High-mechanical-strength bacterial cellulose and fermentation method thereof

Abstract

The invention belongs to the technical field of fermentation engineering, and particularly relates to high-mechanical-strength bacterial cellulose and a fermentation method thereof. The preparation method comprises the following steps: inoculating a Kosakonia oryzendophytic FY-07 seed solution into an HS-XGK fermentation culture medium, so as to obtain a fermentation solution; and performing sealed fermentation on the fermentation liquor at 28-37 DEG C to obtain the bacterial cellulose with high mechanical strength. The FY-07 disclosed by the invention can be used for quickly fermenting and producing gas to increase the pressure in a container, so that a bacterial cellulose product is more compact, and the mechanical strength is improved. Compared with bacterial cellulose prepared through traditional open fermentation, the bacterial cellulose prepared through the fermentation method has the advantages that the tensile strength is remarkably improved by 2.59 times, the Young modulus is remarkably improved by 4.36 times, and it is indicated that a closed fermentation product has better mechanical performance.

Description

Technical Field

[0001] The present invention belongs to the technical field of fermentation engineering, and particularly relates to a bacterial cellulose with high mechanical strength and a fermentation method thereof. Background Art

[0002] As a new material, bacterial cellulose (BC) has been widely used in industries such as food, cosmetics, and biomedicine due to its many excellent properties, such as high purity, high crystallinity, and high mechanical strength.

[0003] Kosakonia oryzendophytica FY-07 (formerly Enterobacter sp. FY-07, FY-07) is the only unique strain reported so far that can rapidly produce bacterial cellulose under aerobic, microaerobic, and anaerobic conditions, and can achieve submerged fermentation for the production of bacterial cellulose. Under optimized fermentation production processes, FY-07 can carry out efficient large-scale production of bacterial cellulose. However, the excessive cell metabolism and fermentation production lead to a large amount of gas production during fermentation. Therefore, the gas content in the obtained bacterial cellulose is relatively high, and the network density is lower than that of the bacterial cellulose produced by traditional Gluconacetobacter, and its corresponding mechanical strength is also lower, which limits the practical application of the product.

[0004] At the present stage, the mechanical strength of bacterial cellulose can be improved by chemical modification such as carboxymethylation and acetylation of bacterial cellulose, using methods such as hot pressing and freeze drying, or preparing bacterial cellulose into composite materials, as well as through post-treatment techniques such as crosslinking and coating. However, these methods all have different degrees of defects, such as cumbersome steps, high costs, and being harmful to the environment. Therefore, there is an urgent need to develop a new method with high efficiency and low cost to solve the problem that the mechanical strength of the bacterial cellulose produced by FY-07 is not high enough. Summary of the Invention

[0005] The purpose of the present invention is to provide a bacterial cellulose with high mechanical strength and a fermentation method thereof, and the mechanical strength of the bacterial cellulose obtained by using the fermentation method of the present invention is significantly improved compared with the traditional fermentation method.

[0006] The present invention provides a fermentation method for improving the mechanical strength of bacterial cellulose, comprising the following steps: inoculating the seed liquid of Kosakonia oryzendophytica FY-07 into the HS-XGK fermentation medium to obtain a fermentation broth; fermenting the fermentation broth in a closed state at 28-37°C to obtain a bacterial cellulose with high mechanical strength;

[0007] The preservation number of Kosakonia oryzendophytica FY-07 is CGMCC No. 6103;

[0008] The HS-XGK fermentation medium per liter includes the following raw materials: 0.5 - 1.5 g of xanthan gum, 7 - 8 g of yeast powder, 8 - 12 g of peptone, 8 - 12 g of Na2HPO4, 0.5 - 1.5 g of KNO3, 15 - 30 g of glucose, and the balance of water.

[0009] As a preferred embodiment, the fermentation time is 24 - 48 h.

[0010] As a preferred embodiment, during the closed fermentation, the injection volume of the fermentation broth is 1 / 6 - 1 times the volume of the fermentation vessel.

[0011] As a preferred embodiment, the volume ratio of the Kosakonia oryzendophytica FY-07 seed liquid to the HS-XGK fermentation medium is 0.5 - 2:100.

[0012] As a preferred embodiment, the bacterial concentration of the Kosakonia oryzendophytica FY-07 seed liquid is ≥10 7 cells / mL.

[0013] As a preferred embodiment, after obtaining the bacterial cellulose with high mechanical strength, it further includes rinsing the bacterial cellulose with sterilized distilled water, soaking the rinsed bacterial cellulose with high mechanical strength in an NaOH aqueous solution, and repeating the processes of rinsing and soaking until the pH value of the bacterial cellulose with high mechanical strength is 6.8 - 7.2, thereby obtaining a wet film of bacterial cellulose with high mechanical strength.

[0014] As a preferred embodiment, the soaking temperature is 90 - 100 °C, and the soaking time for each time is 0.5 - 1 h.

[0015] As a preferred embodiment, the molar concentration of NaOH in the NaOH aqueous solution is 0.5 - 1 M.

[0016] As a preferred embodiment, after obtaining the wet film of bacterial cellulose with high mechanical strength, it further includes drying the wet film of bacterial cellulose with high mechanical strength.

[0017] The present invention also provides the bacterial cellulose with high mechanical strength prepared by the fermentation method described in the above solution. The tensile strength of the bacterial cellulose with high mechanical strength is ≥8 Mpa, and the Young's modulus is ≥20 Mpa.

[0018] Beneficial effects:

[0019] The present invention provides a fermentation method for improving the mechanical strength of bacterial cellulose, comprising the following steps: inoculating the seed solution of Kosakonia oryzendophytica FY-07 into the HS-XGK fermentation medium to obtain a fermentation broth; fermenting the fermentation broth in a sealed manner at 28-37 °C to obtain bacterial cellulose with high mechanical strength; the preservation number of Kosakonia oryzendophytica FY-07 is CGMCC No. 6103; based on 1 L, the HS-XGK fermentation medium comprises the following raw materials: 0.5-1.5 g of xanthan gum, 7-8 g of yeast powder, 8-12 g of peptone, 8-12 g of Na2HPO4, 0.5-1.5 g of KNO3, 15-30 g of glucose, and the balance of water. According to the characteristics that the strain Kosakonia orzyendophytica FY-07 (CGMCC No. 6103) can produce bacterial cellulose by submerged fermentation under aerobic, microaerobic and anaerobic conditions, the inoculated fermentation medium is injected into a fermentation container, covered to make it sealed, and the strain is used to quickly ferment and produce gas to increase the pressure in the container, so that the bacterial cellulose product is more dense and the mechanical strength is improved. The bacterial cellulose prepared by the fermentation method of the present invention has a tensile strength significantly increased by 2.59 times and a Young's modulus significantly increased by 4.36 times compared with the bacterial cellulose prepared by traditional open fermentation, indicating that the sealed fermentation product of the present invention has better mechanical properties.

[0020] Biological deposit description

[0021] Kosakonia oryzendophytica FY-07, formerly known as Enterobacter sp., was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on May 11, 2012, with the preservation number of CGMCC No. 6103. In 2022, through 16S rRNA sequence analysis, multilocus sequence analysis based on atpD, gyrB, infB and rpoB genes, in silico DNA-DNA hybridization, average nucleotide similarity, GC content difference and physiological and biochemical property detection, it was re-identified as the genus Kosakonia, species oryzendophytica, and named Kosakonia oryzendophytica FY-07 (see the literature: Ge Gao et al. Reclassification of Enterobacter sp. FY-07 as Kosakonia oryzendophytica FY-07 and Its Potential to Promote Plant Growth. Microorganisms 2022, 10(3):575.). Brief Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments will be briefly introduced below.

[0023] Figure 1 Morphology diagrams of the high mechanical strength bacterial cellulose products prepared for Examples 1 to 3; where A is a side view; B is a top view; C is a cross-sectional view;

[0024] Figure 2 Morphology diagrams of the bacterial cellulose products prepared for Comparative Example 1 and Example 1; where A is the open fermentation product of Comparative Example 1; B is the closed fermentation product of Example 1;

[0025] Figure 3 Characterization diagrams of the bacterial cellulose products prepared for Comparative Example 1 and Example 1; where A is an X-ray diffraction pattern; B is a Fourier transform infrared spectrum; C is a thermogravimetric curve; D is a derivative thermogravimetric analysis curve;

[0026] Figure 4 Scanning electron microscope diagrams of the bacterial cellulose products prepared for Comparative Example 1 and Example 1; where A is the open fermentation product of Comparative Example 1; B is the closed fermentation product of Example 1;

[0027] Figure 5 Pore distribution diagrams of the bacterial cellulose products prepared for Comparative Example 1 and Example 1; where A is the open fermentation product of Comparative Example 1; B is the closed fermentation product of Example 1;

[0028] Figure 6 Water holding rate and swelling rate of the bacterial cellulose products prepared for Comparative Example 1 and Example 1; where A is the water holding rate; B is the swelling rate; ns indicates that the data has no significant difference, * indicates that the data has a significant difference, p < 0.05;

[0029] Figure 7 Tensile strength diagrams of the bacterial cellulose products prepared for Comparative Example 1 and Example 1; where A is the stress; B is the tensile strength; C is the elongation at break; D is the Young's modulus; *** indicates that the data has a highly significant difference, p < 0.001;

[0030] Figure 8 Comparison diagrams of the texture properties of the bacterial cellulose products prepared for Comparative Example 1 and Example 1; where A is the hardness; B is the viscosity; C is the elasticity; D is the chewiness; E is the adhesiveness; F is the cohesiveness; G is the resilience; H is the gel strength; ns indicates that the data has no significant difference, * indicates that the data has a significant difference, p < 0.05; ** indicates that the data has a significant difference, p < 0.01; *** indicates that the data has a highly significant difference, p < 0.001;

[0031] Figure 9 Hemolysis rate graph of the bacterial cellulose products prepared in Comparative Example 1 and Example 1. Detailed implementation mode

[0032] The present invention provides a fermentation method for improving the mechanical strength of bacterial cellulose, comprising the following steps: inoculating the seed liquid of Kosakonia oryzendophytica FY-07 into the HS-XGK fermentation medium to obtain a fermentation broth; fermenting the fermentation broth in a closed state at 28-37 °C to obtain bacterial cellulose with high mechanical strength; the preservation number of Kosakonia oryzendophytica FY-07 is CGMCC No. 6103; based on 1 L, the HS-XGK fermentation medium comprises the following raw materials: 0.5-1.5 g of xanthan gum, 7-8 g of yeast powder, 8-12 g of peptone, 8-12 g of Na2HPO4, 0.5-1.5 g of KNO3, 15-30 g of glucose and the balance of water.

[0033] The seeds of Kosakonia oryzendophytica FY-07 of the present invention were inoculated into the HS-XGK fermentation medium to obtain a fermentation broth. Kosakonia oryzendophytica FY-07 of the present invention, formerly known as Enterobacter sp., was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on May 11, 2012, with the deposit number CGMCC No. 6103 and was disclosed in Patent CN111321184A. In 2022, through 16S rRNA sequence analysis, multilocus sequence analysis based on atpD, gyrB, infB, and rpoB genes, in silico DNA-DNA hybridization, average nucleotide similarity, GC content difference, and physiological and biochemical property detection, it was re-identified as the genus Kosakonia, species oryzendophytica, and named Kosakonia oryzendophytica FY-07. Kosakonia oryzendophytica FY-07 of the present invention can produce bacterial cellulose by submerged fermentation under aerobic, microaerobic, and anaerobic conditions. In the present invention, the inoculated fermentation medium was injected into a fermentation vessel, covered to make it airtight, and the rapid fermentation of the strain to produce gas was used to increase the pressure in the vessel, which can make the bacterial cellulose product denser and the mechanical strength higher. As an embodiment, the method for preparing the seeds of Kosakonia oryzendophytica FY-07 of the present invention comprises the following steps: (1) inoculating Kosakonia oryzendophytica FY-07 into an LB solid medium containing an aqueous solution of congo red, culturing at 28-32°C for 20-30 h, and picking a single colony of Kosakonia oryzendophytica FY-07; (2) inoculating the single colony of Kosakonia oryzendophytica FY-07 into an LB slant medium, culturing at 28-32°C for 20-30 h, rinsing the surface of the LB slant medium with sterilized distilled water, and collecting the rinsing solution to obtain the seeds of Kosakonia oryzendophytica FY-07.

[0034] The HS-XGK fermentation medium of the present invention, based on 1 L, comprises the following raw materials: 0.5 - 1.5 g of xanthan gum, 7 - 8 g of yeast powder, 8 - 12 g of peptone, 8 - 12 g of Na2HPO4, 0.5 - 1.5 g of KNO3, 15 - 30 g of glucose, and the balance of water. As an embodiment, the preparation of the HS-XGK fermentation medium includes: mixing yeast powder, peptone, Na2HPO4, KNO3, glucose, and water to obtain a mixed solution, slowly adding xanthan gum to the mixed solution with stirring, and subjecting it to shaking treatment at 30 °C to obtain the HS-XGK fermentation medium; the present invention has no special limitation on the time in the above process, and it is subject to being uniformly mixed. The xanthan gum in the HS-XGK fermentation medium of the present invention can provide a high-yield bacterial cellulose environment for Kosakonia oryzendophytica FY-07, and can physically or chemically act with bacterial cellulose to improve the properties of bacterial fiber.

[0035] As an embodiment, the bacterial concentration of the Kosakonia oryzendophytica FY-07 seed liquid ≥ 10 7 cells / mL. As an embodiment, the volume ratio of the Kosakonia oryzendophytica FY-07 seed liquid to the HS-XGK fermentation medium is 0.5 - 2:100. In specific embodiments of the present invention, the volume ratio of the Kosakonia oryzendophytica FY-07 seed liquid to the HS-XGK fermentation medium can be 0.5:100, 1:100, 1.5:100, or 2:100.

[0036] After obtaining the fermentation broth, the present invention seals and ferments the fermentation broth at 28 - 37 °C to obtain bacterial cellulose with high mechanical strength. As an embodiment, the injection amount of the fermentation broth is 1 / 6 - 1 times the volume of the fermentation container. In the present invention, the applicant found that when the injection amount of the fermentation broth is less than 1 / 6 of the volume of the fermentation container, the yield of the obtained bacterial cellulose is low, so the injection amount of the fermentation broth is selected to be 1 / 6 - 1 times the volume of the fermentation container. In specific embodiments of the present invention, the injection amount of the fermentation broth can be 1 / 6, 2 / 6, 3 / 6, 4 / 6, 5 / 6, or 1 times the volume of the fermentation container. The results of the examples show that the appearance and properties of the bacterial cellulose products obtained with different injection amounts are similar.

[0037] As an embodiment, the fermentation time is 24 - 48 h; in specific embodiments of the present invention, the fermentation time can be any value within 24 - 48 h. The fermentation temperature of the present invention is 28 - 37 °C; in specific embodiments of the present invention, the fermentation temperature can be any value within 28 - 37 °C.

[0038] As an implementation method, after obtaining the bacterial cellulose with high mechanical strength, it further includes rinsing the bacterial cellulose with sterilized distilled water, soaking the rinsed bacterial cellulose with high mechanical strength in an NaOH aqueous solution, and repeating the processes of rinsing and soaking until the pH value of the bacterial cellulose with high mechanical strength is 6.8 - 7.2, thereby obtaining a wet film of bacterial cellulose with high mechanical strength. The high-mechanical-strength bacterial cellulose produced by the fermentation of FY-07 in the present invention contains a large amount of culture medium. After rinsing and soaking, the culture medium contained in the high-mechanical-strength bacterial cellulose can be replaced with water, and at this time, the high-mechanical-strength bacterial cellulose is called a wet film of bacterial cellulose with high mechanical strength. At the same time, by repeating the processes of rinsing and soaking in the present invention, the residual NaOH solution, lysed bacteria, and other impurities can be removed, and a wet film of high-mechanical-strength bacterial cellulose containing only water can be obtained. As an implementation method, the molar concentration of NaOH in the NaOH aqueous solution is 0.5 - 1M. In a specific embodiment of the present invention, the molar concentration of NaOH in the NaOH aqueous solution can be 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, or 1M. As an implementation method, the soaking temperature is 90 - 100°C, and the soaking time each time is 0.5 - 1h. In a specific embodiment of the present invention, the soaking temperature can be any value within 90 - 100°C; the soaking time each time can be any value within 0.5 - 1h.

[0039] As an implementation method, after obtaining the wet film of bacterial cellulose with high mechanical strength in the present invention, it further includes drying the wet film of bacterial cellulose with high mechanical strength. As an implementation method, the drying method includes drying in an oven or vacuum freeze-drying; the present invention has no special restrictions on the drying temperature and time, and it is subject to the conventional settings in the art. Drying the wet film of bacterial cellulose with high mechanical strength in the present invention can broaden the application scenarios of bacterial cellulose. Taking the application as a facial mask as an example, the wet film can be directly attached to the skin; after drying, the user can soak it in essence according to their own needs and then use it.

[0040] The present invention also provides the high-mechanical-strength bacterial cellulose prepared by the fermentation method described in the above solution, and the tensile strength of the high-mechanical-strength bacterial cellulose ≥ 8 Mpa, and the Young's modulus ≥ 20 Mpa.

[0041] To further illustrate the present invention, the following will describe in detail a high-mechanical-strength bacterial cellulose and its fermentation method provided by the present invention with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] Example 1

[0043] (1) Preparation of Kosakonia oryzendophytica FY-07 seed liquid:

[0044] A. Streak the Kosakonia oryzendophytica FY-07 bacterial liquid on an LB solid medium plate containing 0.1% congo red, and culture it at 30 °C for 24 h;

[0045] B. Pick a large red single colony and streak it densely on an LB slant medium, and culture it at 30 °C for 24 h;

[0046] C. Pick a part of the thin bacterial cellulose membrane containing a large amount of active bacteria from the LB slant medium obtained in step B and streak it densely on a new LB slant medium, and culture it at 30 °C for 24 h;

[0047] D. Rinse the LB slant medium obtained in step C with 100 mL of pre-sterilized distilled water, and rinse repeatedly until the bacterial cellulose membrane containing a large amount of bacteria on the slant medium detaches, and place it in the distilled water and mix evenly to obtain the Kosakonia oryzendophytica FY-07 seed liquid; the components of the LB medium are: yeast powder 5 g / L, peptone 10 g / L, and NaCl 10 g / L, pH 7.4 - 7.6.

[0048] (2) Preparation of HS-XGK fermentation medium: Based on 1 L, the components are: xanthan gum 1.0 g / L, yeast powder 7.5 g / L, peptone 10 g / L, Na2HPO4 10 g / L, KNO3 1.0 g / L, and glucose 25 g / L, and the pH does not need to be adjusted. Xanthan gum needs to be added last, and when adding xanthan gum, it should be slow and accompanied by stirring. After adding, place it at 30 °C and shake and mix evenly until completely dissolved, and obtain the HS-XGK fermentation medium after autoclaving.

[0049] (3) Inoculation: Take the Kosakonia oryzendophytica FY-07 seed liquid prepared in step (1) and inoculate it into the HS-XGK fermentation medium prepared in step (2) at a volume ratio of 1%, and mix evenly to obtain the fermentation broth.

[0050] (4) Fermentation: Inject the fermentation broth obtained in step (3) into a fermentation container, seal it with a lid, and place it in an incubator, and culture and ferment it at 30 °C for 24 h; the fermentation broth fills the fermentation container, that is, the volume of the injected fermentation broth is equal to the volume of the fermentation container.

[0051] (5) Treatment: After the fermentation is completed, slowly release the gas in the fermentation container, then fish out the high mechanical strength bacterial cellulose membrane produced by the fermentation in step (4), wash it with water to remove most of the bacteria and impurities, and then soak it in 0.5M NaOH solution at 100 °C for 0.5 h to remove the bacteria and residual medium in the high mechanical strength bacterial cellulose hydrate. After that, wash and soak it multiple times until the pH is neutral to obtain a high mechanical strength bacterial cellulose wet membrane.

[0052] Example 2

[0053] A fermentation method of high mechanical strength bacterial fiber similar to that in Example 1, the steps are as follows:

[0054] (1) Preparation of Kosakonia oryzendophytica FY-07 seed liquid: Operate according to step (1) of Example 1.

[0055] (2) Preparation of HS-XGK fermentation medium: Operate according to step (2) of Example 1.

[0056] (3) Inoculation: Operate according to step (3) of Example 1.

[0057] (4) Fermentation: Operate according to step (4) of Example 1, the difference is that the volume of the fermentation broth is 2 / 3 times the volume of the fermentation container.

[0058] (5) Treatment: Operate according to step (5) of Example 1.

[0059] Example 3

[0060] A fermentation method of high mechanical strength bacterial fiber similar to that in Example 1, the steps are as follows:

[0061] (1) Preparation of Kosakonia oryzendophytica FY-07 seed liquid: Operate according to step (1) of Example 1.

[0062] (2) Preparation of HS-XGK fermentation medium: Operate according to step (2) of Example 1.

[0063] (3) Inoculation: Operate according to step (3) of Example 1.

[0064] (4) Fermentation: Operate according to step (4) of Example 1, the difference is that the volume of the fermentation broth is 1 / 3 times the volume of the fermentation container.

[0065] (5) Treatment: Operate according to step (5) of Example 1.

[0066] Comparative Example 1

[0067] A fermentation method of high mechanical strength bacterial cellulose similar to that in Example 1 is as follows:

[0068] (1) Prepare the Kosakonia oryzendophytica FY-07 seed liquid: Operate according to step (1) of Example 1.

[0069] (2) Prepare the HS-XGK fermentation medium: Operate according to step (2) of Example 1.

[0070] (3) Inoculate: Operate according to step (3) of Example 1.

[0071] (4) Ferment: Inject the fermentation broth obtained in step (3) into a 500 mL volumetric beaker, add a breathable bottle sealing film, and place it in an incubator. Ferment at 30 °C for 24 h.

[0072] (5) Treat: Operate according to step (5) of Example 1.

[0073] Application Example 1

[0074] Take pictures of the high mechanical strength bacterial cellulose obtained in Examples 1 to 3, and observe the state of the high mechanical strength bacterial cellulose products prepared from the fermentation broth with different injection amounts in the fermentation container. The results are as Figure 1 shown.

[0075] According to Figure 1 it can be seen that the high mechanical strength bacterial cellulose prepared in Examples 1 to 3 is dense, bubble-free, has a tighter texture, and is white in overall color. The states of the high mechanical strength bacterial cellulose products with different injection amounts are similar, indicating that the injection amount of the fermentation broth in the fermentation container has no obvious effect on the high mechanical strength bacterial cellulose.

[0076] Application Example 2

[0077] (1) Select the high mechanical strength bacterial cellulose prepared by closed fermentation in Example 1 (denoted as BC-P) and the bacterial cellulose prepared by open fermentation in Comparative Example 1 (denoted as BC) for product morphology comparison. The results are as Figure 2 shown. According to Figure 2 it can be seen that the bacterial cellulose product obtained by closed fermentation is denser, bubble-free, has a tighter texture, and is whiter in overall color.

[0078] (2) Take partial fermentation products (bacterial cellulose wet films) from Example 1 and Comparative Example 1 respectively, and conduct vacuum freeze-drying treatment to obtain high mechanical strength bacterial cellulose after drying (Example 1, denoted as BC-P) and bacterial cellulose (Comparative Example 1, denoted as BC). The sealed fermentation product (BC-P) and open fermentation product (BC) after drying in Example 1 and Comparative Example 1 were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM) and thermogravimetric analysis (TGA). The detection method was referred to the literature (X. Zhao, Y. Shi, M. Yang, et al., “Barnacle-Inspired and Polyphenol-Assisted Modification of Bacterial Cellulose-Based Wound Dressings for Promoting Infectious Wound Healing,” International Journal of Biological Macromolecules 279 (2024): 135291, https: / / doi.org / 10.1016 / j.ijbiomac.2024.135291.) and the results are as Figures 3 - 4 shown.

[0079] (3) Detect the porosity, water holding rate, swelling degree, ultraviolet light transmittance, water vapor transmittance, tensile strength, texture analysis, blood compatibility and other characteristics of the sealed fermentation products (BC-P) and open fermentation products (BC) in Example 1 and Comparative Example 1. Among them, the fermentation products (bacterial cellulose wet films) obtained in Example 1 and Comparative Example 1 were used for tensile strength, texture analysis and water holding rate detection; the high mechanical strength bacterial cellulose (BC-P) and bacterial cellulose (BC) prepared in step (2) after drying were used for porosity, swelling degree, ultraviolet light transmittance, water vapor transmittance and blood compatibility detection.

[0080] A. The porosity detection method refers to GB / T 21650.1 "Determination of pore size distribution and porosity of solid materials by mercury intrusion method and gas adsorption method - Part 1: Mercury intrusion method", and the detection results are shown in Table 1 and Figure 5 shown.

[0081] Table 1 Comparison of porosity detection data of fermentation products in Example 1 and Comparative Example 1

[0082] Item Comparative Example 1 (BC) Example 1 (BC-P) Total Intrusion Volume 12.42 mL / g 7.59 mL / g Total Pore Area <![CDATA[18.39m 2 / g]]> <![CDATA[60.06m 2 / g]]> Mesopore Diameter (Volume) 57276.65 nm 7813.77 nm Mesopore Diameter (Area) 159.76 nm 69.95 nm Average Pore Diameter (4V / A) 2700.49 nm 505.35 nm Volume Density at an Absolute Pressure of 0.49 psia. 0.07 g / mL 0.11 g / mL Apparent (Skeletal) Density 0.58 g / mL 0.80 g / mL Porosity 87.76％ 85.86％

[0083] B. The detection methods of water holding rate and swelling degree refer to the literature (G. Gao, Z. Liao, Y. Cao et al., “Highly efficient production of bacterial cellulose from corn stover total hydrolysate by Enterobacter sp. FY-07” Bioresource Technology 341 (2021): 125781 https: / / doi.org / 10.1016 / j.biortech.2021.125781) and the detection results are as Figure 6 shown in and Table 2.

[0084] C. The detection method of tensile strength refers to the literature (S. Chen, L. Patricia, D. Wang, et al., “Mechanical properties of bacterial cellulose synthesised by diverse strains of the genus Komagataeibacter” Food Hydrocolloids 81 (2018): 87-95 https: / / doi.org / 10.1016 / j.foodhyd.2018.02.031) and the detection results are as Figure 7 shown in and Table 2.

[0085] Table 2 Comparison of water holding rate and tensile strength of the fermentation products of Example 1 and Comparative Example 1

[0086]

[0087] D. The water vapor transmission rate was detected by referring to the standard test method for water vapor permeability of ASTM E96 materials; the ultraviolet light transmission rate was detected by referring to the literature (Y. Zhang, G. Chen, W. Qin et al., “In Situ Fermentation of an Ultra-Strong, Microplastic-Free, and Biodegradable Multilayer Bacterial Cellulose Film for Food Packaging” ACS Applied Materials & Interfaces 15(37)(2023):44354–44363 https: / / doi.org / 10.1021 / acsami.3c10563). The test results are shown in Table 3.

[0088] Table 3 Comparison of Ultraviolet Light Transmission Rate and Water Vapor Transmission Rate of Fermentation Products of Example 1 and Comparative Example 1

[0089] Group T280(％) T200-280(％) T600(％) <![CDATA[WVTR (g / (m 2 ·day))]]> Comparative Example 1 (BC) 0.05 0.04 5.5732 1168.6 Example 1 (BC-P) 0.05 0.03 2.652 358.8

[0090] E. The texture analysis method was referred to the literature (G. Gao, Y. Cao, Y. Zhang et al., “In situ production of bacterial cellulose / xanthan gum nanocomposites with enhanced productivity and properties using Enterobacter sp. FY-07” Carbohydrate Polymers 248(2020):116788 https: / / doi.org / 10.1016 / j.carbpol.2020.116788). The test results are as Figure 8 and shown in Table 4.

[0091] Table 4 Comparison of Texture Analysis of Fermentation Products of Example 1 and Comparative Example 1

[0092]

[0093] F. The blood compatibility test method was referred to the literature (G. Gao, Z. Liao, Y. Cao et al., “Enhancing Wound Healing and Bactericidal Efficacy: A Hydrogel Membrane of Bacterial Cellulose and Sanxan Gel for Accelerating the Healing of Infected Wounds” Advanced Healthcare Materials 13(8)(2024): 2303216 https: / / doi.org / 10.1002 / adhm.2303216.) and the test results are as Figure 9 shown in and Table 5.

[0094] Table 5 Comparison of Hemolysis Rates of Fermentation Products of Example 1 and Comparative Example 1

[0095] Group Comparative Example 1 (BC) Example 1 (BC-P) PBS <![CDATA[H2O]]> Hemolysis Rate (%) 0.60％ 2.27％ 0％ 100％

[0096] According to Tables 1 - 5 and Figures 3 - 9 the results, it was found that compared with the normal fermentation product, the crystallinity ( Figure 3 A in), the functional group composition ( Figure 3 B in), and the thermogravimetric curve ( Figure 3 C, D in) of the closed - fermentation product had no significant differences; Scanning electron microscopy observation found that the fiber network density of the closed - fermentation product was greater and the pore size was smaller ( Figure 4 ); The mercury intrusion experiment showed that the porosity of the closed - fermentation product had no significant change but the pore size was smaller ( Figure 5 ); There was no significant difference in the water - holding capacity between the open - fermentation product and the closed - fermentation product, but the swelling rate decreased ( Figure 6 ), which might be due to the large network density; The slightly lower ultraviolet transmittance of the closed - fermentation product indicated that the closed - fermentation bacterial cellulose product was denser and had higher ultraviolet resistance (Table 2), and the significantly lower water vapor transmittance indicated that the denser membrane had better water vapor barrier performance and had better application potential in food preservation, etc. (Table 2); The tensile strength of the closed - fermentation product increased significantly by 2.59 times and the Young's modulus increased significantly by 4.36 times, indicating that the closed - fermentation product had better mechanical properties ( Figure 7 ); The elasticity, chewiness, and cohesiveness of the closed - fermentation product increased significantly, while the hardness, adhesiveness, resilience, and gel strength decreased significantly ( Figure 8 ), indicating that there were differences in the effects of the closed - fermentation product and the open - fermentation product when used to improve the food taste; Both products had extremely low hemolysis rates ( Figure 9 ), indicating good biocompatibility.

[0097] In summary, the present invention prepares bacterial cellulose films with high mechanical strength through closed fermentation, and tests their basic properties, tensile strength, texture properties and other performance. The results show that the products of closed fermentation have significantly improved in mechanical strength and other aspects compared with ordinary products. It can be seen that closed fermentation can be used to produce bacterial cellulose with high mechanical strength, and this strategy has broad application prospects.

[0098] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fermentation method for improving the mechanical strength of bacterial cellulose, characterized in that: The following steps are involved: The seed liquid of Kosakonia oryzendophytica FY-07 is inoculated into the HS-XGK fermentation medium to obtain a fermentation liquid; the fermentation liquid is sealed and fermented at 28 to 37° C. to obtain bacterial cellulose with high mechanical strength; The deposit number of Kosakonia oryzendophytica FY-07 is CGMCC No.6103; The HS-XGK fermentation medium, based on 1L, includes the following raw materials: 0.5-1.5g of xanthan gum, 7-8g of yeast powder, 8-12g of peptone, 8-12g of Na2HPO4, 0.5-1.5g of KNO3, 15-30g of glucose and the balance of water.

2. The fermentation method according to claim 1, characterized in that The fermentation time is 24 to 48 hours.

3. The fermentation method according to claim 1, characterized in that During the closed fermentation, the amount of fermentation liquid injected is 1 / 6 to 1 times the volume of the fermentation container.

4. The fermentation method according to claim 1, characterized in that The volume ratio of the Kosakonia oryzendophytica FY-07 seed solution to the HS-XGK fermentation medium is 0.5 to 2:

100.

5. The fermentation method according to claim 1 or 4, characterized in that: The bacterial concentration of the Kosakonia oryzendophytica FY-07 seed solution is ≥10 7 cells / mL.

6. The fermentation method according to claim 1, characterized in that After obtaining the high mechanical strength bacterial cellulose, the method further includes rinsing the bacterial cellulose with sterile distilled water, soaking the rinsed high mechanical strength bacterial cellulose in a NaOH aqueous solution, repeating the rinsing and soaking process until the pH value of the high mechanical strength bacterial cellulose is 6.8-7.2, and obtaining a high mechanical strength bacterial cellulose wet film.

7. The fermentation method according to claim 6, characterized in that The soaking temperature is 90-100° C., and the soaking time is 0.5-1 hour.

8. The fermentation method according to claim 6, characterized in that The molar concentration of NaOH in the NaOH aqueous solution is 0.5-1M.

9. The fermentation method according to claim 6, characterized in that: After obtaining the high mechanical strength bacterial cellulose wet film, the method further includes drying the high mechanical strength bacterial cellulose wet film.

10. The high mechanical strength bacterial cellulose prepared by the fermentation method according to any one of claims 1 to 9, characterized in that: The tensile strength of the high mechanical strength bacterial cellulose is ≥8Mpa, and the Young's modulus is ≥20Mpa.

Images