Bacterial cellulose modified by Sanzan collagen site and preparation method thereof

Abstract

The invention provides bacterial cellulose modified in situ by utilizing Sanzan gum and a preparation method thereof, and belongs to the technical field of biopolymer materials and biological fermention.The preparation method comprises the following steps that yeast powder, peptone, Na2HPO4, KNO3 and glucose are mixed with water, then the Sanzan gum is slowly added and stirred until the Sanzan gum is completely dissolved, and a mixture is obtained; then adding vegetable oil and a fat-soluble modifier to prepare an HS-SG fermentation culture medium; inoculating the FY-07 seed solution into an HS-SG fermentation culture medium for culture and fermentation; fishing out a bacterial cellulose membrane generated by fermentation, washing with water, and removing thalli and a residual culture medium in the bacterial cellulose hydrate by using a NaOH solution under a high-temperature condition, so as to obtain a bacterial cellulose wet membrane. Therefore, the emulsification effect of the Sanzan gum can realize uniform dispersion of fat-soluble components, so that uniform in-situ modification of the bacterial cellulose is realized, and the in-situ modified bacterial cellulose with good oxidation resistance and good biocompatibility is further obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of biopolymer materials and biofermentation, and in particular relates to bacterial cellulose modified in situ using trisaccharide gum 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 application scenarios usually require it to have different performance focuses, so it is usually modified in situ to improve its original performance or add new functions. In situ modification refers to changing the morphology, mechanical properties, crystallinity, rehydration rate and other important functional properties of bacterial cellulose by changing the culture conditions or adding additional modifiers (such as additives or materials) to the culture medium during the fermentation process.

[0004] Although in-situ modification is low-cost and simple, the bacterial cellulose-producing strains currently used are strictly aerobic bacteria, which can only produce BC on the contact surface between the culture medium and the air, resulting in uneven modification of the final product. In addition, only water-soluble modifiers are suitable for in-situ modification, because water-insoluble modifiers will precipitate or aggregate in the culture medium, making in-situ modification difficult to achieve, resulting in a large number of functional fat-soluble natural products being unable to be used as modifiers.

[0005] Kosakonia oryzednophytica FY-07 (formerly known as Enterobacter sp. FY-07, FY-07) is a special bacterial cellulose-producing strain that has a unique deep fermentation mode because it can quickly produce large amounts of bacterial cellulose under aerobic, oxygen-limited and anaerobic conditions. Previous studies have shown that the products obtained by in situ modification of FY-07 are more uniform based on the deep fermentation mode. However, water-insoluble modifiers cannot be evenly distributed in the culture medium, so even using the deep fermentation mode, it is impossible to obtain a uniform modified product.

[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 by in-situ modification of tri-zan gum, comprising the following steps: S1, preparing Kosakonia oryzendophytica FY-07 seed solution; S2. Preparation of HS-SG fermentation medium: yeast powder, peptone, Na 2 HPO 4 , KNO 3 and glucose with water, without pH adjustment; then slowly add trisammonium gum with stirring, after adding, shake and mix until completely dissolved, then add the modifier oil solution formed by vegetable oil and fat-soluble modifier, mix evenly to form an emulsified system, and obtain HS-SG fermentation medium after high-pressure sterilization; 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, washed 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, and then washed and soaked with water for multiple times until the pH is neutral, thereby obtaining a bacterial cellulose wet membrane.

[0009] Further, step S1 includes: S11, streak the bacterial suspension of Kosakonia oryzendophytica FY-07 on LB plate medium containing 0.1% Congo red and culture at 30°C for 24 h; S12, picking up the red and larger single colonies cultured in step S11 and streaking densely on the first LB slant culture medium, and culturing at 30° C. for 24 h; S13, picking up the thin bacterial cellulose membrane 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 h; S14, rinse the second LB slant culture medium cultured in step S13 with 100 ml of pre-sterilized distilled water and recover it, rinse repeatedly until the bacterial cellulose film containing a large number of bacteria on the second LB slant culture medium is detached, and mix the bacterial cellulose containing a large number of bacteria in the recovered distilled water as a seed solution.

[0010] Furthermore, 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.

[0011] Further, in step S2, Sanzan gum is added at a speed of 1 g / min.

[0012] Further, in step S2, the HS-SG fermentation medium includes 7.5 g / L yeast powder, 10 g / L peptone, 10 g / L Na 2 HPO 4 、1.0 g / L KNO 3 , glucose 25 g / L, 0.1~0.3% (w / w) triglyceride, 8~12% (w / w) vegetable oil and 1~3g / L fat-soluble modifier.

[0013] Furthermore, in step S2, the vegetable oil is preferably sunflower oil, peanut oil or soybean oil.

[0014] Furthermore, in step S2, the fat-soluble modifier is preferably carotene, astaxanthin, silymarin, or vitamin E.

[0015] Furthermore, in step S3, the seed solution 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.

[0016] Furthermore, in step S4, the bacterial cellulose membrane is rinsed with water and then treated with a 0.5M NaOH solution at 100°C for 0.5h.

[0017] The present invention also provides a bacterial cellulose in situ modified by using tri-zan gum.

[0018] Due to the adoption of the above technical solution, the present invention has the following beneficial effects: Trisammonia gum is an extracellular polymer synthesized by Sphingomonas trisammonia. 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.

[0019] 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 usually used in the food, medicine, and cosmetics industries. The present invention uses them as excellent modifiers for the in-situ modification of bacterial cellulose.

[0020] The present invention uses trisammonium gum as an emulsifier and suspending agent to dissolve the fat-soluble active substance in vegetable oil, and uniformly emulsifies, suspends and disperses it in a culture medium. Then, the emulsified suspension system and FY-07 as a bacterial cellulose production strain are used to carry out in-situ modified bacterial cellulose fermentation production, thereby obtaining a bacterial cellulose product with different functional properties and good biocompatibility.

[0021] It can be seen that the emulsifying effect of Sanzan gum can achieve uniform dispersion of fat-soluble components and thus achieve uniform in-situ modification of bacterial cellulose, thereby obtaining in-situ modified bacterial cellulose with good antioxidant properties and good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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 methods of the present invention will become more obvious. The contents shown in the accompanying drawings are only used to explain the present invention and do not constitute any limitation to the present invention in any sense. In the accompanying drawings: Figure 1 The diagram shows the emulsification effect of the Sanzan gum of the present invention on different fat-soluble modifiers.

[0023] Figure 2 This is a diagram of bacterial cellulose products to which different fat-soluble modifiers are added in the present invention.

[0024] Figure 3 It is a bar graph of the yield of FY-07 bacterial cellulose at different addition amounts of Sanzan gum of the present invention.

[0025] Figure 4 This is a bar graph showing the product structure and properties of FY-07 bacterial cellulose at different addition amounts of Sanzan gum of the present invention.

[0026] 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.

[0027] Figure 6 It is a release curve diagram of the in situ modified bacterial cellulose of the present invention during in vitro digestion. DETAILED DESCRIPTION

[0028] The present invention discloses a method for preparing bacterial cellulose by in-situ modification of tri-glucan, comprising the following steps: S1, preparing Kosakonia oryzendophytica FY-07 seed solution; S11, streak the bacterial suspension of Kosakonia oryzendophytica FY-07 on LB plate medium containing 0.1% Congo red and culture at 30°C for 24 h; S12, picking up the red and larger single colonies cultured in step S11 and streaking densely on the first LB slant culture medium, and culturing at 30° C. for 24 h; S13, picking up the thin bacterial cellulose membrane 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 h; S14, rinse the second LB slant culture medium cultured in step S13 with 100 ml of pre-sterilized distilled water and recover it, rinse repeatedly until the bacterial cellulose film containing a large number of bacteria on the second LB slant culture medium is detached, and mix the bacterial cellulose containing a large number of bacteria in the recovered distilled water as a seed solution.

[0029] S2. Preparation of HS-SG fermentation medium: yeast powder, peptone, Na 2 HPO 4 , KNO 3 and glucose with water, and the pH does not need to be adjusted; then, trisammonium gum is added at a rate of 1 g / min with stirring, and after the addition is completed, it is placed under 30°C for oscillation and mixing until it is completely dissolved, and then a modifier oil solution formed by vegetable oil and a fat-soluble modifier is added, and mixed evenly to form an emulsified system, and the HS-SG fermentation medium is obtained after high-pressure sterilization; The HS-SG fermentation medium includes 7.5 g / L yeast powder, 10 g / L peptone, 10 g / L Na 2 HPO 4 、1.0 g / L KNO 3 , glucose 25 g / L, 0.1~0.3% (w / w) triglyceride, 8~12% (w / w) vegetable oil and 1~3g / L fat-soluble modifier.

[0030] S3. Take the seed solution 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.

[0031] 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, and then washed and soaked with water for multiple times until the pH is neutral, thereby obtaining a bacterial cellulose wet membrane.

[0032] S5. Drying the bacterial cellulose wet film to obtain a dry film, and calculating the yield.

[0033] The Kosakonia oryzendophytica FY-07 was isolated from oilfield produced fluid and deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.6103 and a deposit date of May 11, 2012.

[0034] 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.

[0035] Among them, the Sanzan gum used as an emulsifier comes from Meihua Group.

[0036] Wherein, the vegetable oil is sunflower oil, peanut oil, soybean oil and the like.

[0037] Wherein, the fat-soluble modifier is carotene, astaxanthin, silymarin, vitamin E and the like.

[0038] The present invention also provides a bacterial cellulose in situ modified by using tri-zan gum.

[0039] Emulsification effect of Sanzan gum on fat-soluble modifiers: The method of preparing HS-SG fermentation medium in step S2 is used to compare the emulsification effect of trisammonium gum on different fat-soluble modifiers, such as Figure 1 As shown, the results indicate that 0.2% (w / w) tri-zan gum has a good emulsifying effect on carotene, astaxanthin and silymarin dissolved in sunflower oil (from left to right in the figure, the final concentration is 2 g / L), and can remain stable after high temperature and high pressure sterilization.

[0040] Effects of different amounts of trisammonium gum added to HS-SG fermentation medium on the yield of FY-07 bacterial cellulose: Experiment 1: Step S1 is the same as in the above embodiment.

[0041] S2. Prepare three groups of HS-SG fermentation medium: yeast powder, peptone, Na 2 HPO 4 , KNO 3, glucose was mixed with water, and the pH did not need to be adjusted; then 0.1% (w / w), 0.2% (w / w), and 0.3% (w / w) of tri-zan gum (i.e., 1, 2, and 3 g / L of tri-zan gum) were added to the three groups respectively, at a rate of 1 g / min and accompanied by stirring. After the addition was completed, the mixture was placed under 30°C for oscillation and mixing until it was completely dissolved. Subsequently, carotene was added to each group to a final concentration of 0.2% (w / w) and dissolved in sunflower seed oil to form 3 ml of a modifier oil solution, which was evenly mixed to form an emulsified system. After high-pressure sterilization, 30 ml of HS-SG fermentation medium was obtained for each group; S3, taking the seed solution prepared in step S1, inoculating it into the three groups of HS-SG fermentation medium prepared in step S2 at a ratio of 1%, mixing them evenly, placing them in an incubator, and culturing and fermenting them at 30° C. for 24 hours; S4, taking out the bacterial cellulose membrane produced by the fermentation in step S3, washing 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, and then washing and soaking with water for multiple times until the pH is neutral, thereby obtaining three groups of bacterial cellulose wet membranes; S5. Dry the three groups of bacterial cellulose wet films to obtain dry films, and calculate the yield.

[0042] Experiment 2: Replace the carotene in Experiment 1 with astaxanthin, and keep the rest the same.

[0043] In Experiment 3, the carotene in Experiment 1 was replaced with silymarin, and the rest remained the same.

[0044] like Figure 2 As shown, through Experiments 1, 2 and 3, it can be seen that the HS-SG fermentation medium with the addition of different lipophilic modifiers can produce in situ modified bacterial cellulose films with good morphology.

[0045] like Figure 3 As shown, the effect of different amounts of triazole gum added on the bacterial cellulose yield was further measured. It can be seen that with the increase of triazole gum 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, but when the amount of triazole gum added is higher than 0.3% (w / w), the yield no longer increases.

[0046] Effects of different amounts of trisammonium gum added to HS-SG fermentation medium on the texture properties of FY-07 bacterial cellulose: Nine groups of bacterial cellulose wet films were obtained by the methods of Experiments 1, 2 and 3, and their texture characteristics were measured by TA. XTC texture analyzer (Shanghai Baosheng Technology Co., Ltd.) using a cylindrical probe (p / 0.5). The 2×2cm (height×width) cylindrical sample was compressed to 50%, with a speed of 2.00 mm / s before the test; a test speed of 1.00 mm / s; a speed of 1.00 mm / s after the test; a strain of 75%; and a trigger-type automatic-5 g. The obtained full texture analysis (TPA) curve can characterize the hardness, gel strength, and adhesiveness of each group of samples.

[0047] like Figure 4 As shown in the results, it was found that adding a final concentration of more than 0.2% (w / w) of tri-zan gum can prepare an in-situ modified bacterial cellulose film with high hardness, adhesion and gel strength, but adding 0.3% (w / w) of tri-zan gum in the experiment of adding astaxanthin as a modifier will cause a certain degree of reduction in texture performance. In addition, in the experiment of adding silymarin as a modifier, adding 0.1% (w / w) of tri-zan gum cannot be fermented normally to produce in-situ modified bacterial cellulose film, which may be due to its certain antibacterial effect. Under low concentrations of tri-zan gum, a good emulsification effect cannot be obtained, resulting in its inability to produce film normally.

[0048] Detection of Antioxidant Properties of Bacterial Cellulose Modified by Sanzan Gum Add 0.2% (w / w) trisaccharide, and obtain three groups of wet bacterial cellulose films by the methods of Experiment 1, Experiment 2 and Experiment 3, and then use the ABTS antioxidant kit to test the antioxidant properties of the three groups of samples. The specific operation steps are as follows: Mix the ABTS solution and the oxidant solution, react in the dark for a certain period of time (usually 12~16h), and then dilute with anhydrous ethanol to make a working solution. Take 280μl of the working solution, add 7μl of the sample to be tested (take 1g of in situ modified bacterial cellulose membrane in each group, extract with 1ml of anhydrous ethanol respectively), mix well, place at room temperature for 6min, and then use an enzyme reader to measure the absorbance of each tube at 734nm. The total antioxidant capacity of the sample is calculated according to the standard curve, and the unit is usually expressed in Trolox equivalents. The ABTS free radical scavenging ability in the sample can be quantitatively analyzed by comparing the changes in absorbance.

[0049] like Figure 5 As shown, it can be found that the free radical scavenging rate of the bacterial cellulose samples modified in situ by adding carotene and astaxanthin can reach more than 10%, while the free radical scavenging rate of the bacterial cellulose samples modified in situ by adding silymarin can reach more than 20%, both of which have certain antioxidant properties.

[0050] Detection of sustained release effect of Sanzan collagen in situ modified bacterial cellulose during in vitro digestion: 0.2% (w / w) triazine gum was added to obtain a BC wet film by the method of Experiment 1. During in vitro digestion, the release kinetics of β-carotene encapsulated in the in situ modified BC were evaluated using simulated gastric fluid (SGF) and simulated intestinal fluid (SIF).

[0051] 10 g of in-situ modified bacterial cellulose was cut into small pieces and transferred to several transparent flasks, and then 30 ml of SGF and SIF were added respectively and mixed thoroughly by stirring. The samples were incubated at 30°C for 24 h at a shaking rate of 200 rpm in a shaker to complete the digestion process. At a specific digestion period, one of the digestion solutions was immediately taken out for further analysis (β-carotene content determination). The β-carotene content in the two simulated digestion solutions was extracted and determined. 21 ml of simulated digestion solution was added with 1 ml of n-hexane, and the β-carotene was completely extracted by vigorous shaking for 2 min. The organic layer was removed after standing for 5 min. The organic layer was centrifuged at 12000 rpm for 2 min to separate the possible mixed particles or aqueous phase. The β-carotene content in each bottle of organic layer was determined by absorbance at a wavelength of 450 nm.

[0052] like Figure 6 As shown in the figure, the release of β-carotene in SIF increased rapidly in the early stage of digestion (within 7h), and then the release gradually decreased, reflecting the gradual transfer of β-carotene from the in situ modified bacterial cellulose to the aqueous phase during digestion. The lipid and polypeptide components contained in Sanzan gum can be digested by the enzymes in SIF, resulting in the gradual release of β-carotene. In contrast, in SGF, Sanzan gum maintains a good gel state, so the release of β-carotene remains at an extremely low level. In summary, Sanzan gum in situ modified bacterial cellulose can be used for pH-responsive controlled release of specific drugs or active ingredients.

[0053] The embodiments of the present invention are described in detail above, but the contents are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation 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 tri-glucan, characterized in that: The following steps are involved: S1, preparing Kosakonia oryzendophytica FY-07 seed solution; S2. Prepare HS-SG fermentation medium: mix yeast powder, peptone, Na2HPO4, KNO3 and glucose with water; then slowly add trisaccharide with stirring, shake and mix until completely dissolved after the addition, then add the modifier oil solution formed by vegetable oil and fat-soluble modifier, mix evenly to form an emulsified system, and obtain HS-SG fermentation medium after high-pressure sterilization; 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, washed 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, and then washed and soaked with water for 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 tri-gluconate according to claim 1, characterized in that: Step S1 includes: S11, streak the bacterial suspension of Kosakonia oryzendophytica FY-07 on LB plate medium containing 0.1% Congo red and culture at 30°C for 24 h; S12, picking up the red single colony cultured in step S11 and streaking densely on the first LB slant medium, and culturing at 30° C. for 24 h; S13, picking up the bacterial cellulose membrane cultured in step S12 and streaking densely on the second LB slant culture medium, and culturing at 30° C. for 24 h; S14, rinse the second LB slant culture medium cultured in step S13 with 100 ml of pre-sterilized distilled water and recover it, rinse repeatedly until the bacterial cellulose film containing a large number of bacteria on the second LB slant culture medium is detached, and mix the bacterial cellulose containing a large number of bacteria in the recovered distilled water as a seed solution.

3. The method for preparing bacterial cellulose by in-situ modification of tri-gluconate according to claim 2, characterized in that: 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 tri-gluconate according to claim 1, characterized in that: In step S2, trimethoprim-gum was added at a rate of 1 g / min.

5. The method for preparing bacterial cellulose by in-situ modification of tri-gluconate according to claim 1, characterized in that: 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.

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

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

8. The method for preparing bacterial cellulose by in-situ modification of tri-gluconate according to claim 1, characterized in that: In step S3, the seed solution 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.

9. The method for preparing bacterial cellulose by in-situ modification of tri-gluconate 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.

10. Bacterial cellulose in situ modified with tri-zan gum obtained by the method for preparing bacterial cellulose in situ modified with tri-zan gum according to any one of claims 1 to 9.

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