Bacterial cellulose producing strain and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a bacterial cellulose production strain and application thereof. Novacetimonas hansenii SZBC067, with a preservation number of CGMCC No. 37080. The strain can ferment and produce bacterial cellulose with defective crown pear juice as a carbon source. The obtained bacterial cellulose membrane has a smooth surface, a high dry membrane yield, and excellent characteristics such as high water content and high purity. The strain solves the inherent defects of uneven surface and uneven thickness of the bacterial cellulose membrane in the prior art, and the product has a significantly better flatness than conventional strains. The defective crown pear juice is used to realize low-cost production, and the bacterial cellulose has a wide application prospect in the fields of medical wound dressings, precise papermaking materials and functional food additives.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a bacterial cellulose-producing strain and its application. Background Technology

[0002] Bacterial cellulose (BC) is a bio-based material produced by microbial fermentation. Compared with plant fibers, BC has many unique properties, such as high crystallinity, high water retention, excellent biocompatibility and mechanical stability, showing broad application prospects in medical materials, food additives, environmental protection materials and other fields.

[0003] Acetic acid bacteria (including *Acetobacter xylinum*, *Komagataeibacter hansenii*, and *Novacetimonas hansenii*) are the main strains for synthesizing bacterial cellulose. Currently, the carbon source required for the fermentation of bacterial cellulose by acetic acid bacteria is generally glucose or fructose, which is costly and thus limits its production and application. Zhang Junna et al. disclosed a method for producing bacterial cellulose (BC) using *Komagataeibacter hansenii* as the strain (Zhang Junna et al., 2012) and apple and pear juice as the fermentation medium. However, the strain CGMCC 3917 used in this method had a low utilization rate of apple or pear juice, and the yield of bacterial cellulose produced by fermentation was only 46.343 g / 100 mL.

[0004] Furthermore, existing research on cellulose production primarily focuses on indicators such as yield, moisture content, and nutrient composition, neglecting the surface smoothness of cellulose membranes. Conventional fermentation-produced bacterial cellulose membranes commonly exhibit problems such as surface protrusions, uneven thickness, and edge curling due to factors like localized bacterial aggregation, uneven nutrient supply at the gas-liquid interface, and non-directional microfibril secretion. This makes bacterial cellulose membranes unsuitable for applications requiring high morphological uniformity, such as medical wound dressings needing close adhesion to the skin, precision papermaking requiring uniform film formation, and biomedical scaffolds requiring structural homogeneity, severely hindering the expansion of high-end applications of bacterial cellulose. Summary of the Invention

[0005] To address the problems of low yield, high cost, uneven membrane surface, and inconsistent thickness in bacterial cellulose production strains, this invention provides a bacterial cellulose production strain and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A bacterial cellulose-producing strain, the strain being... Novacetimonas hanseniiSZBC067 is now deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the deposit date is December 15, 2025; and the accession number is CGMCC No. 37080.

[0007] The present invention also provides the above. Novacetimonas hansenii Application of SZBC067 in bacterial cellulose production.

[0008] Furthermore, the application includes the following steps: (1) Novacetimonas hansenii SZBC067 was inoculated onto slant culture medium and cultured at 26-30℃ for 3-7 days; (2) After the slant culture matures, it is transferred to the seed culture medium and cultured at 26-30℃ and 180 rpm for 20-28 hours to obtain mature seed liquid; (3) Transfer the mature seed liquid to the fermentation medium at an inoculation rate of 2.5% (v / v), and incubate at 26-30℃ until a gel-like film forms on the surface of the medium. Remove the film and purify it to obtain bacterial cellulose.

[0009] Furthermore, the composition of the slant culture medium in step (1) is as follows: 2g glucose, 0.5g yeast powder, 0.1g K2HPO4, 0.15g MgSO4, 1.8g agar, 100mL purified water, and natural pH.

[0010] Furthermore, the seed culture medium in step (2) consists of: 2g glucose, 0.5g yeast powder, 0.1g K2HPO4, 0.15g MgSO4, 100mL purified water, and natural pH.

[0011] Furthermore, the fermentation medium in step (3) consists of: 100 mL of pear juice with a sugar content of 20 g / L, 0.5 g of yeast powder, 1 g of corn steep liquor, purified water to a final volume of 100 mL, and a natural pH. The pear juice is derived from substandard Crown pears.

[0012] Furthermore, in step (3), after transferring the mature seed liquid to the fermentation medium and allowing it to stand for 2-3 days, a feed medium is added. The feed medium has the same components as the fermentation medium, and the amount added is 1 / 20-1 / 10 of the volume of the fermentation medium. After adding the feed medium, the culture is repeated for 1-2 days until the fermentation is completed.

[0013] Furthermore, the purification process described in step (3) is as follows: ①Immerse the removed film in 0.1mol / L NaOH solution and keep it at 80℃ for 2 hours. Replace the NaOH solution with an equal amount every 0.5 hours until the film turns milky white and translucent. ② The film treated in step ① is then immersed in a 0.5% acetic acid solution; ③ Rinse the membrane treated in step ② repeatedly with distilled water until the pH is neutral.

[0014] Furthermore, the bacterial cellulose is suitable for use in artificial skin, wound dressings, drug sustained-release systems, or face masks.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention provides a bacterial cellulose-producing strain Novacetimonas hansenii SZBC067, through its own metabolic characteristics (uniform cell growth and directional arrangement of microfibers), overcomes the problems of uneven thickness and protrusion of the membrane surface of existing bacterial cellulose production strains. The resulting bacterial cellulose membrane has a high degree of smoothness and can be directly applied to high-end scenarios with high requirements for morphological uniformity, such as artificial skin, wound dressings, drug sustained-release systems or face masks. (2) The present invention Novacetimonas hansenii The SZBC067 strain can use the juice of substandard Crown pears as a carbon source to transform agricultural waste into high-value products. The raw material cost is only 1 / 3 to 1 / 5 of that of commercial-grade juice, while solving the resource waste and environmental pressure caused by discarding substandard pears. (3) The present invention Novacetimonas hansenii The bacterial cellulose dry film produced by strain SZBC067 has a high yield, reaching over 19.0 g / L, and maintains excellent characteristics such as high moisture content and high purity. Attached Figure Description

[0016] Picture 1 The process for screening the bacterial strains of this invention is illustrated in Figure A, which shows the typical morphology of the bacterial cellulose membrane; Figure B shows colonies with a clear zone around them obtained after isolation and purification; and Figure C shows the pure culture obtained after further isolation and purification using the streak plate method. Novacetimonas hansenii SZBC067; Figure D is... Novacetimonas hansenii Image of Gram staining results for SZBC067.

[0017] Picture 2 The bacterial cellulose products of Example 4 (Figure A) and Example 5 (Figure B) are shown.

[0018] Picture 3 Figure B shows a comparison of the smoothness of the bacterial cellulose product (Figure A) and the product of Example 3. Detailed Implementation

[0019] The following embodiments further illustrate the present invention. It should be noted that the following embodiments are only for explaining the present invention and do not limit its content. Obvious variations or modifications derived from the spirit of the present invention are still within the protection scope of the present invention. Example 1

[0020] Novacetimonas hansenii Isolation, screening and identification of SZBC067 The culture medium involved in this embodiment is as follows: Enrichment medium: 2g glucose, 0.5g yeast extract, 0.1g K2HPO4, 0.15g MgSO4, 2% ethanol (v / v), pH natural; sterilize at 121℃ for 20min.

[0021] Isolation medium: 2g glucose, 0.5g yeast extract, 0.1g K2HPO4, 0.15g MgSO4, 1g calcium carbonate, 2% ethanol (v / v), 1.8g agar, purified water to 100mL, pH natural; sterilize at 121℃ for 20min.

[0022] Seed culture medium: 2g glucose, 0.5g yeast powder, 0.1g K2HPO4, 0.15g MgSO4, pH natural; sterilize at 121℃ for 20min.

[0023] Fermentation medium: Same as seed culture medium.

[0024] Slant culture medium: glucose 2g, yeast powder 0.5g, K2HPO4 0.1g, MgSO4 0.15g, agar 1.8g, purified water 100mL, pH natural; sterilize at 121℃ for 20min.

[0025] (1) Enrichment culture Collect samples of rotten apples, grapes, pears, etc., dilute with sterile water, and take 1 mL of the diluted solution into a test tube containing 10 mL of enrichment culture medium. After static incubation at 28℃ for 5 days, check the sample. If a milky white gelatinous bacterial film grows on the surface of the liquid in the tube, it is considered positive.

[0026] (2) Separation and purification Take 0.5 mL of the above positive enrichment culture medium and add it to a 10 mL centrifuge tube containing 4.5 mL of sterile water. Mix well to prepare 10 mL of the culture medium. -1 The bacterial suspension was serially diluted using the same procedure to prepare 10... -2 10 -3 10 -4 10 -5 The bacterial suspension was then taken in batches of 10. -3 10 -4 10 -5Spread 0.1 mL of the concentration gradient dilution onto the separation medium, incubate at 28℃ for 3-5 days, and observe.

[0027] Through experiments, positive bacterial films were screened from rotten crown pear samples. These films were elastic, insoluble in water, and conformed to the typical morphology of bacterial cellulose films. Picture 1 A), after separation and purification, yielded a colony with a clear zone around it. Picture 1 B). Fermentation culture proved that this strain could produce bacterial cellulose, and it was initially named A2-002.

[0028] 16S rRNA gene sequencing was performed on strain A2-002 (sequencing primers: 27F: 5-AGAGTTTGATCMTGGCTCAG-3; 1492R: 5-GGTTACCTTGTTACGACTT-3). The sequencing results were compared with those in GenBank. Novacetimonas hansenii The homology was over 99.9%, and combined with physiological and biochemical characteristics, the strain was identified as... Novacetimonas hansenii ; Its physicochemical properties are shown in Table 1: Table 1

[0029] Note: + indicates positive; - indicates negative.

[0030] To ensure the purity of the strain, strain A2-002 was further isolated and purified using the streak plate method to obtain a pure culture, which was named... Novacetimonas hansenii SZBC067 ( Picture 1 C); Gram staining of SZBC067 ( Picture 1 D), microscopic examination revealed short rod-shaped, Gram-negative, and non-spore-forming (consistent with...). Novacetimonas hansenii (morphological characteristics).

[0031] Novacetimonas hansenii The 16S rRNA gene sequence of SZBC067 is shown in SEQ ID NO.1. Example 2

[0032] This embodiment provides a method for utilizing Novacetimonas hansenii The method for producing bacterial cellulose using SZBC067 includes the following steps: (1) Novacetimonas hansenii SZBC067 strain was inoculated onto slant culture medium and cultured at 28℃ for 5 days. (2) After the slant culture matures, it is transferred to the seed culture medium and cultured at 28℃ and 180rpm for 24h to obtain mature seed liquid; (3) Transfer the mature seed liquid described in step (2) to the fermentation medium at an inoculation rate of 2.5%, and incubate at 28°C for 5 days until a gel-like film forms on the surface of the medium; remove the film and purify it to obtain the cellulose membrane. The seed culture was conducted using a 100mL conical flask with a volume of 20mL; the fermentation culture was conducted using a 1000mL beaker with a volume of 200mL.

[0033] The slant culture medium consisted of 2g glucose, 0.5g yeast extract, 0.1g K₂HPO₄, 0.15g MgSO₄, 1.8g agar, and 100mL purified water at natural pH. Sterilization was performed at 121℃ for 20min.

[0034] The seed culture medium consisted of 2g glucose, 0.5g yeast extract, 0.1g K₂HPO₄, 0.15g MgSO₄, and natural pH. It was sterilized at 121℃ for 20 minutes.

[0035] The fermentation medium is the same as the seed culture medium.

[0036] The specific purification steps are as follows: After fermentation, the bacterial cellulose membrane was removed and immersed in a 0.1 mol / L NaOH solution at 80℃ for 2 hours, with the NaOH solution replaced every 0.5 hours, until the membrane became milky white and translucent. Then, it was immersed in a 0.5% acetic acid solution and rinsed with distilled water until the surface pH paper showed neutrality. The surface water was then drained, yielding a wet cellulose membrane. The yield of the wet cellulose membrane was 624.63 g / L. The wet cellulose membrane was then dried in a 60℃ oven to constant weight, yielding a dry cellulose membrane. The yield of the dry cellulose membrane was 9.03 g / L, and the calculated water content was 98.55%. Example 3

[0037] This embodiment provides a method for utilizing Novacetimonas hansenii The method for producing bacterial cellulose by fermentation of SZBC067 is the same as that in Example 2, except for the components of the fermentation culture.

[0038] The fermentation medium components described in this embodiment are: pear juice with a final total sugar concentration of 20 g / L, 0.5 g yeast powder, 1 g corn steep liquor, purified water to a final volume of 100 mL, and natural pH. The pear juice preparation method is as follows: Select 10 kg of deformed or damaged Crown pears, remove rotten parts, peel, juice using a juicer, centrifuge at 4000 rpm for 5 min, and collect the supernatant for later use.

[0039] The yield of bacterial cellulose wet film obtained in this embodiment was 788.96 g / L; the yield of bacterial cellulose dry film was 14.67 g / L, and the calculated water content was 98.14%.

[0040] As can be seen from the comparison of Examples 2 and 3, Example 3 optimized the fermentation medium and used inexpensive pear juice as the main carbon source, which significantly improved the bacterial cellulose yield. The wet film yield was 26.31% higher than that of Example 2, and the dry film yield was 62.46% higher than that of Example 2.

[0041] The inventors found that the bacterial cellulose membranes obtained by fermentation using the strain described in this invention in Examples 2 and 3 had significantly smoother surfaces than those obtained by other methods. Therefore, to further quantitatively analyze the smoothness of the bacterial cellulose membranes, the surface roughness Ra of the bacterial cellulose membranes obtained in this example was analyzed using a contact surface roughness meter, as follows: A small contact surface roughness tester (such as TR200 or SJ-210) should be selected, and the operation should be standardized according to the soft characteristics of the BC film, ensuring that the test results are accurate and repeatable. The probe type is a diamond probe (tip radius ≤ 5μm, probe angle 60°); before testing, the instrument should be calibrated with a standard roughness sample (Ra known, such as 0.5μm, 1.0μm, 3.0μm) to ensure that the indication error is ≤ ±5%.

[0042] Take the purified BC wet membrane and gently wipe away the free moisture on the surface with filter paper (keep the membrane moist but without water droplets to avoid drying and shrinkage). Lay the BC membrane flat on a clean glass slide and fix the edges of the membrane with double-sided tape (to prevent the membrane from shifting or wrinkling during detection), ensuring that the membrane surface is free of protrusions and tension (keep it in its natural state). The fixed sample was placed in an environment of 25℃±2℃ and relative humidity of 50%±5% for 30 minutes to eliminate the influence of ambient temperature and humidity on the membrane morphology.

[0043] After setting the detection parameters of the surface roughness tester, multiple different regions of the cellulose membrane sample obtained in this embodiment were tested, and the arithmetic mean of the Ra values ​​of the five regions was taken as the final test result.

[0044] The surface roughness Ra of the BC membrane obtained by fermentation in this embodiment was tested to be 1.2 μm. Example 4

[0045] This embodiment provides a fermentation method for increasing bacterial cellulose membrane yield. It includes the following steps: (1) Novacetimonas hansenii SZBC067 strain was inoculated onto slant culture medium and cultured at 28℃ for 5 days. (2) After the slant culture matures, it is transferred to the seed culture medium and cultured at 28℃ and 180rpm for 24h to obtain mature seed liquid; (3) Transfer the mature seed culture from step (2) to the fermentation medium at an inoculation rate of 2.5%. After static culture at 28°C for 2 days, start adding 5 mL of feed medium. After culturing for 1 day, continue adding 5 mL of feed medium (the same as the fermentation medium) for a total of 10 times. After fermentation, remove the bacterial cellulose membrane and purify it to obtain the cellulose membrane. The fed culture fermentation process is as follows: Picture 2 As shown in (A).

[0046] The slant culture medium, seed culture medium, and fermentation culture medium are the same as in Example 2. The seed culture device is a 100mL conical flask with a volume of 20mL, and the fermentation culture device is a 1000mL beaker with a volume of 100mL.

[0047] The specific purification steps are the same as in Example 2.

[0048] The bacterial cellulose wet film yield obtained in this embodiment was 904.95 g / L, the bacterial cellulose dry film yield was 9.14 g / L, and the calculated water content was 98.99%. Example 5

[0049] This embodiment provides a fermentation method to increase bacterial cellulose membrane production. The specific fermentation method and culture medium are the same as in Example 4, except that the feeding method used in step (3) of this embodiment is as follows: After two days of static fermentation, 10 mL of fed-batch culture medium was added. This was repeated every two days for a total of five additions. The fed-batch fermentation process was as follows: Picture 2 As shown in (B).

[0050] The bacterial cellulose wet film yield obtained in this embodiment was 989.95 g / L, the bacterial cellulose dry film yield was 19.72 g / L, and the calculated moisture content was 98.01%.

[0051] Comparative Example In this comparative example, the standard strains *Acetobacter xylinum* ATCC 23767 and *Komagataeibacter hansenii* ATCC 23769 were used as experimental strains. Bacterial cellulose production and testing were carried out according to the fermentation method described in Example 3. The yields of wet bacterial cellulose films were 78.96 g / L and 96.83 g / L, respectively; the yields of dry bacterial cellulose films were 2.32 g / L and 2.71 g / L, respectively; the calculated moisture contents were 97.06% and 97.20%, respectively; and the surface roughness Ra were 3.46 μm and 3.94 μm, respectively.

[0052] like Picture 3The image shows a comparison of the smoothness of the bacterial cellulose membrane obtained by fermentation using the standard strain of *Glucosium acetylene* in this comparative example (Figure A) and the bacterial cellulose membrane obtained by fermentation using the strain described in this invention in Example 3 (Figure B) after being placed in the culture flask and removed from the culture vessel.

[0053] The above embodiments are only some embodiments of the present invention, and not all embodiments. The detailed description of the embodiments is merely for the purpose of clearly illustrating the present invention, and is not intended to limit the implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A bacterial cellulose-producing strain, wherein the strain is Novacetimonas hansenii SZBC067, with accession number CGMCC No.37080.

2. The claim 1 Novacetimonas hansenii Application of SZBC067 in bacterial cellulose production.

3. The application according to claim 2, characterized in that, Includes the following steps: (1) Novacetimonas hansenii SZBC067 was inoculated onto slant culture medium and cultured at 26-30℃ for 3-7 days; (2) After the slant culture matures, it is transferred to the seed culture medium and cultured at 26-30℃ and 180 rpm for 20-28 hours to obtain mature seed liquid; (3) Transfer the mature seed liquid to the fermentation medium at an inoculation rate of 2.5% (v / v), and incubate at 26-30℃ until a gel-like film forms on the surface of the medium. Remove the film and purify it to obtain bacterial cellulose.

4. The application according to claim 3, characterized in that, The composition of the slant culture medium in step (1) is as follows: 2g glucose, 0.5g yeast powder, 0.1g K2HPO4, 0.15g MgSO4, 1.8g agar, and 100mL purified water.

5. The application according to claim 3, characterized in that, The seed culture medium in step (2) consists of: 2g glucose, 0.5g yeast powder, 0.1g K2HPO4, 0.15g MgSO4, and 100mL purified water.

6. The application according to claim 3, characterized in that, The fermentation medium in step (3) consists of: pear juice with a sugar content of 20 g / L, 0.5 g yeast powder, 1 g corn steep liquor, and purified water to 100 mL.

7. The application according to claim 6, characterized in that, The pear juice is derived from substandard Crown pears.

8. The application according to claim 3, characterized in that, In step (3), after transferring the mature seed liquid to the fermentation medium and allowing it to stand for 2-3 days, the feed medium is added. The feed medium has the same components as the fermentation medium, and the amount added is 1 / 20-1 / 10 of the volume of the fermentation medium. After adding the feed medium, the culture is repeated for 1-2 days until the fermentation is completed.

9. The application according to claim 3, characterized in that, The purification process described in step (3) is as follows: ①Immerse the removed film in 0.1mol / L NaOH solution and keep it at 80℃ for 2 hours. Replace the NaOH solution with an equal amount every 0.5 hours until the film turns milky white and translucent. ② The film treated in step ① is then immersed in a 0.5% acetic acid solution; ③ Rinse the membrane treated in step ② repeatedly with distilled water until the pH is neutral.

10. The application according to claim 2, characterized in that, The bacterial cellulose is suitable for use in artificial skin, wound dressings, drug release systems, or face masks.

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