Bacterial cellulose modified in situ using tri-glucan and preparation method thereof

Abstract

The present invention provides a bacterial cellulose in situ modified by tri-zan gum and a preparation method thereof, which belongs to the field of biopolymer materials and biofermentation technology, comprising the following steps: yeast powder, peptone, Na2HPO4, KNO3 and glucose are mixed with water, and then tri-zan gum is slowly added with stirring until completely dissolved, followed by adding vegetable oil and a fat-soluble modifier to prepare an HS-SG fermentation medium; FY-07 seed liquid is inoculated into the HS-SG fermentation medium for culturing and fermentation; the bacterial cellulose film produced by fermentation is fished out, rinsed with water, and then the bacteria and residual culture medium in the bacterial cellulose hydrate are removed with a NaOH solution under high temperature conditions, thereby obtaining a bacterial cellulose wet film. It can be seen that the emulsification effect of tri-zan gum can achieve uniform dispersion of fat-soluble components, thereby achieving uniform in situ modification of bacterial cellulose, and then obtaining in situ modified bacterial cellulose with good antioxidant properties and good biocompatibility.

Description

Technical Field

[0001] The invention belongs to the technical field of biopolymer materials and biofermentation, and in particular relates to bacterial cellulose in situ modified with tri-glucan and a preparation method thereof. Background Art

[0002] Bacterial cellulose (BC) is a biomacromolecule polymer with many excellent properties, such as high purity, high crystallinity, high mechanical strength and good biocompatibility. The unique advantages of bacterial cellulose make it widely used in food, biomedicine and other industries.

[0003] Although bacterial cellulose has excellent performance, different applications often require different performance priorities. Therefore, in situ modification is often used to enhance its original performance or add new functions. In situ modification refers to the process of changing the culture conditions or adding additional modifiers (such as additives or materials) to the culture medium during the fermentation process to alter the morphology, mechanical properties, crystallinity, rehydration rate, and other important functional properties of bacterial cellulose.

[0004] While in situ modification offers low cost and a simple process, the currently used bacterial cellulose-producing strains are strictly aerobic and can only produce BC at the interface between the culture medium and air, resulting in uneven modification of the final product. Furthermore, only water-soluble modifiers are suitable for in situ modification, as water-insoluble modifiers precipitate or aggregate in the culture medium, making in situ modification difficult. This, in turn, precludes the use of a large number of functional, lipid-soluble natural products as modifiers.

[0005] Kosakonia oryzednophytica FY-07 (formerly known as Enterobacter sp. FY-07, FY-07) is a unique bacterial cellulose-producing strain. Its ability to rapidly produce large quantities of BC under aerobic, oxygen-limited, and anaerobic conditions necessitates a unique submerged fermentation mode. Previous studies have shown that in situ modification of FY-07 using this submerged fermentation mode results in more uniform products. However, water-insoluble modifiers cannot be evenly distributed in the culture medium, making it difficult to obtain a uniform modified product even using this submerged fermentation mode.

[0006] Therefore, there is currently no effective method to solve the problem of using fat-soluble modifiers to modify bacterial cellulose, which seriously limits the further application of bacterial cellulose. Summary of the Invention

[0007] The problem to be solved by the present invention is to provide a bacterial cellulose in situ modified by using tri-zan gum and a preparation method thereof.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is: a preparation method of bacterial cellulose using tri-zan gum for in-situ modification, comprising the following steps:

[0009] S1, preparing Kosakonia oryzendophytica FY-07 seed solution;

[0010] S2. Prepare HS-SG fermentation medium: Mix yeast powder, peptone, Na2HPO4, KNO3, and glucose with water; no pH adjustment is required; then slowly add triglyceride with stirring, shake and mix until completely dissolved; then add a modifier oil solution formed by vegetable oil and a fat-soluble modifier, mix well to form an emulsion, and sterilize by autoclaving to obtain HS-SG fermentation medium;

[0011] S3, taking the seed solution prepared in step S1, inoculating it into the HS-SG fermentation medium prepared in step S2, mixing evenly, and placing it in an incubator for culture and fermentation;

[0012] S4. The bacterial cellulose membrane produced by the fermentation in step S3 is fished out, rinsed with water to remove most of the bacteria and impurities, and then treated with NaOH solution under high temperature conditions to remove the bacteria and residual culture medium in the bacterial cellulose hydrate. Thereafter, the membrane is washed and soaked with water multiple times until the pH is neutral, thereby obtaining a bacterial cellulose wet membrane.

[0013] Furthermore, step S1 includes:

[0014] S11. Streak the bacterial suspension of Kosakonia oryzendophytica FY-07 onto LB plates containing 0.1% Congo red and incubate at 30°C for 24 h.

[0015] S12, picking up the red and larger single colonies cultured in step S11 and streaking densely onto the first LB slant medium, and culturing at 30°C for 24 hours;

[0016] S13, picking up the thin bacterial cellulose film containing a large number of active bacteria cultured in step S12 and streaking densely on the second LB slant culture medium, and culturing at 30°C for 24 hours;

[0017] S14. Rinse the second LB slant culture medium cultured in step S13 with 100 ml of pre-sterilized distilled water and recover it. Repeat the rinsing until the bacterial cellulose film containing a large number of bacteria on the second LB slant culture medium is detached. Mix the bacterial cellulose containing a large number of bacteria in the recovered distilled water to serve as the seed solution.

[0018] Furthermore, in step S1, the components of the LB plate culture medium, the first LB slant culture medium, and the second LB slant culture medium are: 5 g / L yeast powder, 10 g / L peptone, and 10 g / L NaCl; and the pH is 7.4-7.6.

[0019] Furthermore, in step S2, Sanzan gum is added at a rate of 1 g / min.

[0020] Furthermore, in step S2, the HS-SG fermentation medium includes 7.5 g / L yeast powder, 10 g / L peptone, 10 g / L Na2HPO4, 1.0 g / L KNO3, 25 g / L glucose, 0.1~0.3% (w / w) triglyceride, 8~12% (w / w) vegetable oil and 1~3 g / L fat-soluble modifier.

[0021] Furthermore, in step S2, the vegetable oil is preferably sunflower oil, peanut oil, or soybean oil.

[0022] Furthermore, in step S2, the fat-soluble modifier is preferably carotene, astaxanthin, silymarin, or vitamin E.

[0023] Furthermore, in step S3, the seed liquid prepared in step S1 is taken and inoculated into the HS-SG fermentation medium prepared in step S2 at a ratio of 1%, mixed evenly, and placed in an incubator for fermentation at 30°C for 24 hours.

[0024] Furthermore, in step S4, the bacterial cellulose membrane is rinsed with water and then treated with a 0.5 M NaOH solution at 100° C. for 0.5 h.

[0025] The present invention also provides a bacterial cellulose in situ modified by using tri-glucan.

[0026] Due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0027] Trisammonium gum is an extracellular polymer synthesized by Sphingomonas trisammonium. Compared with other polysaccharide polymers, it has low extraction cost, high viscosity, and shear thinning properties. It also has a unique amphiphilic structure composed of polysaccharides, polypeptides and lipids. The present invention uses it as an excellent emulsifier.

[0028] Natural active ingredients (such as carotene, astaxanthin, silymarin, vitamin E, etc.) are well-known for their unique biological activities, such as antibacterial, antioxidant, anti-inflammatory, etc., as well as safety, non-toxicity, environmental friendliness, and biocompatibility. They are commonly used in the food, medicine, and cosmetics industries. In the present invention, they are used as excellent modifiers for the in-situ modification of bacterial cellulose.

[0029] The present invention dissolves fat-soluble active substances in vegetable oil using tri-zan gum as an emulsifier and suspending agent, and uniformly emulsifies, suspends and disperses the substances in a culture medium. Then, the emulsified suspension system is used and FY-07 is used as a bacterial cellulose production strain to carry out in-situ modified bacterial cellulose fermentation production, thereby obtaining bacterial cellulose products with different functional properties and good biocompatibility.

[0030] It can be seen that the emulsification effect of Sanzan gum can achieve uniform dispersion of fat-soluble components, thereby achieving uniform in situ modification of bacterial cellulose, and then obtaining in situ modified bacterial cellulose with good antioxidant properties and good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be described in detail below with reference to the accompanying drawings and in combination with examples, and the advantages and implementation modes of the present invention will become more apparent. The contents shown in the accompanying drawings are only used to illustrate the present invention and do not constitute any limitation to the present invention. In the accompanying drawings:

[0032] Figure 1 This is a diagram showing the emulsification effect of the Sanzan gum of the present invention on different fat-soluble modifiers.

[0033] Figure 2 This is a diagram of bacterial cellulose products to which different fat-soluble modifiers are added in the present invention.

[0034] Figure 3 This is a bar graph showing the yield of FY-07 bacterial cellulose at different addition amounts of Sanzan gum of the present invention.

[0035] Figure 4 This is a bar chart showing the structural properties of FY-07 bacterial cellulose produced with different addition amounts of Sanzan gum according to the present invention.

[0036] Figure 5 The antioxidant performance bar graph of the FY-07 bacterial cellulose product prepared by adding different fat-soluble modifiers in the present invention.

[0037] Figure 6 This is a release curve of the in situ modified bacterial cellulose of the present invention during in vitro digestion. DETAILED DESCRIPTION

[0038] The present invention provides a method for preparing bacterial cellulose by in-situ modification of tri-glucan gum, comprising the following steps:

[0039] S1, preparing Kosakonia oryzendophytica FY-07 seed solution;

[0040] S11. Streak the bacterial suspension of Kosakonia oryzendophytica FY-07 onto LB plates containing 0.1% Congo red and incubate at 30°C for 24 h.

[0041] S12, picking up the red and larger single colonies cultured in step S11 and streaking densely onto the first LB slant medium, and culturing at 30°C for 24 hours;

[0042] S13, picking up the thin bacterial cellulose film containing a large number of active bacteria cultured in step S12 and streaking densely on the second LB slant culture medium, and culturing at 30°C for 24 hours;

[0043] S14. Rinse the second LB slant culture medium cultured in step S13 with 100 ml of pre-sterilized distilled water and recover it. Repeat the rinsing until the bacterial cellulose film containing a large number of bacteria on the second LB slant culture medium is detached. Mix the bacterial cellulose containing a large number of bacteria in the recovered distilled water to serve as the seed solution.

[0044] S2. Prepare HS-SG fermentation medium: Mix yeast powder, peptone, Na2HPO4, KNO3, and glucose with water; no pH adjustment is required; then, add triglyceride at a rate of 1 g / min with stirring. After addition, shake and mix at 30°C until completely dissolved. Then, add a modifier oil solution formed by vegetable oil and a fat-soluble modifier, mix well to form an emulsified system, and sterilize by high pressure to prepare HS-SG fermentation medium.

[0045] The HS-SG fermentation medium includes 7.5 g / L yeast powder, 10 g / L peptone, 10 g / L Na2HPO4, 1.0 g / L KNO3, 25 g / L glucose, 0.1-0.3% (w / w) triglyceride, 8-12% (w / w) vegetable oil and 1-3 g / L fat-soluble modifier.

[0046] S3. Take the seed liquid prepared in step S1 and inoculate it into the HS-SG fermentation medium prepared in step S2 at a ratio of 1%. After mixing evenly, place it in an incubator and culture and ferment at 30° C. for 24 hours.

[0047] S4. The bacterial cellulose membrane produced by the fermentation in step S3 is fished out, rinsed with water to remove most of the bacteria and impurities, and then treated with 0.5M NaOH solution at 100°C for 0.5h to remove the bacteria and residual culture medium in the bacterial cellulose hydrate. The membrane is then washed and soaked with water several times until the pH is neutral, thereby obtaining a bacterial cellulose wet membrane.

[0048] S5. Drying the wet bacterial cellulose film to obtain a dry film, and calculating the yield.

[0049] The Kosakonia oryzendophytica FY-07 was isolated from oilfield produced fluid and deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with a deposit number of CGMCC No. 6103 and a deposit date of May 11, 2012.

[0050] The components of the LB plate culture medium, the first LB slant culture medium, and the second LB slant culture medium are: 5 g / L yeast powder, 10 g / L peptone, and 10 g / L NaCl; and the pH is 7.4-7.6.

[0051] Among them, the Sanzan gum as an emulsifier comes from Meihua Group.

[0052] Wherein, the vegetable oil is sunflower oil, peanut oil, soybean oil and the like.

[0053] Wherein, the fat-soluble modifier is carotene, astaxanthin, silymarin, vitamin E and the like.

[0054] The present invention also provides a bacterial cellulose in situ modified by using tri-glucan.

[0055] Emulsification effect of Sanzan gum on fat-soluble modifiers:

[0056] The method of preparing HS-SG fermentation medium in step S2 was used to compare the emulsification effect of tri-zan gum on different fat-soluble modifiers, such as Figure 1 As shown, the results showed that 0.2% (w / w) triamcinol had a good emulsifying effect on carotene, astaxanthin and silymarin dissolved in sunflower oil (from left to right in the figure, the final concentration was 2 g / L), and could remain stable after high temperature and high pressure sterilization.

[0057] Effects of different amounts of triglycerides added to HS-SG fermentation medium on the yield of FY-07 bacterial cellulose:

[0058] Experiment 1: Step S1 is the same as in the above embodiment.

[0059] S2. Prepare three groups of HS-SG fermentation medium: in each group, mix yeast powder, peptone, Na2HPO4, KNO3, and glucose with water, and do not adjust the pH; then add 0.1% (w / w), 0.2% (w / w), and 0.3% (w / w) of triamcinol (i.e., 1, 2, and 3 g / L of triamcinol) to the three groups, respectively, at a rate of 1 g / min with stirring. After addition, shake and mix at 30°C until completely dissolved. Then, add carotene to each group to a final concentration of 0.2% (w / w) and dissolve it in sunflower oil to form 3 ml of a modifier oil solution. Mix well to form an emulsified system, and sterilize under high pressure to prepare 30 ml of HS-SG fermentation medium per group;

[0060] S3. Take the seed solution prepared in step S1 and inoculate it into the three groups of HS-SG fermentation medium prepared in step S2 at a ratio of 1%, mix them evenly, and place them in an incubator for fermentation at 30°C for 24 hours;

[0061] S4, removing the bacterial cellulose membrane produced by the fermentation in step S3, rinsing with water to remove most of the bacterial cells and impurities, and then treating with 0.5M NaOH solution at 100°C for 0.5h to remove the bacterial cells and residual culture medium in the bacterial cellulose hydrate, followed by multiple washing and soaking until the pH is neutral, thereby obtaining three groups of bacterial cellulose wet membranes;

[0062] S5. Dry the three groups of bacterial cellulose wet films to obtain dry films, and calculate the yield.

[0063] Experiment 2: Replace the carotene in Experiment 1 with astaxanthin, and keep the rest the same.

[0064] In Experiment 3, the carotene in Experiment 1 was replaced with silymarin, and the rest of the ingredients remained the same.

[0065] like Figure 2 As shown in the figure, through experiments 1, 2 and 3, it can be seen that the HS-SG fermentation medium with the addition of different liposoluble modifiers can produce in situ modified bacterial cellulose films with good morphology.

[0066] like Figure 3 As shown in the figure, the effect of different amounts of tri-gluconolactone added on the bacterial cellulose yield was further measured. It can be seen that with the increase of tri-gluconolactone addition, the yield of in situ modified bacterial cellulose also increased significantly. It is speculated that this is because more fat-soluble modifiers are incorporated into the three-dimensional network structure of bacterial cellulose to form a composite material. However, when the amount of tri-gluconolactone added is higher than 0.3% (w / w), the yield no longer increases.

[0067] Effects of different amounts of trisammonium gum added to HS-SG fermentation medium on the textural properties of FY-07 bacterial cellulose:

[0068] Nine sets of bacterial cellulose wet films were obtained using the methods of Experiments 1, 2, and 3. Their textural properties were measured using a TA.XTC Texture Analyzer (Shanghai Baosheng Technology Co., Ltd.) using a cylindrical probe (p / 0.5). Cylindrical samples measuring 2 × 2 cm (height × width) were compressed to 50% at a pre-test speed of 2.00 mm / s, a test speed of 1.00 mm / s, and a post-test speed of 1.00 mm / s. The strain was 75% and the trigger-activated automatic pressure was -5 g. The resulting texture profile analysis (TPA) curves characterized the hardness, gel strength, and adhesiveness of each sample.

[0069] like Figure 4 As shown in the results, it was found that adding a final concentration of greater than 0.2% (w / w) of tri-zan gum could produce in-situ modified bacterial cellulose membranes with high hardness, adhesion, and gel strength. However, in the experiment where astaxanthin was added as a modifier, adding 0.3% (w / w) of tri-zan gum resulted in a certain degree of reduction in texture properties. In addition, in the experiment where silymarin was added as a modifier, adding 0.1% (w / w) of tri-zan gum did not allow for normal fermentation to produce in-situ modified bacterial cellulose membranes. This may be because tri-zan gum has a certain antibacterial effect, and a good emulsification effect could not be achieved under low concentrations of tri-zan gum, resulting in the inability to produce membranes normally.

[0070] Detection of the Antioxidant Properties of Bacterial Cellulose Modified by Sanzan Glue

[0071] Three sets of wet bacterial cellulose membranes were prepared using the methods of Experiments 1, 2, and 3, with the addition of 0.2% (w / w) trisaccharin. The antioxidant properties of these samples were then tested using an ABTS antioxidant kit. The specific steps are as follows: The ABTS solution and the oxidant solution were mixed and allowed to react in the dark for a specified period of time (usually 12–16 hours). The solution was then diluted with anhydrous ethanol to form a working solution. 280 μl of the working solution was added to 7 μl of the sample to be tested (1 g of in situ modified bacterial cellulose membrane per set was extracted with 1 ml of anhydrous ethanol). The mixture was then allowed to stand at room temperature for 6 minutes. The absorbance of each tube was then measured at 734 nm using a microplate reader. The total antioxidant capacity of the sample was calculated using a standard curve, typically expressed as Trolox equivalents. The ABTS free radical scavenging activity of the sample was quantified by comparing the changes in absorbance.

[0072] like Figure 5 As shown in the results, it can be found that the free radical scavenging rate of the bacterial cellulose samples modified in situ with the addition of carotene and astaxanthin can reach more than 10%, while the free radical scavenging rate of the bacterial cellulose samples modified in situ with the addition of silymarin can reach more than 20%, both of which have certain antioxidant properties.

[0073] Detection of sustained-release effect of Sanzan collagen in situ modified bacterial cellulose during in vitro digestion:

[0074] 0.2% (w / w) triazan gum was added to obtain a bacterial cellulose wet film by the method of experiment 1. During in vitro digestion, the release kinetics of β-carotene encapsulated in the in situ modified bacterial cellulose were evaluated using simulated gastric fluid (SGF) and simulated intestinal fluid (SIF).

[0075] 10 g of in situ modified bacterial cellulose was cut into small pieces and transferred to several transparent flasks. 30 ml of SGF and SIF were then added to each flask and thoroughly mixed by stirring. The samples were incubated at 30°C for 24 hours in a shaker at 200 rpm to complete the digestion process. At the designated digestion stage, one of the digestion solutions was immediately removed for further analysis (β-carotene content determination). β-carotene content in the two simulated digestion solutions was extracted and determined. 21 ml of the simulated digestion solution was added to 1 ml of n-hexane and vigorously shaken for 2 minutes to completely extract the β-carotene. After standing for 5 minutes, the organic layer was removed. The organic layer was centrifuged at 12,000 rpm for 2 minutes to separate any contaminants or the aqueous phase. The β-carotene content in each organic layer was determined by absorbance at 450 nm.

[0076] like Figure 6 As shown, the release of β-carotene in SIF increases rapidly during the initial digestion phase (within 7 hours), followed by a gradual decrease, reflecting the gradual transfer of β-carotene from the in situ modified BC to the aqueous phase during digestion. The lipid and peptide components of triamcinol are digested by enzymes in SIF, resulting in the gradual release of β-carotene. In contrast, triamcinol maintains a well-defined gel state in SGF, resulting in minimal β-carotene release. In summary, triamcinol in situ modified BC can be used for pH-responsive controlled release of specific drugs or active ingredients.

[0077] The embodiments of the present invention are described in detail above, but the contents are only preferred embodiments of the present invention and should not be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A method for preparing bacterial cellulose by in situ modification of triglycerides, characterized in that: The following steps are involved: S1. Prepare Kosakonia oryzendophytica FY-07 seed solution; the Kosakonia oryzendophytica FY-07 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No.6103; S2. Prepare HS-SG fermentation medium: The HS-SG fermentation medium comprises 7.5 g / L yeast powder, 10 g / L peptone, 10 g / L Na2HPO4, 1.0 g / L KNO3, 25 g / L glucose, 0.1-0.3% w / w triglyceride, 8-12% w / w vegetable oil, and 1-3 g / L fat-soluble modifier; the yeast powder, peptone, Na2HPO4, KNO3, and glucose are mixed with water; triglyceride is then slowly added with stirring, and after the addition is completed, the mixture is shaken and mixed until completely dissolved, and then a modifier oil solution formed by the vegetable oil and the fat-soluble modifier is added, triglyceride is used as an emulsifier and suspending agent to achieve uniform dispersion of the fat-soluble modifier, the mixture is evenly mixed to form an emulsified system, and the mixture is sterilized by high pressure to obtain the HS-SG fermentation medium; S3, taking the seed solution prepared in step S1, inoculating it into the HS-SG fermentation medium prepared in step S2, mixing evenly, and placing it in an incubator for culture and fermentation; S4. The bacterial cellulose membrane produced by the fermentation in step S3 is fished out, rinsed with water to remove most of the bacteria and impurities, and then treated with NaOH solution under high temperature conditions to remove the bacteria and residual culture medium in the bacterial cellulose hydrate. Thereafter, the membrane is washed and soaked with water multiple times until the pH is neutral, thereby obtaining a bacterial cellulose wet membrane.

2. The method for preparing bacterial cellulose by in situ modification of triglycerides according to claim 1, wherein: Step S1 includes: S11. Streak the bacterial suspension of Kosakonia oryzendophytica FY-07 onto LB plate containing 0.1% Congo red and incubate at 30°C for 24 h. S12, picking up the red single colony cultured in step S11 and streaking densely onto the first LB slant medium, and culturing at 30°C for 24 hours; S13, picking up the bacterial cellulose membrane cultured in step S12 and streaking densely on the second LB slant medium, and culturing at 30°C for 24 hours; S14. Rinse the second LB slant culture medium cultured in step S13 with 100 ml of pre-sterilized distilled water and recover it. Repeat the rinsing until the bacterial cellulose film containing a large number of bacteria on the second LB slant culture medium is detached. Mix the bacterial cellulose containing a large number of bacteria in the recovered distilled water to serve as the seed solution.

3. The method for preparing bacterial cellulose by in situ modification of triglycerides according to claim 2, wherein: In step S1, the ingredients of the LB plate culture medium, the first LB slant culture medium, and the second LB slant culture medium are: 5 g / L yeast powder, 10 g / L peptone, and 10 g / L NaCl; and the pH is 7.4-7.

6.

4. The method for preparing bacterial cellulose by in situ modification of triglycerides according to claim 1, wherein: In step S2, triamcinolone was added at a rate of 1 g / min.

5. The method for preparing bacterial cellulose by in situ modification of triglycerides according to claim 1, characterized in that: In step S2, the vegetable oil is sunflower oil, peanut oil or soybean oil.

6. The method for preparing bacterial cellulose by in situ modification of triglycerides according to claim 1, characterized in that: In step S2, the fat-soluble modifier is carotene, astaxanthin, silymarin or vitamin E.

7. The method for preparing bacterial cellulose by in situ modification of triglycerides according to claim 1, characterized in that: In step S3, the seed liquid prepared in step S1 is taken and inoculated into the HS-SG fermentation medium prepared in step S2 at a ratio of 1%, mixed evenly, and placed in an incubator for fermentation at 30°C for 24 hours.

8. The method for preparing bacterial cellulose by in situ modification of triglycerides according to claim 1, characterized in that: In step S4, the bacterial cellulose membrane is rinsed with water and then treated with a 0.5 M NaOH solution at 100° C. for 0.5 h. 9 . Bacterial cellulose in situ modified with tri-glucan gum obtained by the method for preparing bacterial cellulose in situ modified with tri-glucan gum according to any one of claims 1 to 8 .

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