Application and method for preparing bacterial cellulose through fermentation of brevibacillus mesosporus

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to application and a method for preparing bacterial cellulose based on fermentation of brevibacillus mesosporus. According to the brevibacillus mesosporus ZF-9, the screened brevibacillus mesosporus ZF-9 is used as a bacterial cellulose producing strain, a new process for preparing the bacterial cellulose through fermentation is developed, the brevibacillus mesosporus ZF-9 has high bacterial cellulose producing capacity, the yield of the bacterial cellulose is high, and new industrial application is provided for the brevibacillus mesosporus ZF-9.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and in particular relates to an application and method for preparing bacterial cellulose based on fermentation of Brevibacillus mesosporus. Background Art

[0002] Bacterial cellulose (BC) is a natural polymer material, an extracellular polysaccharide secreted by various bacteria. It possesses excellent biocompatibility, biodegradability, strong water-holding capacity, and high mechanical properties. It excels in adhesion, skin elasticity, softness, and moisturizing properties, and is considered a novel environmentally friendly biomaterial. Compared to traditional plant fibers, BC offers advantages such as high crystallinity, high water-holding capacity, high Young's modulus, high purity, and excellent biocompatibility. BC has broad applications in biomedical materials such as medical dressings, food, papermaking, and textiles.

[0003] However, due to the low productivity and high production cost of bacterial cellulose, the industrial large-scale application of bacterial cellulose is still limited. In recent years, the process of synthesizing bacterial cellulose by microbial fermentation has been widely studied, especially the process of producing bacterial cellulose based on gluconic acid acetobacter xylonyli has been widely developed. However, the types of microorganisms currently reported to be applicable to bacterial cellulose fermentation processes are still relatively small, and the fermentation efficiency is not ideal. Therefore, this field expects to develop more microorganisms that can efficiently ferment and produce bacterial cellulose, which has positive significance for the large-scale industrial production of bacterial cellulose. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a method for preparing bacterial cellulose based on fermentation of Brevibacillus mesosporus;

[0005] The second technical problem to be solved by the present invention is to provide an application of Brevibacillus mesosporus in a process for fermenting and preparing bacterial cellulose.

[0006] To solve the above technical problems, the present invention provides a use of Brevibacillus centrosporus for fermenting and preparing bacterial cellulose. The Brevibacillus centrosporus is Brevibacillus centrosporus ZF-9, which is classified as Brevibacillus centrosporus and has been deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms on June 21, 2012, with a deposit number of CGMCC No. 6267.

[0007] The present invention also discloses a method for preparing bacterial cellulose by fermentation, comprising the steps of inoculating bacterial cellulose-producing bacteria into a suitable fermentation medium for fermentation and culturing;

[0008] The bacterial cellulose-producing bacterium is Brevibacillus centrosporus ZF-9, which is classified as Brevibacillus centrosporus and has been deposited in the General Microbiology Center of the China Culture Collection Administration on June 21, 2012, with a deposit number of CGMCC No. 6267.

[0009] Specifically, the method for preparing bacterial cellulose by fermentation, wherein the fermentation medium comprises the following components by mass content: 20-100 g / L of carbon source, 10-50 g / L of nitrogen source, 5-15 g / L of inorganic salt, and the pH is adjusted to 6.5-7.5;

[0010] Preferably, the carbon source is selected from one or more of glucose, sucrose, fructose, maltose, molasses, anhydrous ethanol or starch hydrolyzate;

[0011] Preferably, the nitrogen source is selected from one or more of beef extract, peptone, yeast extract, corn steep liquor, soybean meal powder, cottonseed meal, urea, (NH4)2SO4 or NH4Cl;

[0012] Preferably, the inorganic salt is selected from one or more of sodium salt, phosphate and dihydrogen phosphate.

[0013] Specifically, in the method for preparing bacterial cellulose by fermentation, the fermentation and culturing step includes a step of static fermentation or shaking fermentation;

[0014] The conditions of the static fermentation step include: fermentation culture at 25-32° C. for 5-8 days;

[0015] The conditions of the shaking fermentation step include: controlling the rotation speed to 100-150 rpm and performing fermentation culture at 25-32° C. for 5-8 days.

[0016] Specifically, the method for preparing bacterial cellulose by fermentation further comprises the step of inoculating the bacterial cellulose-producing bacteria into a seed culture medium for seed liquid culture;

[0017] The seed culture medium comprises the following components by mass content: 10-30 g / L of carbon source, 10-20 g / L of nitrogen source, 3-8 g / L of inorganic salt, and the pH is adjusted to 6.5-7.5;

[0018] Preferably, the carbon source is selected from one or more of glucose, sucrose, fructose, maltose, molasses, anhydrous ethanol or starch hydrolyzate;

[0019] Preferably, the nitrogen source is selected from one or more of beef extract, peptone, yeast extract, corn steep liquor, soybean meal powder, cottonseed meal, urea, (NH4)2SO4 or NH4Cl;

[0020] Preferably, the inorganic salt is selected from one or more of sodium salt, phosphate and dihydrogen phosphate.

[0021] Specifically, in the method for preparing bacterial cellulose by fermentation, the seed liquid culture step includes a static culture or a shaking culture step;

[0022] The conditions of the static culture step include: culturing the seed solution at 25-32° C. for 1-2 days;

[0023] The conditions of the shaking culture step include: controlling the rotation speed to 80-120 rpm and carrying out seed liquid culture at 25-32° C. for 1-2 days.

[0024] Specifically, the method for preparing bacterial cellulose by fermentation further comprises the step of inoculating the bacterial cellulose-producing bacteria into a slant culture medium for activation;

[0025] The slant culture medium comprises the following components by mass content: 10-30 g / L carbon source, 10-20 g / L nitrogen source, 3-8 g / L inorganic salt, 15-25 g / L agar, and the pH is adjusted to 6.5-7.5;

[0026] Preferably, the carbon source is selected from one or more of glucose, sucrose, fructose, maltose, molasses, anhydrous ethanol or starch hydrolyzate;

[0027] Preferably, the nitrogen source is selected from one or more of beef extract, peptone, yeast extract, corn steep liquor, soybean meal powder, cottonseed meal, urea, (NH4)2SO4 or NH4Cl;

[0028] Preferably, the inorganic salt is selected from one or more of sodium salt, phosphate and dihydrogen phosphate.

[0029] Specifically, in the method for preparing bacterial cellulose by fermentation, the conditions of the slant culture medium activation step include: constant temperature cultivation at 25-32° C. for 18-30 hours.

[0030] Specifically, the method for preparing bacterial cellulose by fermentation further comprises the steps of collecting the fermentation product and separating the bacterial cellulose;

[0031] The separation step includes the steps of performing solid-liquid separation on the fermentation product and collecting the bacterial cellulose gel in the solid portion.

[0032] Specifically, the method for preparing bacterial cellulose by fermentation further comprises the step of purifying the bacterial cellulose gel;

[0033] The purification step includes the steps of washing, soaking in alkali solution and drying the cellulose gel;

[0034] Preferably, the alkali solution comprises 0.1-1 mol / L NaOH solution;

[0035] Preferably, the temperature of the alkali solution impregnation step is 60-100° C., and the impregnation time is 30-90 minutes;

[0036] Preferably, the temperature of the drying step is 60-80°C.

[0037] The present invention utilizes the screened Brevibacillus centrosporus ZF-9 as a bacterial cellulose-producing bacterium to develop a new fermentation process for preparing bacterial cellulose. The Brevibacillus centrosporus ZF-9 has a high bacterial cellulose production capacity, which not only increases the yield of bacterial cellulose but also provides a new industrial application for the Brevibacillus centrosporus ZF-9.

[0038] The method for preparing bacterial cellulose by fermentation of the present invention utilizes a new bacterial cellulose fermentation strain Brevibacillus mesosporus ZF-9 and can obtain a high yield of bacterial cellulose product using only a basic culture medium system, which is conducive to industrial large-scale production and has significant economic and social benefits. DETAILED DESCRIPTION

[0039] In the following examples of the present invention, a bacterial cellulose fermentation process was carried out based on the previously screened Brevibacillus centrosporus ZF-9 strain.

[0040] The Brevibacillus centrosporus ZF-9 described in the present invention is classified as Brevibacillus centrosporus and has been deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 6267 and a deposit date of June 21, 2012 at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0041] The colony morphology, physiological and biochemical characteristics, and nutritional characteristics of the Brevibacillus centrosporus ZF-9 strain described in the present invention are described in Chinese Patent No. CN103074288A. In the following examples of the present invention, this strain is referred to as Brevibacillus centrosporus ZF-9 or simply ZF-9.

[0042] In the following examples of the present invention, the activation of Brevibacillus centosporus ZF-9 was performed using a conventional slant culture medium, that is, a solid slant culture medium conventionally used in the art containing a carbon source, a nitrogen source, an inorganic salt, and agar.

[0043] In the following examples of the present invention, as an exemplary slant culture medium scheme, the slant culture medium may include the following components by mass content: 10-30 g / L carbon source, 10-20 g / L nitrogen source, 3-8 g / L inorganic salt, 15-25 g / L agar, and adjusted to pH 6.5-7.5. The preparation method thereof may refer to conventional methods in the art. As an exemplary embodiment, the carbon source is selected from one or more of glucose, sucrose, fructose, maltose, molasses, anhydrous ethanol, or starch hydrolyzate; the nitrogen source is selected from one or more of beef extract, peptone, yeast extract, corn steep liquor, soybean meal, cottonseed cake powder, urea, (NH4)2SO4, or NH4Cl; and the inorganic salt is selected from one or more of sodium salt, phosphate, and dihydrogen phosphate. The activation performance of the slant culture medium under the above-mentioned carbon source, nitrogen source, and inorganic salt combination on the Brevibacillus centrosporus ZF-9 is basically similar, with no significant difference.

[0044] In the following examples of the present invention, as an exemplary activation scheme for slant culture medium, the conditions of the slant culture medium activation step include: constant temperature culture at 25-32°C for 18-30 hours. Those skilled in the art are capable of adaptively adjusting the activation parameters based on experimental results.

[0045] In the following embodiments of the present invention, the seed liquid culture step of Brevibacillus centrosporus ZF-9 can be performed using a conventional seed liquid culture medium in the art, for example, a conventional liquid culture medium containing a carbon source, a nitrogen source, and an inorganic salt. It should be noted that the seed liquid culture step of Brevibacillus centrosporus ZF-9 of the present invention is intended to amplify bacterial cellulose-producing bacteria. In fact, even without seed liquid amplification, the Brevibacillus centrosporus ZF-9 of the present invention can be directly inoculated into the fermentation medium for fermentation to produce bacterial cellulose.

[0046] In the following examples of the present invention, as an exemplary seed liquid culture medium, the seed culture medium includes the following components by mass: 10-30 g / L of carbon source, 10-20 g / L of nitrogen source, 3-8 g / L of inorganic salts, and adjusted to pH 6.5-7.5; the preparation method thereof can be referred to conventional methods in the art. As an exemplary embodiment, the carbon source is selected from one or more of glucose, sucrose, fructose, maltose, molasses, anhydrous ethanol, or starch hydrolyzate; the nitrogen source is selected from one or more of beef extract, peptone, yeast extract, corn steep liquor, soybean meal, cottonseed meal, urea, (NH4)2SO4, or NH4Cl; and the inorganic salt is selected from one or more of sodium salt, phosphate, and dihydrogen phosphate. The seed liquid culture medium with the above-mentioned carbon source, nitrogen source, and inorganic salt combination has a similar effect on the seed liquid amplification of the Brevibacillus mesosporus ZF-9, with no significant difference.

[0047] In the following examples of the present invention, the seed solution culture method can adopt conventional static culture or shaking culture methods in the art, both of which can achieve the amplification of Brevibacillus centrosporus ZF-9. As an exemplary seed solution amplification scheme, the conditions for the static seed solution culture step may include: culturing the seed solution at 25-32°C for 1-2 days; the conditions for the shaking seed solution culture step may include: controlling the rotation speed at 80-120 rpm and culturing the seed solution at 25-32°C for 1-2 days. Those skilled in the art are capable of adaptively adjusting the seed solution amplification parameters based on experimental results.

[0048] In the following examples of the present invention, the fermentation culture step of Brevibacillus centrosporus ZF-9 can adopt a conventional fermentation medium in the art, for example, a conventional liquid culture medium containing a carbon source, a nitrogen source, and an inorganic salt in the art.

[0049] In the following examples of the present invention, as an exemplary fermentation medium, the fermentation medium includes the following components by mass: 20-100 g / L of carbon source, 10-50 g / L of nitrogen source, 5-15 g / L of inorganic salt, and adjusted to pH 6.5-7.5; the preparation method thereof can be referred to conventional methods in the art. As an exemplary embodiment, the carbon source is selected from one or more of glucose, sucrose, fructose, maltose, molasses, anhydrous ethanol, or starch hydrolyzate; the nitrogen source is selected from one or more of beef extract, peptone, yeast extract, corn steep liquor, soybean meal, cottonseed meal, urea, (NH4)2SO4, or NH4Cl; and the inorganic salt is selected from one or more of sodium salt, phosphate, and dihydrogen phosphate. The fermentation medium with the above-mentioned carbon source, nitrogen source, and inorganic salt combination has a substantially similar ability and effect on the bacterial cellulose production of Brevibacillus centrosporus ZF-9.

[0050] Regarding the fermentation process described herein, it should be noted that the fermentation process can be performed using either static or oscillating fermentation methods to produce the target bacterial cellulose product. In the following examples of the present invention, as exemplary seed solution amplification protocols, the conditions for the static fermentation step may include: fermentation at 25-32°C for 5-8 days; and the conditions for the oscillating fermentation step may include: controlling the rotation speed at 100-150 rpm and fermentation at 25-32°C for 5-8 days. Those skilled in the art are capable of adaptively adjusting the fermentation process parameters based on the bacterial cellulose production effect.

[0051] In the following embodiments of the present invention, the steps of separation, extraction and purification of bacterial cellulose in the fermentation broth can refer to the steps of the prior art. For example, the extraction method of bacterial cellulose in the fermentation broth described in Chinese patent CN102994430A, since bacterial cellulose is a gel-like solid in the fermentation broth after fermentation, the fermentation broth after fermentation is subjected to solid-liquid separation, the solid is the bacterial cellulose gel, and the liquid is the fermentation waste liquid. The bacterial cellulose gel is taken out and rinsed with distilled water several times to remove the remaining bacteria and culture medium. The bacterial cellulose gel is soaked in 0.1-1mol / L NaOH solution, kept warm at 60-100℃ for 30-90min, and then repeatedly rinsed with deionized water. At this time, the gel is milky white and translucent, and the bacterial cellulose gel is dried at 60-80℃ to constant weight to obtain bacterial cellulose.

[0052] The following examples of the present invention are mainly used to verify the ability of Brevibacillus centosporus ZF-9 to produce bacterial cellulose, and the composition systems of the slant culture medium, seed culture solution and fermentation medium involved are only used as exemplary implementation methods.

[0053] In the following embodiments of the present invention, exemplary slant culture medium, seed culture medium and fermentation medium used include:

[0054] Slant medium A: glucose 20 g / L, yeast extract 15 g / L, potassium dihydrogen phosphate 5 g / L, agar 20 g / L, pH 7.0;

[0055] Slant medium B: sucrose 10 g / L, peptone 20 g / L, potassium dihydrogen phosphate 3 g / L, agar 20 g / L, adjusted to pH 6.5;

[0056] Slant medium C: molasses 30 g / L, corn steep liquor 10 g / L, sodium dihydrogen phosphate 8 g / L, agar 20 g / L, pH 7.5;

[0057] Seed liquid medium A: glucose 15g / L, sucrose 5g / L, yeast extract 10g / L, corn steep liquor 5g / L, dipotassium hydrogen phosphate 2g / L, sodium dihydrogen phosphate 3g / L, adjusted to pH 7.2;

[0058] Seed liquid medium B: maltose 5g / L, glucose 5g / L, beef extract 10g / L, soybean meal 10g / L, disodium hydrogen phosphate 3g / L, adjusted to pH 6.5;

[0059] Seed liquid medium C: molasses 10 g / L, starch hydrolyzate 10 g / L, fructose 10 g / L, peptone 10 g / L, disodium hydrogen phosphate 3 g / L, sodium dihydrogen phosphate 5 g / L, adjusted to pH 7.5;

[0060] Fermentation medium A: glucose 30 g / L, sucrose 20 g / L, molasses 30 g / L, peptone 20 g / L, corn steep liquor 20 g / L, disodium hydrogen phosphate 5 g / L, dipotassium hydrogen phosphate 5 g / L, pH adjusted to 7.0;

[0061] Fermentation medium B: fructose 20 g / L, starch hydrolyzate 20 g / L, glucose 10 g / L, beef extract 20 g / L, NH4Cl20 g / L, cottonseed meal 10 g / L, disodium hydrogen phosphate 3 g / L, potassium hydrogen phosphate 2 g / L, pH adjusted to 7.5;

[0062] Fermentation medium C: glucose 40 g / L, maltose 30 g / L, anhydrous ethanol 30 g / L, (NH4)2SO4 10 g / L, yeast extract 20 g / L, disodium hydrogen phosphate 5 g / L, potassium hydrogen phosphate 10 g / L, adjusted to pH 7.5.

[0063] The slant culture medium, seed culture solution and fermentation medium can be sterilized by conventional high-pressure sterilization, for example, sterilization at 121° C. for 10-30 minutes.

[0064] The following examples of the present invention are mainly used to verify the ability of Brevibacillus centrosporus ZF-9 to produce bacterial cellulose. The specific fermentation process and extraction process refer to the general fermentation method of the prior art, such as the fermentation operation process and extraction process described in Chinese Patent CN102994430A.

[0065] Example 1

[0066] The preserved Brevibacillus mesosporus ZF-9 was streaked into the slant culture medium A and cultured at a constant temperature of 28° C. for 24 h.

[0067] Pick a loop of the above slant culture and inoculate it into a 250 ml Erlenmeyer flask containing 40 ml of seed liquid medium A. Culture it at 28 °C and 100 r / min with shaking for 2 days.

[0068] The cultured seed liquid was taken and inoculated into a 500 ml Erlenmeyer flask containing 100 ml of fermentation medium A at a 5% inoculum size. The culture was carried out at 28° C. and 120 rpm with shaking for 6 days, and the fermentation was stopped.

[0069] The fermentation liquid was subjected to solid-liquid separation, and the bacterial cellulose gel was collected. The weight (wet weight) of the bacterial cellulose was measured to be 452.2 g / L.

[0070] The bacterial cellulose gel was rinsed three times with distilled water, immersed in a 0.8 mol / L NaOH solution, kept warm at 80°C for 60 min, and then rinsed three times with deionized water. The bacterial cellulose gel was dried at 80°C to a constant weight and weighed on a balance. The yield (dry weight) of the bacterial cellulose gel was 4.5 g / L.

[0071] Example 2

[0072] The preserved Brevibacillus mesosporus ZF-9 was streaked into the slant culture medium A and cultured at a constant temperature of 28° C. for 24 h.

[0073] Pick a loop of the above slant culture and inoculate it into a 250 ml Erlenmeyer flask containing 40 ml of seed liquid medium A, and culture it statically at 28°C for 2 days.

[0074] The cultured seed liquid was taken and inoculated into a 500 ml Erlenmeyer flask containing 100 ml of fermentation medium A at a 5% inoculum size. The culture was allowed to stand at 28°C for 6 days and the fermentation was stopped.

[0075] The fermentation liquid was subjected to solid-liquid separation, and the bacterial cellulose gel was collected. The weight (wet weight) of the bacterial cellulose was measured to be 561.4 g / L.

[0076] The bacterial cellulose gel was rinsed three times with distilled water, immersed in a 0.8 mol / L NaOH solution, kept warm at 80°C for 60 min, and then rinsed three times with deionized water. The bacterial cellulose gel was dried at 80°C to a constant weight and weighed on a balance. The yield (dry weight) of the bacterial cellulose gel was 5.1 g / L.

[0077] Example 3

[0078] The preserved Brevibacillus mesosporus ZF-9 was streaked into the slant culture medium C and cultured at 28° C. for 24 h.

[0079] A loopful of the above-mentioned slant strain was picked and inoculated into a 500 ml Erlenmeyer flask containing 100 ml of fermentation medium B. The culture was shaken and fermented at 28°C and 120 rpm for 6 days, and then the fermentation was stopped.

[0080] The fermentation liquid was subjected to solid-liquid separation, and the bacterial cellulose gel was collected. The weight (wet weight) of the bacterial cellulose was measured to be 423.9 g / L.

[0081] The bacterial cellulose gel was rinsed three times with distilled water, immersed in a 0.8 mol / L NaOH solution, kept warm at 80°C for 60 min, and then rinsed three times with deionized water. The bacterial cellulose gel was dried at 80°C to a constant weight and weighed on a balance. The yield (dry weight) of the bacterial cellulose gel was 3.9 g / L.

[0082] Example 4

[0083] The preserved Brevibacillus mesosporus ZF-9 was streaked into the slant culture medium B and cultured at 28° C. for 24 h.

[0084] Pick a loop of the above-mentioned slant strain and inoculate it into a 500 ml Erlenmeyer flask containing 100 ml of fermentation medium C. Perform static fermentation at 28°C for 6 days and then stop the fermentation.

[0085] The fermentation liquid was subjected to solid-liquid separation, and the bacterial cellulose gel was collected. The weight (wet weight) of the bacterial cellulose was measured to be 555.9 g / L.

[0086] The bacterial cellulose gel was rinsed three times with distilled water, immersed in a 0.8 mol / L NaOH solution, kept warm at 80°C for 60 min, and then rinsed three times with deionized water. The bacterial cellulose gel was dried at 80°C to a constant weight and weighed on a balance. The yield (dry weight) of the bacterial cellulose gel was 4.8 g / L.

[0087] Example 5

[0088] The preserved Brevibacillus mesosporus ZF-9 was streaked into the slant culture medium B and cultured at a constant temperature of 25° C. for 30 h.

[0089] Pick a loop of the above slant culture and inoculate it into a 250 ml Erlenmeyer flask containing 40 ml of seed liquid medium B. Culture it at 25°C and 120 r / min with shaking for 2 days.

[0090] The cultured seed liquid was taken and inoculated into a 500 ml Erlenmeyer flask containing 100 ml of fermentation medium B at a 5% inoculum size. The culture was carried out at 25° C. and 100 rpm for 8 days, and the fermentation was stopped.

[0091] The fermentation liquid was subjected to solid-liquid separation, and the bacterial cellulose gel was collected. The weight (wet weight) of the bacterial cellulose was measured to be 502.6 g / L.

[0092] The bacterial cellulose gel was rinsed three times with distilled water, immersed in a 0.8 mol / L NaOH solution, kept warm at 80°C for 60 min, and then rinsed three times with deionized water. The bacterial cellulose gel was dried at 80°C to a constant weight and weighed on a balance. The yield (dry weight) of the bacterial cellulose gel was 4.7 g / L.

[0093] Example 6

[0094] The preserved Brevibacillus mesosporus ZF-9 was streaked into the slant culture medium C and cultured at 32° C. for 18 h.

[0095] Pick a loop of the above slant culture and inoculate it into a 250 ml Erlenmeyer flask containing 40 ml of seed liquid medium C, and culture it at 32°C for 1 day.

[0096] The cultured seed liquid was taken and inoculated into a 500 ml Erlenmeyer flask containing 100 ml of fermentation medium C at a 5% inoculum size. The culture was allowed to stand at 32° C. for 7 days and the fermentation was stopped.

[0097] The fermentation liquid was subjected to solid-liquid separation, and the bacterial cellulose gel was collected. The weight (wet weight) of the bacterial cellulose was measured to be 574.3 g / L.

[0098] The bacterial cellulose gel was rinsed three times with distilled water, immersed in a 0.8 mol / L NaOH solution, kept warm at 80°C for 60 min, and then rinsed three times with deionized water. The bacterial cellulose gel was dried at 80°C to a constant weight and weighed on a balance. The yield (dry weight) of the bacterial cellulose gel was 5.1 g / L.

[0099] In summary, the present invention utilizes the screened Brevibacillus centrosporus ZF-9 as a bacterial cellulose-producing bacterium to develop a new fermentation process for preparing bacterial cellulose. The Brevibacillus centrosporus ZF-9 has a high bacterial cellulose production capacity. Even if the conventional basic culture system in the field is used, high bacterial cellulose yield can still be achieved, with significant economic and social benefits.

[0100] It should be understood that the embodiments described in this application are merely illustrative of the principles of the embodiments of this application. Other variations may also fall within the scope of this application. Therefore, as examples and not limitations, alternative configurations of the embodiments of this application may be considered consistent with the teachings of this application. Accordingly, the embodiments of this application are not limited to the embodiments explicitly introduced and described in this application.

Claims

1. A method for preparing bacterial cellulose by fermentation using Brevibacillus mesosporus, characterized in that: The Brevibacillus centrosporus is Brevibacillus centrosporus ZF-9, which is classified as Brevibacillus centrosporus and has been deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms. Its deposit number is: CGMCC No.6267.

2. A method for preparing bacterial cellulose by fermentation, characterized in that: The method comprises the steps of inoculating bacterial cellulose-producing bacteria into a suitable fermentation medium for fermentation culture; The bacterial cellulose producing bacterium is Brevibacillus centrosporus ZF-9, which is classified as Brevibacillus centrosporus and has been deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms. Its deposit number is: CGMCC No.6267.

3. The method for preparing bacterial cellulose by fermentation according to claim 2, characterized in that: The fermentation medium includes the following components by mass content: 20-100 g / L of carbon source, 10-50 g / L of nitrogen source, 5-15 g / L of inorganic salt, and the pH is adjusted to 6.5-7.5; Preferably, the carbon source is selected from one or more of glucose, sucrose, fructose, maltose, molasses, anhydrous ethanol or starch hydrolyzate; Preferably, the nitrogen source is selected from one or more of beef extract, peptone, yeast extract, corn steep liquor, soybean meal powder, cottonseed meal, urea, (NH4)2SO4 or NH4Cl; Preferably, the inorganic salt is selected from one or more of sodium salt, phosphate and dihydrogen phosphate.

4. The method for preparing bacterial cellulose by fermentation according to claim 2 or 3, characterized in that: The fermentation and culturing step includes a static fermentation step or a shaking fermentation step; The conditions of the static fermentation step include: fermentation culture at 25-32° C. for 5-8 days; The conditions of the shaking fermentation step include: controlling the rotation speed to 100-150 rpm and performing fermentation culture at 25-32° C. for 5-8 days.

5. The method for preparing bacterial cellulose by fermentation according to any one of claims 2 to 4, characterized in that: The method further comprises the step of inoculating the bacterial cellulose-producing bacteria into a seed culture medium for seed liquid culture; The seed culture medium comprises the following components by mass content: 10-30 g / L of carbon source, 10-20 g / L of nitrogen source, 3-8 g / L of inorganic salt, and the pH is adjusted to 6.5-7.5; Preferably, the carbon source is selected from one or more of glucose, sucrose, fructose, maltose, molasses, anhydrous ethanol or starch hydrolyzate; Preferably, the nitrogen source is selected from one or more of beef extract, peptone, yeast extract, corn steep liquor, soybean meal powder, cottonseed meal, urea, (NH4)2SO4 or NH4Cl; Preferably, the inorganic salt is selected from one or more of sodium salt, phosphate and dihydrogen phosphate.

6. The method for preparing bacterial cellulose by fermentation according to claim 5, characterized in that: The seed liquid culture step includes a static culture or a shaking culture step; The conditions of the static culture step include: culturing the seed solution at 25-32° C. for 1-2 days; The conditions of the shaking culture step include: controlling the rotation speed to 80-120 rpm and carrying out seed liquid culture at 25-32° C. for 1-2 days.

7. The method for preparing bacterial cellulose by fermentation according to any one of claims 2 to 6, characterized in that: The method further comprises the step of inoculating the bacterial cellulose-producing bacteria into a slant culture medium for activation; The slant culture medium comprises the following components by mass content: 10-30 g / L carbon source, 10-20 g / L nitrogen source, 3-8 g / L inorganic salt, 15-25 g / L agar, and the pH is adjusted to 6.5-7.5; Preferably, the carbon source is selected from one or more of glucose, sucrose, fructose, maltose, molasses, anhydrous ethanol or starch hydrolyzate; Preferably, the nitrogen source is selected from one or more of beef extract, peptone, yeast extract, corn steep liquor, soybean meal powder, cottonseed meal, urea, (NH4)2SO4 or NH4Cl; Preferably, the inorganic salt is selected from one or more of sodium salt, phosphate and dihydrogen phosphate.

8. The method for preparing bacterial cellulose by fermentation according to claim 7, characterized in that: The conditions of the slant culture medium activation step include: constant temperature culture at 25-32° C. for 18-30 hours.

9. The method for preparing bacterial cellulose by fermentation according to any one of claims 2 to 8, characterized in that: The method further comprises the steps of collecting the fermentation product and separating the bacterial cellulose; The separation step includes the steps of performing solid-liquid separation on the fermentation product and collecting the bacterial cellulose gel in the solid portion.

10. The method for preparing bacterial cellulose by fermentation according to claim 9, characterized in that: The method further comprises the step of purifying the bacterial cellulose gel; The purification step includes the steps of washing, soaking in alkali solution and drying the cellulose gel; Preferably, the alkali solution comprises 0.1-1 mol / L NaOH solution; Preferably, the temperature of the alkali solution impregnation step is 60-100°C, and the impregnation time is 30-90 minutes; Preferably, the temperature of the drying step is 60-80°C.