded polytetrafluoroethylene (ePTFE) and silk and other polymers One or more combinations of materials and inorganic organic materials such as plastics. This experiment focuses on choosing wood, glass, and silica gel for research. Three different types of materials were placed on the turntables respectively, cultured, and the growth and shape of the cellulose were observed (see Figure 3). The experimental results show that within the same number of days, the tubular materials formed are wooden rods, frosted glass rods, and silicone tubes in order from thick to thin. See Figure 4 for the real photo of the bioreactor with the frosted glass rod as the mold. The physical pictures of the prepared bacterial cellulose tubes of different sizes are shown in Fig. 5 .

Example 2

As shown in Figure 1, a device for dynamically preparing bacterial cellulose materials with special-shaped cavities, a simple and efficient fermentation device for dynamically producing bacterial cellulose materials with special-shaped cavities, including a thermometer port 1, an acid and alkali liquid addition port 2, Handle 3, ventilation port 4, pH meter port 5, nutrient feeding port 6, rotating shaft 8, fermentation tank 9, motor 10, water bath device 11, mold, characterized in that: one end of the rotating shaft 8 is connected to the output shaft of the fixed motor 10 The other end of the rotating shaft 8 extends into the fermenting tank 9 from the center of one end of the fermenting tank 9; a mold is fixed on the rotating shaft 8 in the fermenting tank 9.

The material of mold 12 is selected from glass, pottery, purple sand, metal, silica gel, wood, cellulose, rubber, polyester, nylon, Orlon, polyvinyl alcohol, polyvinyl alcohol, polyester, Teflon, expanded polytetrafluoroethylene , silk, plastic in one or a combination of several.

The rotating shaft 8 is installed and fixed with a turntable 7 on the inner cavity of the fermenter 9 close to one end of the fermenter 9, and the rotating shaft 8 is symmetrically fixed with a stick-shaped tubular mold 12 through the periphery of the turntable 7, and the stick-shaped tubular mold 12 is solid or hollow. structure.

Thermometer port 1, acid liquid alkali liquid addition port 2, pH meter port 5, nutrient feeding port 6, ventilation port 4, and handle 3 are all located on the outer surface of fermenter 9.

The cross-sectional shape of the stick-shaped tubular mold 12 is circular, square, elliptical, triangular, heart-shaped or five-pointed star-shaped.

When in use, by transferring the liquid culture medium containing the production strains to the fermentation device of the present invention, and then rotating the mold at a speed of 3-20rpm for disturbing culture, the cavity special-shaped cellulose material can be harvested.

Example 3

As shown in Figure 2, the difference between this embodiment and embodiment 2 is only:

(1) The shape of the mold is different. In the present embodiment, the mold is a glove-shaped mold 12, and the glove-shaped mold 12 is a solid or hollow structure. The cross-sectional shape of the glove-shaped mold 12 is circular, square, oval, triangle, heart shape or pentagram;

(2) In the device of the present embodiment, there is no need for the turntable to fix the glove-shaped molds, and only one or more glove-shaped molds need to be inserted sequentially on the rotating shaft.

Example 4

The difference between this embodiment and embodiment 2 only lies in:

The shapes of the moulds are different. In the present embodiment, the mold is a glove-shaped mold 12, and the rotating shaft 8 is symmetrically fixed with more than two glove-shaped moulds; the glove-shaped mold 12 is a solid or hollow structure. The cross-sectional shape is round, square, oval, triangular, heart-shaped or pentagram.

Example 5

(1) Bacteria culture

Acetobacter xylinum (Acetobacter xylinum) was inserted into 300mL liquid culture medium (this embodiment takes the following culture medium formula as example: every 1L of water, mannitol, glucose, maltose, sucrose or fructose 20-200g, peptone or tryptone 3g, Yeast extract 5g, pH3.0-7.5, sterilized at 121°C for 20min; or mannitol, glucose, sucrose or fructose 20-200g, yeast extract 5g, peptone or tryptone 5g, citric acid 115g, Na ₂ HPO ₄ 2.7 g, water 1L, pH 3.0-7.5, sterilized at 121°C for 20 minutes) for expansion, cultured on a shaking table at 20-30°C, 100-250r/min or standing for 12-48 hours before use;

(2) Fermentation preparation of hollow shaped bacterial cellulose material

The liquid medium containing the production strain prepared in step (1) was transferred to a bioreactor equipped with a rod-shaped mold, and then the mold was rotated to disturb the cultivation at a speed of 7, 15, 30 and 60 rpm, and the 4- After 20 days, the cavity shaped cellulose material can be harvested;

Or get the activated bacterial classification of step (1) to insert in 300mL fermentation medium with 10% inoculum size, place in 500mL Erlenmeyer flask, cultivate 4 hours under the condition of 30 ℃ and 160rpm, then ferment The solution was transferred to the bioreactor, and then the mold was rotated to disturb the culture at the speed of 7, 15, 30 and 60rpm, and cultured at 30°C for different days to prepare tubular bacterial cellulose materials with different thicknesses.

The fermentation device is composed of a horizontal stirring reactor and a rotating shaft on which several stick-shaped molds are fixed on a turntable, forming a horizontally disturbed bioreactor. Through the rotation of the mold on the turntable around the axis of the rotating shaft, the mold wraps the bacterial cellulose in the fermentation broth to form a hollow bacterial cellulose material of a certain shape on the surface of the mold.

Since Acetobacter xylinum is an aerobic microorganism and is sensitive to external shear force, it is not only necessary to consider the supply of oxygen, but also to consider the impact of shear force (ie rotational speed) on the bacteria and bacteria when designing the bioreactor. For the influence of cellulose winding forming, four lower rotational speeds of 7, 15, 30, and 60rpm were selected in this experiment to study the influence of rotational speed on cellulose forming.

Table 1 Effect of rotational speed on the forming of tubular materials

It can be seen from Table 1 that at 7rpm and 15rpm, the tubular material can be formed, but when it is increased to 30rpm or above, the tubular material is difficult to form. When the rotational speed was 7rpm, more gel-like cellulose was formed at the bottom of the bioreactor during the fermentation process, but less when the rotational speed was 15rpm and higher. This is because when the rotational speed is low, the shear force in the bioreactor is insufficient, resulting in the aggregation of cellulose, and the aggregation of cellulose is unfavorable for the shaping of the tubular material.

(3) Material handling

Remove the prepared cavity special-shaped BC material from the mold, rinse it with distilled water several times, then soak it in 0.5-2wt% NaOH solution, and treat it in a water bath at 70-100°C for 30-120min, so that the BC tubular material is white and translucent After that, it can be washed with an aqueous solution containing 0.5mol/L acetic acid for 4 to 5 times, then repeatedly washed with pure water until neutral, and then refrigerated to obtain the special-shaped bacterial cellulose product.

(4) Characterization of tubular BC materials

A. Scanning electron microscope observation of tubular bacterial cellulose

The samples obtained by freeze-drying were respectively subjected to scanning electron microscope (SEM) observation of four parts: the outer surface of the tube, the inner surface of the tube, the section perpendicular to the direction of the central axis of the tube (cross section), and the section along the direction of the central axis of the tube (longitudinal section) (Fig. 6 ).

From the scanning electron microscope photos of tubular bacterial cellulose, it can be seen that the bacterial cellulose dynamically synthesized by Acetobacter xylinum has the same ultra-fine network structure as the statically prepared film, which is formed by cross-linking high-density microfibers. As shown in the photographs, the outer surface has relatively regular but indistinct trends. It can be seen from Figure (6A) that the growth of bacterial cellulose tends to extend in a certain direction. In the SEM photo of the inner surface of the tubular bacterial cellulose in Figure (6B), it can be found that the growth direction of the cellulose is basically irregular. This may be that the growth of the bacterial cellulose in the early stage was mainly grown on the glass column by adsorption, and the growth was irregular. . From the electron micrographs of the cross-section and longitudinal section of the tubular bacterial cellulose material (Figure 6C and 6D), it can be seen that the growth of cellulose has obvious regularity, and the cellulose grows intertwined in a certain direction, and each layer of cellulose There is also interweaving among them, forming a three-dimensional network structure, which is obviously different from the static cultured tubular cellulose material, and the cross-sectional electron microscope of bacterial cellulose under static culture clearly shows layering. However, the tubular material prepared by the dynamic method in this experiment did not appear delamination.

B. X-ray diffraction analysis

The X-ray diffraction analysis of the static film sample and the dynamic tube sample was carried out by using the X-ray diffractometer of Japan RIGAKU Company. It is calculated that the crystallinity of the bacterial cellulose film prepared by static fermentation is 86.54%, and the crystallinity of the bacterial cellulose tube prepared by dynamic fermentation is 89.36%.

C. Determination of mechanical properties

The prepared tubular bacterial cellulose was subjected to axial and radial strength tests using a universal material tester. (A) Axial strength test (Figure 7): Cut the sample into a 4cm-long tube, set the clamping distance between the two chucks to 3cm, the moving speed to 100mm/min, and the maximum measuring strength of the probe of the testing machine to be 10N . (B) Radial strength test (Fig. 7): the sample is cut into a 1cm long tube, using two U-shaped iron wires (diameter 3mm) through the BC tube, set the clamping distance between the two chucks to 5cm, and the moving speed to 100mm/min. The maximum measuring strength of the probe of the testing machine is 10N.

Figure 8 shows that the axial strength of the material prepared by fermentation in the reactor increases continuously with the increase of the number of days of culture. This is because the thickness of the bacterial cellulose increases with the increase of the number of days of culture, and the amount of cellulose produced is also more many. Through the observation of the process, it can be found that the growth rate of the tubular material is gradually accelerated as the bacterial cellulose is wound on the glass rod. On the first day, the amount of cellulose winding was relatively small, but one day later, when a certain amount of bacterial cellulose was wound on the glass rod, the growth of bacterial cellulose was obviously accelerated, and the thickness of the second day was significantly larger than that of the first day. One day, and so on, this may be because at the beginning of the culture, the glass rod is not conducive to the winding of cellulose, but when there is some cellulose on the glass rod, the winding becomes easier, and the speed of winding thickening becomes faster.

Figure 9 shows that the changing law of radial strength is similar to the results of axial strength. As the number of days of cultivation increases, the radial strength of the tubular material gradually strengthens, and the increase in the strength of the material in the later period is larger than that in the early stage, which is in line with the increase in the thickness of the tubular material as the number of days increases. unanimous. N/g in the figure represents the ratio of the measured actual force to the dry weight of cellulose. Since the dry weight of the tubular material is relatively small, less than 10mg, the change in strength relative to the dry weight will be relatively large, resulting in a relatively large deviation.

Example 5

(1) Bacteria culture

Put kombucha (kombucha) into 300mL liquid seed culture medium according to the inoculation amount of 1-10 discs with a diameter of 0.5cm containing bacteria BC film (this embodiment takes the following medium formula as an example: in every 1L of water, green tea or 1-10g of black tea (5g of tea leaves is optimal), 10-200g of glucose, sucrose or fructose, 3g of peptone or tryptone, 5g of yeast extract, pH3.0-7.5, pasteurized for 30min; per 1L of water, mannitol , glucose, sucrose or fructose 20-200g, peptone or tryptone 3g, yeast extract 5g, pH3.0-7.5, sterilized at 121°C for 20min) to expand culture, shake at 20-30°C, 100-250r/min Bed culture or static culture for 12 to 48 hours before use;

(2) Fermentation preparation of hollow shaped bacterial cellulose material

Transfer the liquid culture medium containing the production strain prepared in step (1) to a bioreactor equipped with a rod-shaped mold, then rotate the mold at a speed of 10 rpm for disturbed culture, and culture it dynamically at 30°C for 4-20 days before harvesting Hollow profiled cellulose material;

Or take the activated strain of step (1) and transfer it to 300mL liquid fermentation medium according to the inoculation amount of 1-10 discs with a diameter of 0.5-1cm containing bacteria BC film, place in a 500mL Erlenmeyer flask, at 30°C and 160rpm for 4 hours, and then transfer the fermentation broth to the bioreactor, and then rotate the mold at a speed of 10rpm to disturb the culture, and culture at 30°C for different days, and different thicknesses can be prepared. Tubular bacterial cellulose material.

The fermentation device consists of a horizontal stirring reactor and a rotating shaft on which several glove-shaped molds are fixed on a turntable, forming a horizontally disturbed bioreactor. Through the rotation of the mold on the turntable around the axis of the rotating shaft, the mold wraps the bacterial cellulose in the fermentation broth to form a hollow bacterial cellulose material of a certain shape on the surface of the mold. The product obtained is similar to that in Figure 5.

(3) Material handling

Remove the prepared cavity special-shaped BC material from the mold, rinse it with distilled water several times, then soak it in 0.5-2wt% NaOH solution, and treat it in a water bath at 70-100°C for 30-120min, so that the BC tubular material is white and translucent After that, it can be washed repeatedly with pure water until neutral, and then freeze-dried to obtain the special-shaped bacterial cellulose product.

Claims (8)

1. A method for dynamically preparing special-shaped cavity bacterial cellulose materials, comprising: (1) Bacteria culture Insert the bacterial cellulose production strain into the liquid medium for expansion, and cultivate it on a shaking table under the conditions of 20-30°C and 100-250r/min or static culture for 12-48 hours before use; (2) Fermentation preparation of special-shaped cavity BC materials Transfer the liquid medium containing the production strain prepared in step (1) to the fermentation device, then rotate the mold at a speed of 3-20rpm for disturbed culture, and after dynamic culture at 20-32°C for 4-20 days, the cavity can be harvested Special-shaped cellulose material; wherein, the mold is a stick-shaped or glove-shaped mold, and the rotating shaft is installed and fixed in the inner cavity of the fermenter with two turntables, and the rotating shaft is symmetrically fixed with more than two molds through the peripheral edge of the turntable; (3) Material handling The prepared hollow special-shaped bacterial cellulose material is removed from the mold, then soaked in 0.5-2wt% NaOH solution, treated in a water bath at 70-100°C for 30-120min, and the bacterial cellulose material is white and translucent. Then wash until neutral to obtain the special-shaped bacterial cellulose product. 2. a kind of method for dynamically preparing special-shaped cavity bacterial cellulose material according to claim 1 is characterized in that: the BC production bacterial strain in the described step (1) is Acetobacter sp., Gluconobacter Gluconobacter sp., Gluconacetobacter sp., Rhizobium sp., Sarcina sp., Pseudomounas sp., Achromobacter sp., Alcaligenes sp., Aerobacter sp., Azotobacter sp., Agrobacterium sp., Pseudomonas cepacia, Campylobacter jejuni or kombucha. 3. a kind of method for dynamically preparing special-shaped cavity bacterial cellulose material according to claim 2, is characterized in that: wherein BC production bacterial strain is Acetobacter xylinum or kombucha. 4. A method for dynamically preparing special-shaped cavity bacterial cellulose material according to claim 1 or 2, characterized in that: bacterial classifications other than kombucha are inserted into liquid seed culture with an inoculum size of 2 to 3 inoculation loops base; Kombucha fungus is inserted into the liquid seed medium according to the inoculation amount of 1-10 discs with a diameter of 0.5cm containing bacteria BC film; Or prepare the liquid seeds of the production bacteria first, and then transfer them to the liquid fermentation medium. The specific steps are as follows: Bacteria except kombucha are inserted into liquid seed culture medium to prepare seed solution according to the inoculation amount of 2 to 3 inoculation loops, and then transferred to liquid fermentation medium according to the inoculum amount of 3vol% to 20vol%; kombucha is inserted into 1 - The inoculum of 10 discs with a diameter of 0.5 cm containing bacteria BC membranes is inserted into the liquid seed medium, and the inoculum of 1 to 10 discs with a diameter of 0.5-1 cm containing bacteria BC films is transferred to the liquid fermentation medium. 5. a kind of method for dynamically preparing special-shaped cavity bacterial cellulose material according to claim 1 is characterized in that: the components of described liquid seed medium and liquid fermentation medium except kombucha are: For every 1L of water, 20-200 g of mannitol, glucose, maltose, sucrose or fructose, 3 g of peptone or tryptone, 5 g of yeast extract, pH 3.0-7.5, sterilized at 121°C for 20 minutes; or mannitol, glucose, sucrose or Fructose 20-200g, yeast extract 5g, peptone or tryptone 5g, citric acid 1.15g, Na ₂ HPO ₄ 2.7g, water 1L, pH 3.0-7.5, sterilized at 121°C for 20min; Kombucha liquid seed medium and liquid fermentation medium are composed of: (1) per 1L of water, 1-10g of green tea or black tea, 10-200g of glucose, sucrose or fructose, 3g of peptone or tryptone, and 5g of yeast extract , pH3.0-7.5, pasteurized for 30min; (2) glucose, sucrose or fructose, green tea or black tea, and water are made into medium, wherein the mass ratio of sugar, tea, and water is 5:0.1-0.4: 100-200, pH3.0-7.5, pasteurized for 30 minutes; or (3) per 1L of water, mannitol, glucose, sucrose or fructose 20-200g, peptone or tryptone 3g, yeast extract 5g, pH3.0 -7.5, sterilized at 121°C for 20 minutes. 6. A kind of method for dynamically preparing special-shaped cavity bacterial cellulose material according to claim 5, characterized in that: 5g of green tea or black tea in the kombucha liquid seed culture medium and liquid fermentation medium (1). 7. a kind of method for dynamically preparing special-shaped cavity bacterial cellulose material according to claim 1, is characterized in that: the liquid culture medium that contains the production strain in the described step (2) is the liquid culture medium that contains the bacterial strain that step (1) prepares Liquid seed medium for production strains or liquid fermentation medium containing production strains. 8. a kind of method for dynamically preparing special-shaped cavity bacterial cellulose material according to claim 1 is characterized in that: in the process of the fermentation preparation of special-shaped cavity BC material, by injecting containing Air with an oxygen content of 1-100vol% to accelerate the formation of the cellulose film on the mold.