阅读说明：本技术 一种快速发酵、工业化生产细菌纤维素的方法 (Method for rapidly fermenting and industrially producing bacterial cellulose ) 是由 凌泽兴 柴谦 唐志刚 房丽丽 尚波 张学宏 梁赫 刘景君 于 2020-12-29 设计创作，主要内容包括：本发明属于微生物领域,具体涉及一种快速发酵、工业化生产细菌纤维素的方法,该方法在种子培养基中添加纤维素酶,利用发酵罐大量快速地进行菌株培养,在静置发酵产膜阶段添加纤维素酶抑制剂,使菌株产生的纤维素可以快速大量累积,进而形成纤维素膜。本发明通过改良培养基成分,使用发酵罐在减少纤维素产生的情况下快速增加菌体量,在静置发酵时通过抑制纤维素酶的作用,保证了纤维素膜的快速产生,并可以保证纤维素膜表面平整,大幅度减少发酵时间,可广泛应用于工业化生产之中。(The invention belongs to the field of microorganisms, and particularly relates to a method for rapidly fermenting and industrially producing bacterial cellulose. According to the invention, through improving the components of the culture medium, the bacterial mass is rapidly increased under the condition of reducing the cellulose production by using the fermentation tank, and through the action of inhibiting the cellulase during standing fermentation, the rapid production of the cellulose membrane is ensured, the surface smoothness of the cellulose membrane can be ensured, the fermentation time is greatly reduced, and the method can be widely applied to industrial production.)

1. A method for rapidly fermenting and industrially producing bacterial cellulose is characterized by comprising the following steps: a seed expanding culture stage and a standing fermentation stage; adding cellulase in the seed culture stage, and adding cellulase inhibitor in the standing fermentation stage.

2. The method for rapidly fermenting and industrially producing bacterial cellulose according to claim 1, wherein the cellulase inhibitor is pepsin inhibitor, sodium carboxymethyl cellulose solution, sodium hyaluronate or oat glucan.

3. The method for rapidly fermenting and industrially producing bacterial cellulose according to claim 1, wherein the concentration of the cellulase inhibitor in the seed fermentation liquid is 1 to 1.5 g/L.

4. The method for rapidly fermenting and industrially producing bacterial cellulose according to claim 1, wherein the stationary fermentation comprises:

after the fermentation of the strain is finished, adding a cellulase inhibitor into the fermented seed liquid;

inoculating the seed liquid added with the cellulase inhibitor into a culture medium for producing cellulose by standing fermentation, uniformly mixing, and then pouring into a tray for standing fermentation to produce a film.

5. The method for rapidly fermenting and industrially producing bacterial cellulose according to claim 1, wherein the stationary fermentation membrane-producing culture stage is performed in an environment with cleanliness of hundreds.

6. The method for rapidly fermenting and industrially producing bacterial cellulose as claimed in claim 1, wherein the static fermentation culture is carried out for 72-96 h at 28-32 ℃ in the tray culture, and the adding depth of the fermentation liquid in the tray is 2-3 cm.

7. The method for rapidly fermenting and industrially producing bacterial cellulose as claimed in claim 1, wherein the cellulase is added into the culture medium in an amount of 1 to 1.5 per mill during the seed expanding culture stage.

8. The method for rapidly fermenting and industrially producing bacterial cellulose according to claim 1, wherein the bacterial species is acetobacter xylinum.

9. A bacterial cellulose membrane prepared by the method of any one of claims 1 to 8.

10. Use of a bacterial cellulose membrane according to claim 9 in the paper, textile, cosmetic or medical fields.

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to a method for shortening the fermentation period of acetobacter xylinum and producing a bacterial cellulose membrane in a large-scale and industrialized manner.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Cellulose is one of the most abundant biomacromolecules on earth and is a valuable renewable natural resource, and the cellulose existing in the nature is classified into plant cellulose and microbial cellulose according to the source of the cellulose. Plant cellulose constitutes the major component of plants, and microbial cellulose is a specific product of primary metabolism of certain microbial cells, and mainly serves as a protective layer. The Bacterial Cellulose (BC) is synthesized by bacteria of some genera such as Acetobacter (Acetobacter), Rhizobium (Rhizobium), Agrobacterium (Agrobacterium) and Sarcina (Sarcina), has the same chemical structural unit with plant Cellulose, is a macromolecular high polymer formed by linking glucopyranose residues through beta-1, 4-glycosidic bonds, is found to have very different macroscopic structure and certain characteristics from the plant Cellulose through research, has extremely high crystallinity, polymerization degree and chemical purity, does not contain heteropolysaccharides such as hemicellulose, lignin and the like which are difficult to remove in the plant fiber, and has wide application prospect in the fields of papermaking, textile, cosmetics, medical treatment and the like. However, the existing bacterial cellulose production enterprises cannot industrially produce bacterial cellulose in a large scale, and the quality of the produced bacterial cellulose membrane is not controllable, so that the produced bacterial cellulose membrane cannot reach a more uniform standard.

At present, the fermentation of bacterial cellulose adopts a standing fermentation method, the period is generally 5-10 days, the method is mainly focused on places such as Guangdong, Fujian and the like, and a newly generated cellulose membrane is cut and then put into a coconut milk culture medium for continuous culture, so that the bacterial cellulose membrane is continuously generated. The method has long culture period, and the inoculation amount and the strain activity cannot be determined, so that the quality of the produced bacterial cellulose membrane cannot be well controlled.

It has been shown that cellulase is added to the seed culture medium during the seed expansion stage to expand the strain amount for the expansion culture. However, the inventor finds that: the method has limitation on the concentration of the added cellulase, and the cellulase in the seed culture solution can influence the toughness of the membrane formed by standing culture in the later period and has great influence on the quality of the cellulose membrane.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the invention provides a method for culturing acetobacter xylinum at high density and quickly producing a bacterial cellulose membrane at high quality.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, a method for rapidly fermenting and industrially producing bacterial cellulose is provided, which comprises: a seed expanding culture stage and a standing fermentation stage; adding cellulase in the seed culture stage, and adding cellulase inhibitor in the standing fermentation stage.

According to the invention, through improving the components of the culture medium, the bacterial mass is rapidly increased under the condition of reducing the cellulose production by using the fermentation tank, and through the action of inhibiting the cellulase during standing fermentation, the rapid production of the cellulose membrane is ensured, the surface smoothness of the cellulose membrane can be ensured, the fermentation time is greatly reduced, and the method can be widely applied to industrial production.

In a second aspect of the invention, there is provided a bacterial cellulose membrane prepared by any of the above methods.

The cellulose membrane prepared by the method not only can be rapidly produced, but also has a smooth surface, greatly reduces the fermentation time, and can be widely applied to industrial production.

In a third aspect of the invention, there is provided the use of a bacterial cellulose membrane as described above in the paper, textile, cosmetic or medical fields.

The method can culture acetobacter xylinum at high density, and can rapidly produce bacterial cellulose membrane with high quality, so that the method is expected to be widely applied to the fields of papermaking, spinning, cosmetics or medical treatment.

The invention has the beneficial effects that:

(1) the invention provides a method for culturing acetobacter xylinum at high density and quickly producing a bacterial cellulose membrane at high quality.

(2) The invention provides a culture method for shortening the fermentation period of bacterial cellulose, which comprises two stages of seed expanding culture and standing fermentation, wherein cellulase is added in the seed expanding culture stage, and different cellulase inhibitors are added in the standing fermentation stage for inhibiting the action of the cellulase and ensuring that a bacterial cellulose membrane can be rapidly generated.

(3) According to the invention, through improving the components of the culture medium, the bacterial mass is rapidly increased under the condition of reducing the cellulose production by using the fermentation tank, and through the action of inhibiting the cellulase during standing fermentation, the rapid production of the cellulose membrane is ensured, the surface smoothness of the cellulose membrane can be ensured, the fermentation time is greatly reduced, and the method can be widely applied to industrial production.

(4) The research of the invention finds that: the pepsin inhibitor can be combined with some groups on the cellulase active center, so that the cellulase activity is reduced or even disappears, and carboxymethyl cellulose, sodium hyaluronate, oat glucan and the like have groups which can be combined with the enzyme active center, so that enzyme conformation molecules are changed to be unfavorable for combining cellulose; meanwhile, the substances have small adverse effect on the bacterial cellulose membrane, and the membrane forming rate and strength are excellent.

(5) The method is simple, convenient to operate, high in practicability and easy to popularize.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The technical scheme of the invention provides a culture method for shortening the fermentation period of bacterial cellulose, which comprises two stages of seed expanding culture and standing fermentation, wherein cellulase is added in the seed expanding culture stage, and different cellulase inhibitors are added in the standing fermentation stage for inhibiting the action of the cellulase and ensuring that a bacterial cellulose membrane can be rapidly generated.

The cellulase inhibitor is pepsin inhibitor, sodium carboxymethylcellulose solution, sodium hyaluronate or oat glucan.

The research finds that: some inhibitors, while capable of inhibiting cellulase activity, also adversely affect the formation of bacterial cellulose membranes. In order to obtain a better bacterial cellulose membrane generation rate, the invention carries out systematic research and experimental screening on various cellulase inhibitors, and finds that: the adverse effect of pepsin inhibitor, carboxymethyl cellulose, sodium hyaluronate, oat glucan and the like on bacterial cellulose film forming is small, and the film forming rate and strength are excellent. This is probably due to the fact that pepsin inhibitors can bind to groups on the cellulase active center, which leads to a reduction or even elimination of the cellulase activity. Carboxymethyl cellulose, sodium hyaluronate, oat glucan and the like have groups capable of being combined with an enzyme activity center, so that enzyme conformation molecules are changed to be unfavorable for combining with cellulose.

In some embodiments of the invention, the addition amount of the cellulase is 1 per mill, the enzyme activity is 500-1000 EGU/mL, and the sterilization mode is filter sterilization.

In some embodiments of the invention, the seed expansion culture is to inoculate the slant strain in a culture medium containing cellulase for culture, and then inoculate the cultured bacterial liquid in a seeding tank for culture, and simultaneously add the cellulase.

In some embodiments of the present invention, the rotation speed of the shaking culture is 100-300 rpm, the temperature is 25-35 ℃, and the culture time is 16-24 h.

In some embodiments of the invention, the bacterial species is selected from acetobacter xylinum.

In some embodiments of the invention, the stirring speed of the seeding tank culture is 100-300 r/min, the temperature is 28-32 ℃, the ventilation volume is 2-5vvm, and the culture time is 16-24 h.

In some embodiments of the invention, a cellulase inhibitor is added to the fermented seed liquor at a suitable concentration after fermentation of the strain is complete.

In some embodiments of the invention, the standing fermentation film-producing stage is to inoculate the seed solution added with the cellulase inhibitor after the fermentation is finished into a culture medium for cellulose production by standing fermentation, mix the seed solution with the cellulose inhibitor, pour the seed solution into a tray for standing fermentation, terminate the fermentation after the fermentation time, and fish out the film.

In some embodiments of the invention, the stationary fermentation membrane-producing culture stage is performed in an environment with a cleanliness of hundreds.

In some embodiments of the invention, the static fermentation culture is carried out for 72-96 h at 28-32 ℃ in the tray culture, and the adding depth of the fermentation liquor in the tray is 2-3 cm.

In some embodiments of the invention, the media components of the individual components of the seed medium (g/L) glucose, 15; peptone, 15; sodium hydrogen phosphate, 2.7; citric acid monohydrate, 1.15; the pH is adjusted to 5 using HCl or NaOH. 150rpm, 30 ℃ and 24 h. The fermentation medium comprises (g/L) glucose, 20; sucrose, 10; peptone, 5; sodium hydrogen phosphate, 2.7; citric acid monohydrate, 1.15; the pH is adjusted to 5 using HCl or NaOH.

The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.

In the following control examples and examples, the cellulase activity was 700 EGU/mL.

The strain number of acetobacter xylinum is as follows: ATCC23767, purchased from China microbial species Inquiry network.

Comparative example

6L of shake flask seed culture medium and 60L of seeding tank culture medium are prepared.

Wherein the culture medium formulas of the seed shake flask and the seed tank are consistent, and the components (g/L) of the culture medium are as follows: glucose, 15; peptone, 15; sodium hydrogen phosphate, 2.7; citric acid monohydrate, 1.15; the pH is adjusted to 5 using HCl or NaOH. The seed shake flask adopts a 1L triangular flask with the liquid loading capacity of 300mL, and the seed tank adopts a 100L mechanical stirring fermentation tank with the liquid loading capacity of 60L.

The formula of the culture medium for producing cellulose by standing fermentation is as follows:

media composition (g/L) glucose, 20; sucrose, 10; peptone, 5; sodium hydrogen phosphate, 2.7; citric acid monohydrate, 1.15; the pH is adjusted to 5 using HCl or NaOH.

Firstly, activating a bacterial strain, completely inoculating a test tube inclined surface bacterial lawn of acetobacter xylinum into a flask filled with sterilized 100mL of seed culture medium by using an inoculating shovel, carrying out shake culture at 30 ℃ for 12h, then inoculating into a 20 flask 300mL of seed culture medium shake flask by using an inoculation amount of 1% as a primary seed solution, carrying out shake culture at 30 ℃ for 12h, then inoculating into a fermentation tank filled with sterilized 60L of standing fermentation culture medium, adding cellulase into the completely sterilized culture medium, adding a cellulase solution subjected to filtration sterilization, and adding the cellulase solution with the concentration of 1 ‰. The aeration volume in the seeding tank is 2vvm, the tank pressure is 0.05MPa, the stirring speed is 150r/min, after 24h of culture, the seed liquid stops fermenting, the seed liquid is transferred to a sterilized standing fermentation cellulose production culture medium, the inoculation amount is 6%, meanwhile, the seed liquid is subjected to plate counting, after being uniformly mixed by a mixing tank, the seed liquid is injected into a tray under the aseptic environment, the thickness of the fermentation liquid in the tray is controlled to be about 3cm, after 3 days of culture, the thickness of cellulose membranes of all trays is observed, membrane collecting treatment is carried out, and 4-5 trays are randomly left for 4 days of culture to collect membranes.

The results are shown in Table 1

Example 1: adding pepsin inhibitor.

Preparation of a culture medium:

6L of shake flask seed culture medium and 60L of seeding tank culture medium are prepared.

Wherein the culture medium formulas of the seed shake flask and the seed tank are consistent and are as follows: media composition (g/L) glucose, 20; peptone, 5; sodium hydrogen phosphate, 2.7; citric acid monohydrate, 1.15; the pH is adjusted to 5 using HCl or NaOH. The seed shake flask adopts a 1L triangular flask with the liquid loading capacity of 300mL, and the seed tank adopts a 100L mechanical stirring fermentation tank with the liquid loading capacity of 60L.

The formula of the culture medium for producing cellulose by standing fermentation is as follows:

media composition (g/L) glucose, 20; sucrose, 10; peptone, 5; sodium hydrogen phosphate, 2.7; citric acid monohydrate, 1.15; the pH is adjusted to 5 using HCl or NaOH.

Preparation of pepsin inhibitor solution: pepsin inhibitor (available from Solebao corporation under CAS number 26305-03-3) was weighed, dissolved in methanol at 1mg/mL, and then placed at 4 ℃ for stabilization for one week. The addition amount is 1mL/L, and 60mL of pepsin inhibitor solution is added into 60L of seed fermentation liquor.

The cellulase and pepstatin solutions were filter sterilized using a 0.22 μm filter, which was performed in a clean bench.

Firstly, activating a bacterial strain, completely inoculating a test tube inclined surface bacterial lawn of acetobacter xylinum into a flask filled with sterilized 100mL of seed culture medium by using an inoculating shovel, carrying out shake culture at 30 ℃ for 12h, then inoculating into a 20 flask 300mL of seed culture medium shake flask by using an inoculation amount of 1% as a primary seed solution, carrying out shake culture at 30 ℃ for 12h, then inoculating into a fermentation tank filled with sterilized 60L of standing fermentation culture medium, adding cellulase into the completely sterilized culture medium, adding a cellulase solution subjected to filtration sterilization, and adding the cellulase solution with the concentration of 1 ‰. Adding cellulase into a 100L fermentation tank, wherein the ventilation volume in a seed tank is 2vvm, the tank pressure is 0.05MPa, the stirring speed is 150r/min, after culturing for 24h, stopping fermenting the seed liquid, adding a pepsin inhibitor subjected to filtration sterilization, after uniformly mixing, transferring to a sterilized standing fermentation cellulose production culture medium, wherein the inoculation amount is 6%, simultaneously carrying out plate counting on the seed liquid, after uniformly mixing in the mixing tank, injecting the seed liquid into a tray under an aseptic environment, controlling the thickness of the fermentation liquid in the tray to be about 3cm, culturing for 3 days, observing the thickness of cellulose membranes of all trays, at the moment, completely utilizing all the culture medium, carrying out membrane collecting treatment, and simultaneously, randomly leaving 4-5 trays to culture for 4 days to collect membranes.

The results are shown in Table 1

Example 2: sodium carboxymethyl cellulose solution was added.

6L of shake flask seed culture medium and 60L of seeding tank culture medium are prepared.

Wherein the culture medium formulas of the seed shake flask and the seed tank are consistent, and the components (g/L) of the culture medium are as follows: glucose, 20; peptone, 5; sodium hydrogen phosphate, 2.7; citric acid monohydrate, 1.15; the pH is adjusted to 5 using HCl or NaOH. The seed shake flask adopts a 1L triangular flask with the liquid loading capacity of 300mL, and the seed tank adopts a 100L mechanical stirring fermentation tank with the liquid loading capacity of 60L.

The formula of the culture medium for producing cellulose by standing fermentation is as follows:

media composition (g/L) glucose, 20; sucrose, 10; peptone, 5; sodium hydrogen phosphate, 2.7; citric acid monohydrate, 1.15; the pH is adjusted to 5 using HCl or NaOH.

Preparing a sodium carboxymethylcellulose solution, weighing 63g of sodium carboxymethylcellulose, dissolving the sodium carboxymethylcellulose with a certain amount of purified water, placing the sodium carboxymethylcellulose solution in a 60 ℃ water bath kettle for accelerated dissolution, cooling after complete dissolution, and fixing the volume to 3L by using the purified water. And (3) sterilizing the prepared sodium carboxymethyl cellulose solution, and adding the sterilized sodium carboxymethyl cellulose solution into the seed solution after fermentation is finished, wherein the final addition amount of the sodium carboxymethyl cellulose is 1 g/L.

Firstly, activating a bacterial strain, completely inoculating a test tube inclined surface bacterial lawn of acetobacter xylinum into a flask filled with sterilized 100mL of seed culture medium by using an inoculating shovel, carrying out shake culture at 30 ℃ for 12h, then inoculating into a 20 flask 300mL of seed culture medium shake flask by using an inoculation amount of 1% as a primary seed solution, carrying out shake culture at 30 ℃ for 12h, then inoculating into a fermentation tank filled with sterilized 60L of standing fermentation culture medium, adding cellulase into the completely sterilized culture medium, adding a cellulase solution subjected to filtration sterilization, and adding the cellulase solution with the concentration of 1 ‰. Adding cellulase into a 100L fermentation tank, wherein the ventilation volume in a seed tank is 2vvm, the tank pressure is 0.05MPa, the stirring speed is 150r/min, after culturing for 24h, stopping fermenting the seed liquid, adding a sterilized sodium carboxymethyl cellulose solution, after uniformly mixing, transferring to a sterilized standing fermentation cellulose production culture medium, wherein the inoculation amount is 6%, simultaneously carrying out plate counting on the seed liquid, after uniformly mixing in the mixing tank, injecting into a tray under an aseptic environment, controlling the thickness of the fermentation liquid in the tray to be about 3cm, observing the thickness of cellulose membranes of all trays after culturing for 3 days, and at the moment, completely utilizing all the culture medium, carrying out membrane collecting treatment, and simultaneously, randomly leaving 4-5 trays for culturing for 3 days and collecting membranes.

Example 3: sodium hyaluronate was added.

6L of shake flask seed culture medium and 60L of seeding tank culture medium are prepared.

Wherein the culture medium formulas of the seed shake flask and the seed tank are consistent, and the components (g/L) of the culture medium are as follows: glucose, 20; peptone, 5; sodium hydrogen phosphate, 2.7; citric acid monohydrate, 1.15; the pH is adjusted to 5 using HCl or NaOH. The seed shake flask adopts a 1L triangular flask with the liquid loading capacity of 300mL, and the seed tank adopts a 100L mechanical stirring fermentation tank with the liquid loading capacity of 60L.

The formula of the culture medium for producing cellulose by standing fermentation is as follows:

media composition (g/L) glucose, 20; sucrose, 10; peptone, 5; sodium hydrogen phosphate, 2.7; citric acid monohydrate, 1.15; the pH is adjusted to 5 using HCl or NaOH.

Preparing a sodium hyaluronate solution, firstly weighing 63g of sodium hyaluronate, dissolving the sodium hyaluronate with a certain amount of purified water, continuously stirring, and after the sodium hyaluronate is completely dissolved, fixing the volume to 3L by using the purified water. And sterilizing the prepared sodium hyaluronate solution, and adding the sterilized sodium hyaluronate solution into the seed solution after fermentation is finished, wherein the final addition amount of the sodium hyaluronate is 1 g/L.

Firstly, activating a bacterial strain, completely inoculating a test tube inclined surface bacterial lawn of acetobacter xylinum into a flask filled with sterilized 100mL of seed culture medium by using an inoculating shovel, carrying out shake culture at 30 ℃ for 12h, then inoculating into a 20 flask 300mL of seed culture medium shake flask by using an inoculation amount of 1% as a primary seed solution, carrying out shake culture at 30 ℃ for 12h, then inoculating into a fermentation tank filled with sterilized 60L of standing fermentation culture medium, adding cellulase into the completely sterilized culture medium, adding a cellulase solution subjected to filtration sterilization, and adding the cellulase solution with the concentration of 1 ‰. Adding cellulase into a 100L fermentation tank, wherein the ventilation volume in a seed tank is 2vvm, the tank pressure is 0.05MPa, the stirring speed is 150r/min, after culturing for 24h, stopping fermenting the seed liquid, adding a sterilized sodium hyaluronate solution, after uniformly mixing, transferring to a sterilized standing fermentation cellulose production culture medium, wherein the inoculation amount is 6%, simultaneously carrying out plate counting on the seed liquid, after uniformly mixing in the mixing tank, injecting into a tray under an aseptic environment, controlling the thickness of the fermentation liquid in the tray to be about 3cm, culturing for 3 days, observing the thickness of cellulose membranes of all trays, at the moment, completely utilizing all the culture medium, carrying out membrane collecting treatment, and simultaneously, randomly leaving 4-5 trays to culture for 3 days to collect membranes.

Example 4: adding oat glucan.

6L of shake flask seed culture medium and 60L of seeding tank culture medium are prepared.

Wherein the culture medium formulas of the seed shake flask and the seed tank are consistent, and the components (g/L) of the culture medium are as follows: glucose, 20; peptone, 5; sodium hydrogen phosphate, 2.7; citric acid monohydrate, 1.15; the pH is adjusted to 5 using HCl or NaOH. The seed shake flask adopts a 1L triangular flask with the liquid loading capacity of 300mL, and the seed tank adopts a 100L mechanical stirring fermentation tank with the liquid loading capacity of 60L.

The formula of the culture medium for producing cellulose by standing fermentation is as follows:

media composition (g/L) glucose, 20; sucrose, 10; peptone, 5; sodium hydrogen phosphate, 2.7; citric acid monohydrate, 1.15; the pH is adjusted to 5 using HCl or NaOH.

Preparing an oat glucan solution, namely weighing 63g of oat glucan firstly, dissolving the oat glucan by using a certain amount of purified water, continuously stirring, and after the oat glucan is completely dissolved, fixing the volume to 3L by using the purified water. And (3) sterilizing the prepared oat glucan solution, and adding the sterilized oat glucan solution into the seed solution after fermentation, wherein the final addition amount of the oat glucan is 1 g/L.

Firstly, activating a bacterial strain, completely inoculating a test tube inclined surface bacterial lawn of acetobacter xylinum into a flask filled with sterilized 100mL of seed culture medium by using an inoculating shovel, carrying out shake culture at 30 ℃ for 12h, then inoculating into a 20 flask 300mL of seed culture medium shake flask by using an inoculation amount of 1% as a primary seed solution, carrying out shake culture at 30 ℃ for 12h, then inoculating into a fermentation tank filled with sterilized 60L of standing fermentation culture medium, adding cellulase into the completely sterilized culture medium, adding a cellulase solution subjected to filtration sterilization, and adding the cellulase solution with the concentration of 1 ‰. Adding cellulase into a 100L fermentation tank, wherein the ventilation volume in a seed tank is 2vvm, the tank pressure is 0.05MPa, the stirring speed is 150r/min, after culturing for 24h, stopping fermenting the seed liquid, adding a sterilized oat glucan solution, after uniformly mixing, transferring to a sterilized standing fermentation cellulose production culture medium, wherein the inoculation amount is 6%, simultaneously counting the plates of the seed liquid, after uniformly mixing in the mixing tank, injecting into a tray under an aseptic environment, controlling the thickness of the fermentation liquid in the tray to be about 3cm, culturing for 3 days, observing the thickness of cellulose membranes of all trays, at the moment, completely utilizing all the culture medium, performing membrane collecting treatment, and simultaneously, randomly leaving 4-5 trays for culturing for 3 days to collect membranes.

The relevant post-treatment modes of the film collection in the comparative examples and the examples are as follows: firstly, washing impurities on the surface of the membrane with clear water, then putting the membrane into a 1% sodium hydroxide solution for soaking for 24 hours, changing alkali liquor for three times during the soaking, then using diluted hydrochloric acid to adjust the membrane to be neutral, and finally using purified water to wash the membrane to obtain the bacterial cellulose membrane.

Plate counting method: diluting the bacterial liquid with sterile physiological saline (0.85%) by 10 times, coating solid seed plate culture medium, standing at 30 deg.C for 5 days, counting, and converting into bacterial amount per unit volume of bacterial liquid.

The Young modulus detection method comprises the following steps: and measuring the thickness of the BC film at multiple positions by using a CH-1-SST type plastic film slice thickness gauge and taking an average value. Tensile strength and elongation at break of the BC film were measured using a microcomputer controlled electronic universal tester (RG-5), first intercepting BC having a length of 10cm and a width of 1cm at a speed of 5mm/s, and recording zero stress as the BC pieces begin to draw. The tensile strength was calculated using the following formula:

if the elongation at break of the recording instrument during operation isAccording to hooke's law, within the elastic limits of an object, stress is proportional to strain, with the proportionality coefficient young's modulus, denoted E, then:

the results are shown in Table 1

Table 1 the results of the measurements of the parameters during the cultivation are as follows:

it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.