CN102978254B - Method for culturing bacterial cellulose through pulsation - Google Patents

Abstract

The invention relates to a method for pulsating bacterial cellulose cultivation. During the cultivation process, a hollow oxygen-permeable mold is placed in a culture container, and gas is filled inside the hollow oxygen-permeable mold, and the hollow oxygen-permeable mold is periodically changed. The gas pressure makes the hollow oxygen-permeable mold produce radial contraction/extension alternately. Under the premise of ensuring that the bacterial cellulose does not produce delamination and partial adhesion, the bacterial cellulose material is prepared by a "quasi-static" method. The internal cellulose microfibrils have obvious orientation in the radial direction, and the cellulose microfibrils are combined by intramolecular and intermolecular hydrogen bonds, and the material has good mechanical strength in the radial direction. The density of the internal structure of the cellulose can be controlled, and a dense and smooth inner cavity can be constructed; the radial orientation is regular, and the bacterial cellulose tubular material with a differentiated structure has a simple molding process, a short preparation period for the scaffold material, and a green preparation process. Environmental protection, simple and fast, and low preparation cost.

Description

A kind of method of pulsating culture bacterial cellulose

technical field

The invention relates to a method for pulse culturing bacterial cellulose.

Background technique

Bacterial cellulose refers to the general term for cellulose synthesized by certain microorganisms in the genus Acetobacter, Agrobacterium, Rhizobium and Sarcina under different conditions. Acetobacter xylinum in the genus Acetobacter is widely used as a template microorganism for basic and applied research on bacterial cellulose because of its highest cellulose production efficiency.

The preparation methods of bacterial cellulose are mainly static culture and dynamic culture. Due to the oxygen-dependent survival properties of the Gram-negative primary cell body, bacterial cellulose is formed at the junction of the sugar source and the aerobic region. The bacterial cellulose material obtained by static culture can form regular geometric shapes such as film and tube according to the different shapes of culture containers and application fields. During the static culture process, it is strictly required not to shake the culture container violently to avoid stratification or partial adhesion of bacterial cellulose, which reduces the application value of the material. In addition, the static culture period is long, the cost is too high, and the production of bacterial cellulose cannot meet the requirements of large-scale production; dynamic culture refers to the process of introducing oxygen or air into the culture container in the form of bubbles and combining with mechanical stirring during the culture process. The method is to artificially make the culture solution with very limited oxygen content contain oxygen content close to that in the air to prepare bacterial cellulose. Due to the shear stress in the cultivation process, the bacterial cellulose obtained is in irregular geometric shapes such as flocculent, star, cluster, spherical, elliptical, etc., and its application value is not high. In addition, some studies have found that strain cell variants will be produced under dynamic culture conditions, and the variants do not have the ability to produce cellulose, so too long dynamic culture time will inhibit the production of cellulose.

It has been reported in the prior art that bacterial cellulose is prepared by a "two-step method" that is dynamic first and then static, and is widely used in the "Nata" (coconut) industry in Southeast Asia. The "two-step method" can increase the yield of bacterial cellulose in a single static culture to a certain extent, but in essence, it is still a simple superposition of two different preparation processes, and it is impossible to achieve a bacterial cellulose material that not only guarantees the yield, but also has Regular geometry.

The existing technology uses oxygen-permeable materials to make hollow molds, and utilizes the biological characteristics of bacteria that rely on oxygen to survive, and a certain pressure of air or oxygen is passed into the inner cavity of the mold, and placed in the fermentation culture solution of the bacteria; Fill the hollow mold with the fermented culture solution of the bacteria, and place it in a certain pressure environment. After standing for a period of time, bacteria secrete cellulose to coat the outer wall of the mold or cling to the inner cavity of the mold to form a special-shaped hollow material. This method can obtain special-shaped hollow bacterial cellulose materials whose shapes are not limited to simple films and tubes. However, this method still belongs to the category of static training in essence.

Oxygen permeable materials can be divided into woven type and thermoplastic type according to the molding process. The weaving type material includes polymer fibers such as cotton, yarn, polyethylene fiber, polypropylene fiber or PET fiber; the thermoplastic type material includes silicone rubber, expanded polytetrafluoroethylene, latex or ceramics. It has been reported that in the prior art, the hollow special-shaped bacterial cellulose prepared by using oxygen-permeable materials is applied to the construction of artificial blood vessels, and certain results have been obtained.

An ideal vascular graft should have a large area of connected endothelial cell layer, and a differentiated, quiescent smooth muscle cell layer, which can provide sufficient mechanical integrity and elastic modulus to meet the material maintenance ability after suturing. And adapt to the body's systemic arterial pressure. At present, through tissue engineering methods and methods of culturing artificial blood vessels in vitro, polymer materials with good biodegradability such as polylactic acid PLA, polyglycolic acid PGA and polylactic acid-polyglycolic acid copolymer PLGA are usually selected as scaffold materials, immersed in Into a biological culture vessel filled with culture medium, and insert cells. In the culture stage, the artificial vascular tissue is cultivated by artificially creating a "pulsating" environment through a liquid perfusion device. The study found that the artificial vascular tissue cultured in a simulated human pulsating environment is very similar in appearance to autologous arteries. Histological observation found that smooth muscle cells migrated inward and covered the scaffold material fragments in the artificial blood vessel wall. Some studies pointed out that the presence of scaffold fragments would increase the probability of intimal hyperplasia and thrombosis in the human body when the material was implanted; at the same time, Scaffold fragments also affect the growth and differentiation of smooth muscle cells. When biodegradable materials degrade in the human body, the local area of the artificial tissue will be obviously acidic, which will cause a series of complications such as inflammation and necrosis of surrounding tissues.

Contents of the invention

The purpose of the present invention is to provide a method for pulsating culture of bacterial cellulose, using oxygen-permeable materials, according to the actual shape, diameter and size of human hollow special-shaped tissues such as blood vessels, bile ducts, urethral tubes, and vas deferens to make the outer peripheral diameter consistent with the target inner diameter hollow mold. On the basis of the existing disclosed technology, by precisely adjusting the gas pressure inside the hollow mold and the oxygen volume concentration, the hollow mold can produce alternate changes of contraction/relaxation along the radial direction at a certain frequency. Since the radial contraction/expansion of the hollow mold is very small, generally no more than 5%, the process of pulsating bacterial cellulose cultivation is a "quasi-static" process. In the prior art, under the simulated systolic/diastolic pulsation environment of the human body, two different types of cells, endothelial cells providing the blood vessel anticoagulant interface and smooth muscle cells providing mechanical elasticity, can grow well inside the scaffold.

Bacterial Cellulose (also known as microbial cellulose) is a nanofibrous material. It uses the inside of the bacterial cell as a biosynthesis reactor, and the glucose small molecule undergoes a series of complex allosteric processes under the catalysis of the enzyme, and finally forms a β-1,4-glucose chain through the β-1,4-glucosidic bond. The catalytic site on the side of the lineage cell is extruded. The β-1,4-glucose chains interact with each other through intramolecular and intermolecular hydrogen bonds to form lipopolysaccharide layers, crystal-like aggregates, cellulose microfibrils, and finally cellulose in a stepwise and hierarchical manner. This series of extracellular (Extracellular) forming process is called "cellulose self-assembly".

It is this unique process of microbial participation that endows bacterial cellulose with good physical and chemical properties: ultra-fine three-dimensional network structure; good moisture absorption, moisture retention and breathability; ultra-high water holding capacity and wet strength; high tensile strength and modulus of elasticity, etc. A large number of studies have shown that bacterial cellulose materials have good in vivo and in vitro biocompatibility, coupled with their excellent shape controllability and shape maintenance, they have unique advantages in constructing in vivo and in vitro tissue engineering scaffold materials.

The bacterial cellulose prepared in a pulsating environment has a more obvious orientation in the radial direction of the internal cellulose microfibrils, and the cellulose microfibrils are bonded by intramolecular and intermolecular hydrogen bonds, which endows the material It has good mechanical strength in the radial direction; secondly, while adding "pulsation" during the cultivation process, by adjusting the gas pressure and oxygen volume concentration in the hollow mold, the density of the internal structure of the cellulose can be controlled, and an inner cavity can be constructed. Dense and smooth; bacterial cellulose tubular material with regular radial orientation and differentiated structure.

A method for pulsating bacterial cellulose cultivation of the present invention, in the bacterial cellulose cultivation process, the hollow oxygen-permeable mold is placed in the culture container, and gas is filled inside the hollow oxygen-permeable mold to periodically change the hollow oxygen-permeable mold. The gas pressure in the mold makes the hollow oxygen-permeable mold alternately change in radial contraction/relaxation.

As a preferred technical solution:

A method for pulsating bacterial cellulose culture as described above, the hollow oxygen-permeable mold is made of an oxygen-permeable material according to the actual shape, diameter and size of the human tissue to be repaired; the hollow oxygen-permeable mold The shape of the mold is consistent with the shape of human internal mammary artery, saphenous vein, urethra, duodenum or bile duct.

A method for pulsating bacterial cellulose culture as described above, the oxygen-permeable material refers to an oxygen transmission rate of 100 to 1000 mL.m ^-2 .s ^-1 at normal temperature and pressure, and a pore uniformity deviation of < ±0.3 material.

A kind of method for pulsating bacterial cellulose as described above, described oxygen-permeable material refers to that cotton, yarn, polyethylene fiber, polypropylene fiber and/or PET fiber are made by knitting, weaving or knitting method The hollow porous material, or the hollow oxygen-permeable scaffold made of silicone rubber, expanded polytetrafluoroethylene, latex or ceramic oxygen-permeable material through one-time molding.

According to the method for pulsating bacterial cellulose culture as described above, the gas refers to the gas whose volume concentration of oxygen is in the range of 10% to 100%.

A method for pulsating bacterial cellulose culture as described above, the periodic change of the gas pressure in the hollow oxygen-permeable mold means that the gas pressure is alternately switched between the set lower limit and the set upper limit, and the set upper limit is at most no If the pressure exceeds 1.5 standard atmospheres, the minimum set lower limit should not be lower than 1 standard atmosphere, and the switching frequency is 65 to 165 times per minute.

A method for pulsating bacterial cellulose culture as described above, the method of alternating conversion refers to filling the gas with a set upper limit pressure to expand the hollow oxygen-permeable mold, and deflated to restore the gas pressure in the hollow oxygen-permeable mold To the set lower limit; or refers to continuously and alternately filling the gas with the set upper limit and the set lower limit pressure.

According to a method for pulsating bacterial cellulose culture as described above, the hollow oxygen-permeable mold produces radial shrinkage/relaxation alternately changing outer diameter deformation of no more than 5%.

A kind of method for pulsating culture bacterial cellulose as described above, specifically comprises the following steps:

1) Preparation of fermentation medium;

Components of the fermentation medium, in mass percent, in wt%: glucose, fructose, sucrose or mannitol 2-5, peptone 0.05-0.5, yeast extract 0.05-0.5, citric acid 0.01-0.1, disodium hydrogen phosphate 0.02 ～0.2, potassium dihydrogen phosphate 0.01～0.1, the balance is water;

The pH of the fermentation medium is 4.0-6.0;

The above components are mixed, sterilized by high pressure steam, irradiated with ultraviolet light and cooled to room temperature, and passed through pure oxygen to obtain the fermentation culture solution;

2) Bacteria expansion;

Inoculate and expand the fermentation culture solution; the degree of expansion: the number of strain cells is 2×10 ⁵ to 2×10 ⁷ cells/ml;

3) Static cultivation;

Transfer the expanded bacteria liquid to a culture container filled with fermentation culture liquid and have a hollow oxygen-permeable mold inside, place it in a constant temperature incubator, and culture it at 28-32°C;

By filling air into the hollow oxygen-permeable mold during the cultivation process, the hollow oxygen-permeable mold produces alternating changes of radial contraction/relaxation to realize pulsating culture of bacterial cellulose;

The hollow oxygen-permeable mold produces radial contraction/relaxation alternately in one of two situations:

(1) Constantly alternately change the upper limit and lower limit of the pressure setting of the output gas of the gas generating device, so that the hollow oxygen permeable mold produces an alternating change of radial contraction/relaxation;

(2) Fill the gas with the set upper limit pressure to expand the hollow oxygen permeable mold, and deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit, so that the hollow oxygen permeable mold produces radial contraction/relaxation alternate changes ;

The gas generating device is an oxygen cylinder, a compressed air cylinder or an air pump;

Described pulsating culture bacterial cellulose is one or two or three of the following three situations:

Ⅰ) Pulsating culture of loose layer:

a. The upper limit of the output gas pressure of the gas generating device is set within the range of less than 1.2 standard atmospheric pressure and not lower than 1.1 standard atmospheric pressure; the lower limit of the gas pressure setting is greater than 1 standard atmospheric pressure and not exceeding 1.1 standard atmospheric pressure; Alternately change the set upper limit and set lower limit of the output gas pressure of the gas generating device, the switching frequency is 65-165 times/minute; the oxygen volume concentration in the gas is within the range of 10-15%;

b. Fill the gas with the set upper limit pressure less than 1.2 standard atmospheric pressure and not lower than 1.1 standard atmospheric pressure to make the hollow oxygen permeable mold expand; deflate to restore the gas pressure in the hollow oxygen permeable mold to the set lower limit Pressure, set the lower limit pressure within the range of greater than 1 standard atmospheric pressure and not exceeding 1.1 standard atmospheric pressure; the deflation frequency is 65-165 times/min, and the oxygen volume concentration in the gas is within the range of 10-15%;

The cellulose content of the prepared loose layer is 0.1×10 ^-2 to 0.2×10 ^-2 g/cm ³ , and the thickness is 1 to 5 mm;

Ⅱ) Pulse culture elastic layer:

a. Set the upper limit of the output gas pressure of the gas generating device within the range of less than 1.5 standard atmospheres and not less than 1.3 standard atmospheres, and the lower limit of the gas pressure is greater than 1.1 standard atmospheres and not exceeding 1.3 standard atmospheres; constantly change alternately The set upper limit and set lower limit of the output gas pressure of the gas generating device, the conversion frequency is 65-165 times/min; the oxygen volume concentration in the gas is within the range of 30-50%;

b. Fill the gas with the set upper limit pressure less than 1.5 standard atmospheric pressure and not lower than 1.3 standard atmospheric pressure to make the hollow oxygen permeable mold expand; deflate to restore the gas pressure in the hollow oxygen permeable mold to the set lower limit Pressure, set the lower limit pressure within the range of greater than 1 standard atmospheric pressure and no more than 1.3 standard atmospheric pressure; the deflation frequency is 65-165 times/min, and the oxygen volume concentration in the gas is within the range of 30-50%;

The cellulose content of the prepared elastic layer is 0.3×10 ^-2 to 0.6×10 ^-2 g/cm ³ , and the thickness is 1 to 5 mm;

Ⅲ) Dense layer cultured by pulsation

a. Set the upper limit of the output gas pressure of the gas generating device within the range of less than 2.0 standard atmospheres and no less than 1.5 standard atmospheres, and the lower limit of the gas pressure is greater than 1.3 standard atmospheres and no more than 1.5 standard atmospheres; constantly change alternately The set upper limit and set lower limit of the output gas pressure of the gas generating device, the conversion frequency is 65-165 times/min; the oxygen volume concentration in the gas is not less than 50%;

b. Fill the gas with the set upper limit pressure less than 2.0 standard atmospheric pressure and not lower than 1.5 standard atmospheric pressure to make the hollow oxygen permeable mold expand; deflate to restore the gas pressure in the hollow oxygen permeable mold to the set lower limit Pressure, set the lower limit pressure within the range of greater than 1 standard atmospheric pressure and no more than 1.5 standard atmospheric pressure; the deflation frequency is 65 to 165 times per minute, and the oxygen volume concentration in the gas is not less than 50%;

The prepared dense layer has a cellulose content of 0.7×10 ^-2 to 1.0×10 ^-2 g/cm ³ and a thickness of 1 to 5 mm;

4) Post-processing

After the pulsation culture is over, soak the above-mentioned bacterial cellulose into a sodium hydroxide solution with a concentration of 1-10wt%, boil it for 2-10 hours, wash it with pure water until the pH is 7.0, and the endotoxin of the material is <0.5EU/ml , and then the treated bacterial cellulose is autoclaved and packaged for storage at low temperature.

The bacterial species mentioned above refer to microorganisms capable of biosynthesizing cellulose, including: Acetobacter xylinum, Acetobacter, Acetobacter, Acetobacter pasteurianus, Glucobacterium, Agrobacterium, Rhizobium, Sarcina, Pseudomonas cepacia One or more of Monomonas, Pseudomonas cocos, or Campylobacter jejuni.

A method for pulsating bacterial cellulose culture as described above, the ultraviolet irradiation after the high-pressure steam sterilization refers to placing the fermentation culture solution in an autoclave at 121°C for 30 minutes and then taking it out and placing it in a Irradiate and cool to room temperature under ultraviolet light; said pure oxygen refers to passing medical oxygen into the above-mentioned culture solution at a speed of 1L/min, and maintains it for 30 minutes; Take an appropriate amount of strains stored in test tubes at 4°C, and transfer them to the above-mentioned fermentation broth; the expansion culture refers to putting the fermentation broth after the strains are inserted at 28 to 32°C. Cultivate on a shaker for 8 to 24 hours.

Beneficial effect:

Compared with prior art, the beneficial effect of the present invention is:

(1) The "pulsation" dynamic factor is added to the traditional single static culture of bacterial cellulose to ensure that the bacterial cellulose does not produce delamination, partial adhesion and other negative factors that reduce the application value of the material. Through the "quasi-static" "The way to prepare bacterial cellulose materials in a dynamic and static way.

(2) The bacterial cellulose prepared in a pulsating environment has obvious orientation in the radial direction of the internal cellulose microfibrils, and the cellulose microfibrils are combined by intramolecular and intermolecular hydrogen bonds. The radial direction has good mechanical strength.

(3) By accurately adjusting the gas pressure and oxygen volume concentration in the hollow mold, the density of the internal structure of the cellulose can be controlled, and a dense and smooth inner cavity can be constructed; a bacterial cellulose tubular material with regular radial orientation and a differentiated structure .

(4) The degradation cycle of bacterial cellulose materials in animals is at least two years, which can be considered as a non-degradable biological material. Therefore, during the in vitro culture stage of the stent and after implantation in the human body, the stent fragments will not be covered by cell-forming tissue. Compared with biodegradable stent materials, the probability of postoperative intimal hyperplasia and thrombosis will be greatly reduced.

(5) A method for pulsating bacterial cellulose has a simple forming process, a short scaffold material preparation cycle, an environmentally friendly, simple and fast preparation process, and low preparation costs, making it an ideal biomedical scaffold material.

detailed description

The present invention will be further described below in combination with specific embodiments. 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.

Example 1

1) Preparation of fermentation medium;

Components of the fermentation medium, in mass percent, in wt%: glucose, fructose, sucrose or mannitol 2, peptone 0.05, yeast extract 0.05, citric acid 0.01, disodium hydrogen phosphate 0.02, potassium dihydrogen phosphate 0.01, and amount of water;

The pH of the fermentation broth is 4.0;

The above components are mixed, sterilized by high pressure steam, irradiated with ultraviolet light and cooled to room temperature, and passed through pure oxygen to obtain the fermentation culture solution;

2) Bacteria expansion;

The fermentation culture liquid is inoculated and expanded; the expansion degree: the number of strain cells is 2×10 ⁵ /ml.

3) Static cultivation;

The expanded bacterial solution was transferred to a culture container filled with fermentation culture solution and a hollow oxygen-permeable mold inside, placed in a constant temperature incubator, and cultured at 28°C.

By filling air into the hollow oxygen-permeable mold during the cultivation process, the hollow oxygen-permeable mold produces alternating changes of radial contraction/relaxation to realize pulsating culture of bacterial cellulose;

The hollow oxygen-permeable mold produces radial contraction/relaxation alternately in two situations:

(1) Constantly alternately change the upper limit and lower limit of the pressure setting of the output gas of the gas generating device, so that the hollow oxygen permeable mold produces an alternating change of radial contraction/relaxation;

(2) Fill the gas with the set upper limit pressure to expand the hollow oxygen permeable mold, and deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit, so that the hollow oxygen permeable mold produces radial contraction/relaxation alternate changes ;

Described pulsating culture bacterial cellulose is divided into the first situation:

1) Pulse culture loose layer:

Method 1. The output gas pressure of the gas generating device is set to 1.1 standard atmosphere; the lower limit of the gas pressure setting is 1 standard atmosphere. Continuously alternately change the set upper limit and set lower limit of the output gas pressure of the gas generating device, the conversion frequency is 65 times/min; the oxygen volume concentration in the gas is 10%;

Method 2: Fill the gas with a set upper limit pressure of 1.1 standard atmospheric pressure to expand the hollow oxygen permeable mold; deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit pressure, and set the lower limit pressure to 1 Standard atmospheric pressure; the deflation frequency is 65 times/min, and the oxygen volume concentration in the gas is 10%;

The loose layer refers to a cellulose content of 0.1×10 ^-2 g/cm ³ and a thickness of 1 mm.

4) Post-processing

After the pulsation culture is over, soak the above-mentioned bacterial cellulose into a sodium hydroxide solution with a concentration of 1wt%, boil for 10 hours, wash with pure water until the pH is 7.0, and the endotoxin of the material is <0.5EU/ml, and then the treated The final bacterial cellulose was autoclaved and packaged for storage at low temperature.

Example 2

1) Preparation of fermentation medium;

Components of the fermentation medium, in mass percent, in wt%: 5 glucose, fructose, sucrose or mannitol, 0.5 peptone, 0.5 yeast extract, 0.1 citric acid, 0.2 disodium hydrogen phosphate, 0.1 potassium dihydrogen phosphate, and amount of water;

The pH of the fermentation broth is 6.0;

The above components are mixed, sterilized by high pressure steam, irradiated with ultraviolet light and cooled to room temperature, and passed through pure oxygen to obtain the fermentation culture solution;

2) Bacteria expansion;

The fermentation culture liquid is inoculated and expanded; the expansion degree: the number of strain cells is 2×10 ⁷ /ml.

3) Static cultivation;

Transfer the expanded bacterial solution to a culture container filled with fermentation medium and a hollow oxygen-permeable mold inside, place it in a constant temperature incubator, and culture it at 32°C.

By filling air into the hollow oxygen-permeable mold during the cultivation process, the hollow oxygen-permeable mold produces alternating changes of radial contraction/relaxation to realize pulsating culture of bacterial cellulose;

The hollow oxygen-permeable mold produces radial contraction/relaxation alternately in two situations:

(1) Constantly alternately change the upper limit and lower limit of the pressure setting of the output gas of the gas generating device, so that the hollow oxygen permeable mold produces an alternating change of radial contraction/relaxation;

(2) Fill the gas with the set upper limit pressure to expand the hollow oxygen permeable mold, and deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit, so that the hollow oxygen permeable mold produces radial contraction/relaxation alternate changes ;

Described pulsating culture bacterial cellulose is divided into the first situation:

1) Pulse culture loose layer:

Method 1. The output gas pressure of the gas generating device is set to 1.2 standard atmospheres; the lower limit of the gas pressure setting is 1.1 standard atmospheres. Constantly alternately change the set upper limit and set lower limit of the output gas pressure of the gas generating device, the conversion frequency is 165 times/min; the oxygen volume concentration in the gas is 15%;

Method 2: Fill the gas with the set upper limit pressure at 1.2 standard atmospheric pressure to make the hollow oxygen permeable mold expand; deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit pressure, and set the lower limit pressure to 1.1 Standard atmospheric pressure; the deflation frequency is 165 times/min, and the oxygen volume concentration in the gas is within 15%;

The cellulose content of the prepared loose layer is 0.2×10 ^-2 g/cm ³ , and the thickness is 5 mm;

4) Post-processing

After the pulsation culture is over, soak the above-mentioned bacterial cellulose into a sodium hydroxide solution with a concentration of 10wt%, boil for 2 hours, wash with pure water until the pH is 7.0, and the endotoxin of the material is <0.5EU/ml, and then the treated The final bacterial cellulose was autoclaved and packaged for storage at low temperature.

Example 3

1) Preparation of fermentation medium;

Components of the fermentation medium, in mass percent, in wt%: glucose, fructose, sucrose or mannitol 2, peptone 0.05, yeast extract 0.05, citric acid 0.01, disodium hydrogen phosphate 0.02, potassium dihydrogen phosphate 0.01, and amount of water;

The pH of the fermentation broth is 4.0;

The above components are mixed, sterilized by high pressure steam, irradiated with ultraviolet light and cooled to room temperature, and passed through pure oxygen to obtain the fermentation culture solution;

2) Bacteria expansion;

The fermentation culture liquid is inoculated and expanded; the expansion degree: the number of strain cells is 2×10 ⁵ /ml.

3) Static cultivation;

The expanded bacterial solution was transferred to a culture container filled with fermentation culture solution and a hollow oxygen-permeable mold inside, placed in a constant temperature incubator, and cultured at 28°C.

By filling air into the hollow oxygen-permeable mold during the cultivation process, the hollow oxygen-permeable mold produces alternating changes of radial contraction/relaxation to realize pulsating culture of bacterial cellulose;

The hollow oxygen-permeable mold produces radial contraction/relaxation alternately in two situations:

(1) Constantly alternately change the upper limit and lower limit of the pressure setting of the output gas of the gas generating device, so that the hollow oxygen permeable mold produces an alternating change of radial contraction/relaxation;

(2) Fill the gas with the set upper limit pressure to expand the hollow oxygen permeable mold, and deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit, so that the hollow oxygen permeable mold produces radial contraction/relaxation alternate changes ;

Described pulsating culture bacterial cellulose is the second case:

2) Pulse culture elastic layer

Method 1: The upper limit of the output gas pressure of the gas generating device is set to 1.3 standard atmospheres, and the lower limit of the gas pressure is set to 1.1 standard atmospheres. Constantly alternately change the set upper limit and set lower limit of the output gas pressure of the gas generating device, the conversion frequency is 65 times/min; the oxygen volume concentration in the gas is 30%;

Method 2: Fill the gas with a set upper limit pressure of 1.3 standard atmospheric pressure to make the hollow oxygen permeable mold expand; deflate to restore the gas pressure in the hollow oxygen permeable mold to the set lower limit pressure, and set the lower limit pressure to 1.1 standard atmospheric pressure. The deflation frequency is 65 times/min, and the oxygen volume concentration in the gas is 30%.

The cellulose content of the elastic layer is 0.3×10 ^-2 g/cm ³ , and the thickness is 1mm.

4) Post-processing

After the pulsation culture is over, soak the above-mentioned bacterial cellulose into a sodium hydroxide solution with a concentration of 1wt%, boil for 10 hours, wash with pure water until the pH is 7.0, and the endotoxin of the material is <0.5EU/ml, and then the treated The final bacterial cellulose was autoclaved and packaged for storage at low temperature.

Example 4

1) Preparation of fermentation medium;

Components of the fermentation medium, in mass percent, in wt%: 5 glucose, fructose, sucrose or mannitol, 0.5 peptone, 0.5 yeast extract, 0.1 citric acid, 0.2 disodium hydrogen phosphate, 0.1 potassium dihydrogen phosphate, and amount of water;

The pH of the fermentation broth is 6.0;

The above components are mixed, sterilized by high pressure steam, irradiated with ultraviolet light and cooled to room temperature, and passed through pure oxygen to obtain the fermentation culture solution;

2) Bacteria expansion;

The fermentation culture liquid is inoculated and expanded; the expansion degree: the number of strain cells is 2×10 ⁷ /ml.

3) Static cultivation;

Transfer the expanded bacterial solution to a culture container filled with fermentation medium and a hollow oxygen-permeable mold inside, place it in a constant temperature incubator, and culture it at 32°C.

By filling air into the hollow oxygen-permeable mold during the cultivation process, the hollow oxygen-permeable mold produces alternating changes of radial contraction/relaxation to realize pulsating culture of bacterial cellulose;

The hollow oxygen-permeable mold produces radial contraction/relaxation alternately in two situations:

(1) Constantly alternately change the upper limit and lower limit of the pressure setting of the output gas of the gas generating device, so that the hollow oxygen permeable mold produces an alternating change of radial contraction/relaxation;

(2) Fill the gas with the set upper limit pressure to expand the hollow oxygen permeable mold, and deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit, so that the hollow oxygen permeable mold produces radial contraction/relaxation alternate changes ;

Described pulsating culture bacterial cellulose is the second case:

2) Pulse culture elastic layer

Method 1: The upper limit of the output gas pressure of the gas generating device is set to 1.5 standard atmospheres, and the lower limit of the gas pressure is set to 1.3 standard atmospheres. Constantly alternately change the set upper limit and set lower limit of the output gas pressure of the gas generating device, the conversion frequency is 165 times/min; the oxygen volume concentration in the gas is 50%;

Method 2: Fill the gas with a set upper limit pressure of 1.5 standard atmospheres to expand the hollow oxygen permeable mold; deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit pressure, and set the lower limit pressure to 1.3 standard atmospheric pressure. The deflation frequency is 165 times/min, and the oxygen volume concentration in the gas is 50%.

The cellulose content of the elastic layer is 0.6×10 ^-2 g/cm ³ , and the thickness is 5 mm.

4) Post-processing

After the pulsation culture is over, soak the above-mentioned bacterial cellulose into a sodium hydroxide solution with a concentration of 10wt%, boil for 2 hours, wash with pure water until the pH is 7.0, and the endotoxin of the material is <0.5EU/ml, and then the treated The final bacterial cellulose was autoclaved and packaged for storage at low temperature.

Example 5

1) Preparation of fermentation medium;

Components of the fermentation medium, in mass percent, in wt%: glucose, fructose, sucrose or mannitol 2, peptone 0.05, yeast extract 0.05, citric acid 0.01, disodium hydrogen phosphate 0.02, potassium dihydrogen phosphate 0.01, and amount of water;

The pH of the fermentation broth is 4.0;

The above components are mixed, sterilized by high pressure steam, irradiated with ultraviolet light and cooled to room temperature, and passed through pure oxygen to obtain the fermentation culture solution;

2) Bacteria expansion;

The fermentation culture liquid is inoculated and expanded; the expansion degree: the number of strain cells is 2×10 ⁵ /ml.

3) Static cultivation;

The expanded bacterial solution was transferred to a culture container filled with fermentation culture solution and a hollow oxygen-permeable mold inside, placed in a constant temperature incubator, and cultured at 28°C.

By filling air into the hollow oxygen-permeable mold during the cultivation process, the hollow oxygen-permeable mold produces alternating changes of radial contraction/relaxation to realize pulsating culture of bacterial cellulose;

The hollow oxygen-permeable mold produces radial contraction/relaxation alternately in two situations:

(1) Constantly alternately change the upper limit and lower limit of the pressure setting of the output gas of the gas generating device, so that the hollow oxygen permeable mold produces an alternating change of radial contraction/relaxation;

(2) Fill the gas with the set upper limit pressure to expand the hollow oxygen permeable mold, and deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit, so that the hollow oxygen permeable mold produces radial contraction/relaxation alternate changes ;

Described pulsating culture bacterial cellulose is the third situation:

3) Pulse culture dense layer

Method 1: The upper limit of the output gas pressure of the gas generating device is set to 1.5 standard atmospheres, and the lower limit of the gas pressure is set to 1.3 standard atmospheres. Constantly alternately change the set upper limit and set lower limit of the output gas pressure of the gas generating device, the conversion frequency is 65 times/min; the oxygen volume concentration in the gas is 50%;

Method 2: Fill the gas with a set upper limit pressure of 1.5 standard atmospheres to expand the hollow oxygen permeable mold; deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit pressure, and set the lower limit pressure to 1.3 standard atmospheric pressure. The deflation frequency is 65 times/min, and the oxygen volume concentration in the gas is 50%.

The cellulose content of the elastic layer is 0.7×10 ^-2 g/cm ³ , and the thickness is 1 mm.

4) Post-processing

After the pulsation culture is over, soak the above-mentioned bacterial cellulose into a sodium hydroxide solution with a concentration of 1wt%, boil for 10 hours, wash with pure water until the pH is 7.0, and the endotoxin of the material is <0.5EU/ml, and then the treated The final bacterial cellulose was autoclaved and packaged for storage at low temperature.

Example 6

1) Preparation of fermentation medium;

Components of the fermentation medium, in mass percent, in wt%: 5 glucose, fructose, sucrose or mannitol, 0.5 peptone, 0.5 yeast extract, 0.1 citric acid, 0.2 disodium hydrogen phosphate, 0.1 potassium dihydrogen phosphate, and amount of water;

The pH of the fermentation broth is 6.0;

The above components are mixed, sterilized by high pressure steam, irradiated with ultraviolet light and cooled to room temperature, and passed through pure oxygen to obtain the fermentation culture solution;

2) Bacteria expansion;

The fermentation culture liquid is inoculated and expanded; the expansion degree: the number of strain cells is 2×10 ⁷ /ml.

3) Static cultivation;

Transfer the expanded bacterial solution to a culture container filled with fermentation medium and a hollow oxygen-permeable mold inside, place it in a constant temperature incubator, and culture it at 32°C.

By filling air into the hollow oxygen-permeable mold during the cultivation process, the hollow oxygen-permeable mold produces alternating changes of radial contraction/relaxation to realize pulsating culture of bacterial cellulose;

The hollow oxygen-permeable mold produces radial contraction/relaxation alternately in two situations:

(1) Constantly alternately change the upper limit and lower limit of the pressure setting of the output gas of the gas generating device, so that the hollow oxygen permeable mold produces an alternating change of radial contraction/relaxation;

(2) Fill the gas with the set upper limit pressure to expand the hollow oxygen permeable mold, and deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit, so that the hollow oxygen permeable mold produces radial contraction/relaxation alternate changes ;

Described pulsating culture bacterial cellulose is the third situation:

3) Pulsation culture elastic layer

Method 1: The upper limit of the output gas pressure of the gas generating device is set to 2.0 standard atmospheres, and the lower limit of the gas pressure is set to 1.5 standard atmospheres. Constantly alternately change the set upper limit and set lower limit of the output gas pressure of the gas generating device, the switching frequency is 165 times/min; the oxygen volume concentration in the gas is 100%;

Method 2: Fill the gas with a set upper limit pressure of 2.0 standard atmospheric pressure to make the hollow oxygen permeable mold expand; deflate to restore the gas pressure in the hollow oxygen permeable mold to the set lower limit pressure, and set the lower limit pressure to 1.5 standard atmospheric pressure. The deflation frequency is 165 times/min, and the oxygen volume concentration in the gas is 100%.

The cellulose content of the elastic layer is 1.0×10 ^-2 g/cm ³ , and the thickness is 5 mm.

4) Post-processing

After the pulsation culture is over, soak the above-mentioned bacterial cellulose into a sodium hydroxide solution with a concentration of 10wt%, boil for 2 hours, wash with pure water until the pH is 7.0, and the endotoxin of the material is <0.5EU/ml, and then the treated The final bacterial cellulose was autoclaved and packaged for storage at low temperature.

Example 7

1) Preparation of fermentation medium;

Components of the fermentation medium, in mass percent, in wt%: glucose, fructose, sucrose or mannitol 2, peptone 0.05, yeast extract 0.05, citric acid 0.01, disodium hydrogen phosphate 0.02, potassium dihydrogen phosphate 0.01, and amount of water;

The pH of the fermentation broth is 4.0;

The above components are mixed, sterilized by high pressure steam, irradiated with ultraviolet light and cooled to room temperature, and passed through pure oxygen to obtain the fermentation culture solution;

2) Bacteria expansion;

The fermentation culture liquid is inoculated and expanded; the expansion degree: the number of strain cells is 2×10 ⁵ /ml.

3) Static cultivation;

The expanded bacterial solution was transferred to a culture container filled with fermentation culture solution and a hollow oxygen-permeable mold inside, placed in a constant temperature incubator, and cultured at 28°C.

By filling air into the hollow oxygen-permeable mold during the cultivation process, the hollow oxygen-permeable mold produces alternating changes of radial contraction/relaxation to realize pulsating culture of bacterial cellulose;

The hollow oxygen-permeable mold produces radial contraction/relaxation alternately in two situations:

(1) Constantly alternately change the upper limit and lower limit of the pressure setting of the output gas of the gas generating device, so that the hollow oxygen permeable mold produces an alternating change of radial contraction/relaxation;

(2) Fill the gas with the set upper limit pressure to expand the hollow oxygen permeable mold, and deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit, so that the hollow oxygen permeable mold produces radial contraction/relaxation alternate changes ;

The pulsating culture bacterial cellulose has the structural characteristics of dense inner wall, elastic middle and loose outer wall:

1) Pulse culture loose layer:

Method 1. The output gas pressure of the gas generating device is set to 1.1 standard atmosphere; the lower limit of the gas pressure setting is 1 standard atmosphere. Continuously alternately change the set upper limit and set lower limit of the output gas pressure of the gas generating device, the conversion frequency is 65 times/min; the oxygen volume concentration in the gas is 10%;

Method 2: Fill the gas with a set upper limit pressure of 1.1 standard atmospheric pressure to expand the hollow oxygen permeable mold; deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit pressure, and set the lower limit pressure to 1 Standard atmospheric pressure; the deflation frequency is 65 times/min, and the oxygen volume concentration in the gas is 10%;

The loose layer refers to a cellulose content of 0.1×10 ^-2 g/cm ³ and a thickness of 1 mm.

2) Pulse culture elastic layer

Method 1: The upper limit of the output gas pressure of the gas generating device is set to 1.3 standard atmospheres, and the lower limit of the gas pressure is set to 1.1 standard atmospheres. Constantly alternately change the set upper limit and set lower limit of the output gas pressure of the gas generating device, the conversion frequency is 65 times/min; the oxygen volume concentration in the gas is 30%;

Method 2: Fill the gas with a set upper limit pressure of 1.3 standard atmospheric pressure to make the hollow oxygen permeable mold expand; deflate to restore the gas pressure in the hollow oxygen permeable mold to the set lower limit pressure, and set the lower limit pressure to 1.1 standard atmospheric pressure. The deflation frequency is 65 times/min, and the oxygen volume concentration in the gas is 30%.

The cellulose content of the elastic layer is 0.3×10 ^-2 g/cm ³ , and the thickness is 1 mm.

3) Pulse culture dense layer

Method 1: The upper limit of the output gas pressure of the gas generating device is set to 1.5 standard atmospheres, and the lower limit of the gas pressure is set to 1.3 standard atmospheres. Constantly alternately change the set upper limit and set lower limit of the output gas pressure of the gas generating device, the conversion frequency is 65 times/min; the oxygen volume concentration in the gas is 50%;

Method 2: Fill the gas with a set upper limit pressure of 1.5 standard atmospheres to expand the hollow oxygen permeable mold; deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit pressure, and set the lower limit pressure to 1.3 standard atmospheric pressure. The deflation frequency is 65 times/min, and the oxygen volume concentration in the gas is 50%.

The cellulose content of the elastic layer is 0.7×10 ^-2 g/cm ³ , and the thickness is 1 mm.

4) Post-processing

After the pulsation culture is over, soak the above-mentioned bacterial cellulose into a sodium hydroxide solution with a concentration of 1wt%, boil for 10 hours, wash with pure water until the pH is 7.0, and the endotoxin of the material is <0.5EU/ml, and then the treated The final bacterial cellulose was autoclaved and packaged for storage at low temperature.

Example 8

1) Preparation of fermentation medium;

Components of the fermentation medium, in mass percent, in wt%: 5 glucose, fructose, sucrose or mannitol, 0.5 peptone, 0.5 yeast extract, 0.1 citric acid, 0.2 disodium hydrogen phosphate, 0.1 potassium dihydrogen phosphate, and amount of water;

The pH of the fermentation broth is 5.0;

The above components are mixed, sterilized by high pressure steam, irradiated with ultraviolet light and cooled to room temperature, and passed through pure oxygen to obtain the fermentation culture solution;

2) Bacteria expansion;

The fermentation culture liquid is inoculated and expanded; the expansion degree: the number of strain cells is 2×10 ⁷ /ml.

3) Static cultivation;

Transfer the expanded bacterial solution to a culture container filled with fermentation medium and a hollow oxygen-permeable mold inside, place it in a constant temperature incubator, and culture it at 30°C.

By filling air into the hollow oxygen-permeable mold during the cultivation process, the hollow oxygen-permeable mold produces alternating changes of radial contraction/relaxation to realize pulsating culture of bacterial cellulose;

The hollow oxygen-permeable mold produces radial contraction/relaxation alternately in two situations:

(1) Constantly alternately change the upper limit and lower limit of the pressure setting of the output gas of the gas generating device, so that the hollow oxygen permeable mold produces an alternating change of radial contraction/relaxation;

(2) Fill the gas with the set upper limit pressure to expand the hollow oxygen permeable mold, and deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit, so that the hollow oxygen permeable mold produces radial contraction/relaxation alternate changes ;

The pulsating culture bacterial cellulose has the structural characteristics of dense inner wall, elastic middle and loose outer wall:

1) Pulse culture loose layer:

Method 1. The output gas pressure of the gas generating device is set to 1.1 standard atmosphere; the lower limit of the gas pressure setting is 1 standard atmosphere. Constantly alternately change the set upper limit and set lower limit of the output gas pressure of the gas generating device, the conversion frequency is 80 times/min; the oxygen volume concentration in the gas is 10%;

Method 2: Fill the gas with a set upper limit pressure of 1.1 standard atmospheric pressure to expand the hollow oxygen permeable mold; deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit pressure, and set the lower limit pressure to 1 Standard atmospheric pressure; the deflation frequency is 80 times/min, and the oxygen volume concentration in the gas is 10%;

The loose layer refers to a cellulose content of 0.2×10 ^-2 g/cm ³ and a thickness of 1 mm.

2) Pulse culture elastic layer

Method 1: The upper limit of the output gas pressure of the gas generating device is set to 1.3 standard atmospheres, and the lower limit of the gas pressure is set to 1.1 standard atmospheres. Continuously alternately change the set upper limit and set lower limit of the output gas pressure of the gas generating device, the conversion frequency is 80 times/min; the oxygen volume concentration in the gas is 30%;

Method 2: Fill the gas with a set upper limit pressure of 1.3 standard atmospheric pressure to make the hollow oxygen permeable mold expand; deflate to restore the gas pressure in the hollow oxygen permeable mold to the set lower limit pressure, and set the lower limit pressure to 1.1 standard atmospheric pressure. The deflation frequency is 80 times/min, and the oxygen volume concentration in the gas is 30%.

The cellulose content of the elastic layer is 0.5×10 ^-2 g/cm ³ , and the thickness is 1mm.

3) Pulse culture dense layer

Method 1: The upper limit of the output gas pressure of the gas generating device is set to 1.5 standard atmospheres, and the lower limit of the gas pressure is set to 1.3 standard atmospheres. Constantly alternately change the set upper limit and set lower limit of the output gas pressure of the gas generating device, the conversion frequency is 80 times/min; the oxygen volume concentration in the gas is 50%;

Method 2: Fill the gas with a set upper limit pressure of 1.5 standard atmospheres to expand the hollow oxygen permeable mold; deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit pressure, and set the lower limit pressure to 1.3 standard atmospheric pressure. The deflation frequency is 80 times/min, and the oxygen volume concentration in the gas is 50%.

The cellulose content of the elastic layer is 1.0×10 ^-2 g/cm ³ , and the thickness is 1mm.

4) Post-processing

After the pulsation culture is over, soak the above-mentioned bacterial cellulose into a sodium hydroxide solution with a concentration of 2wt%, boil it for 4 hours, wash it with pure water until the pH is 7.0, and the endotoxin of the material is <0.5EU/ml. The final bacterial cellulose was autoclaved and packaged for storage at low temperature.

Claims (6)

1. a method for pulsating culture bacterial cellulose, is characterized in that, specifically comprises the following steps: 1) the deployment of fermentation broth; Components of the fermentation medium, in terms of mass percentage, in wt%: glucose, fructose, sucrose or mannitol 2-5, peptone 0.05-0.5, yeast extract 0.05-0.5, citric acid 0.01-0.1, disodium hydrogen phosphate 0.02 ～0.2, potassium dihydrogen phosphate 0.01～0.1, the balance is water; The pH of the fermentation medium is 4.0-6.0; The above components are mixed, sterilized by high pressure steam, irradiated with ultraviolet light and cooled to room temperature, and passed through pure oxygen to obtain the fermentation culture solution; 2) strain expansion; Inoculate and expand the fermentation culture solution; the degree of expansion: the number of strain cells is 2×10 ⁵ to 2×10 ⁷ cells/ml; 3) Static cultivation; Transfer the expanded bacterial liquid to a culture container filled with fermentation culture liquid and a hollow oxygen-permeable mold inside, place it in a constant temperature incubator, and cultivate it statically at 28-32°C; By filling air into the hollow oxygen-permeable mold during the cultivation process, the hollow oxygen-permeable mold produces alternating changes of radial contraction/relaxation to realize the pulsating culture of bacterial cellulose; The alternating change of the radial contraction/relaxation produced by the hollow oxygen-permeable mold is: Fill the gas with the set upper limit pressure to expand the hollow oxygen permeable mold, and deflate to restore the gas pressure in the hollow oxygen permeable mold to the set lower limit, so that the hollow oxygen permeable mold will produce radial contraction/relaxation alternately; Described pulsating culture bacterial cellulose is one or two or three of the following three situations: Ⅰ) Pulsating culture of loose layer: a. The upper limit of the output gas pressure setting of the gas generating device is within the range of less than 1.2 standard atmospheric pressure and greater than 1.1 standard atmospheric pressure; the lower limit of the gas pressure setting is within the range of greater than 1 standard atmospheric pressure and less than 1.1 standard atmospheric pressure; constantly alternately change the gas The set upper limit and set lower limit of the output gas pressure of the generating device, the conversion frequency is 65-165 times/min; the oxygen volume concentration in the gas is within the range of 10-15%; b. Fill the gas with the set upper limit pressure within the range of less than 1.2 standard atmospheric pressure and greater than 1.1 standard atmospheric pressure to expand the hollow oxygen permeable mold; deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit pressure, Set the lower limit pressure within the range of greater than 1 standard atmospheric pressure and less than 1.1 standard atmospheric pressure; the deflation frequency is 65-165 times/min, and the oxygen volume concentration in the gas is within the range of 10-15%; The cellulose content of the prepared loose layer is 0.1×10 ^-2 to 0.2×10 ^-2 g/cm ³ , and the thickness is 1 to 5 mm; Ⅱ) Pulsation culture elastic layer: a. Set the upper limit of the output gas pressure of the gas generating device within the range of less than 1.5 standard atmospheres and greater than 1.3 standard atmospheres, and the lower limit of the gas pressure is greater than 1.1 standard atmospheres and less than 1.3 standard atmospheres; constantly alternately change the gas generating device The upper limit and lower limit of the output gas pressure are set, and the conversion frequency is 65-165 times/min; the oxygen volume concentration in the gas is within the range of 30-50%; b. Fill the gas with a set upper limit pressure of less than 1.5 standard atmospheric pressure and greater than 1.3 standard atmospheric pressure to expand the hollow oxygen permeable mold; deflate the gas pressure in the hollow oxygen permeable mold to return to the set lower limit pressure, Set the lower limit pressure within the range of greater than 1 standard atmospheric pressure and less than 1.3 standard atmospheric pressure; the deflation frequency is 65-165 times per minute, and the oxygen volume concentration in the gas is within the range of 30-50%; The cellulose content of the prepared elastic layer is 0.3×10 ^-2 to 0.6×10 ^-2 g/cm ³ , and the thickness is 1 to 5 mm; Ⅲ) Dense layer cultured by pulsation a. Set the upper limit of the output gas pressure of the gas generating device within the range of less than 2.0 standard atmospheres and greater than 1.5 standard atmospheres, and the lower limit of the gas pressure is greater than 1.3 standard atmospheres and less than 1.5 standard atmospheres; constantly alternately change the gas generating device The upper limit and lower limit of the output gas pressure are set, and the switching frequency is 65 to 165 times per minute; the oxygen volume concentration in the gas is not less than 50%; b. Fill the gas with the set upper limit pressure within the range of less than 2.0 standard atmospheric pressure and greater than 1.5 standard atmospheric pressure to make the hollow oxygen permeable mold expand; deflate to restore the gas pressure in the hollow oxygen permeable mold to the set lower limit pressure, Set the lower limit pressure within the range of greater than 1 standard atmospheric pressure and less than 1.5 standard atmospheric pressure; the deflation frequency is 65 to 165 times per minute, and the oxygen volume concentration in the gas is not less than 50%; The prepared dense layer has a cellulose content of 0.7×10 ^-2 to 1.0×10 ^-2 g/cm ³ and a thickness of 1 to 5 mm; 4) Post-processing After the pulsation culture is over, soak the above-mentioned bacterial cellulose into a sodium hydroxide solution with a concentration of 1-10wt%, boil for 2-10 hours, wash with pure water until the pH is 7.0, and the endotoxin of the material is <0.5EU/ml , and then the treated bacterial cellulose is autoclaved and packaged for storage at low temperature. the 2. The method for pulsating bacterial cellulose according to claim 1, wherein the hollow oxygen-permeable mold is made of an oxygen-permeable material. the 3. A method for pulsating bacterial cellulose cultivation according to claim 2, characterized in that said oxygen-permeable material means that the oxygen transmission rate at normal temperature and pressure is 100-1000mL.m ^-2 . s ^-1 material. 4. a kind of pulsating method for cultivating bacterial cellulose according to claim 2 or 3, is characterized in that, described oxygen-permeable material refers to by the method for knitting cotton, yarn, polyethylene fiber, polypropylene fiber and / Or a hollow porous material made of PET fiber, or a hollow oxygen permeable support made of silicone rubber, expanded polytetrafluoroethylene, latex or ceramic oxygen permeable material through a mold. the 5 . A method for pulsating bacterial cellulose cultivation according to claim 1 , wherein the hollow oxygen-permeable mold produces radial shrinkage/relaxation alternately changing outer diameter deformation of no more than 5%. 6 . the 6. the method for a kind of pulsating culture bacterial cellulose according to claim 1, is characterized in that, described strain refers to the microorganism that can biosynthesize cellulose, comprises: Acetobacter xylinum, Acetobacter acetobacter, acetobacter Bacillus, Acetobacter pasteurianus, Glucobacterium, Agrobacterium, Rhizobium, Sarcina, Pseudomonas cepacia, Pseudomonas cocos or Campylobacter jejuni; Ultraviolet irradiation after bacteria refers to placing the fermentation culture liquid in an autoclave at 121°C for 30 minutes and then taking it out and placing it under an ultraviolet lamp to irradiate and cool to room temperature; Oxygen was passed into the above-mentioned culture solution at a rate of 1 L/min, and maintained for 30 minutes; the inoculation refers to using a sterilized inoculation loop to take an appropriate amount of bacterial species stored in a test tube at 4°C, and transfer it to In the above-mentioned fermentation culture liquid; the expansion culture refers to culturing the fermentation culture liquid after adding the strains on a shaking table at 28-32° C. for 8-24 hours. the