Method for improving yield of bacterial cellulose by utilizing low-dose ultraviolet radiation

Abstract

The invention provides a method for increasing the yield of bacterial cellulose by utilizing low-dose ultraviolet radiation, and belongs to the field of microbial fermentation. The method comprises the following steps: inoculating a fermentation culture medium with a Taonella.mepensis seed solution, carrying out fermentation treatment, and carrying out ultraviolet radiation under set conditions in the fermentation process, so as to obtain a fermentation solution containing a bacterial cellulose membrane; and taking out the bacterial cellulose membrane in the fermentation liquor, and purifying the bacterial cellulose membrane to obtain the bacterial cellulose. On the basis of an HS culture medium formula, the ultraviolet irradiation time is regulated and controlled at the same time, so that the activity of cellulose synthesis related enzymes, such as the activity of UDP-glucose pyrophosphorylase and glucose phosphate mutase, is improved, the activity of glucose decomposition pathway related enzymes, such as pyruvate kinase and phosphofructokinase, is inhibited, and the activity of cellulose synthesis related enzymes is improved. The yield of the bacterial cellulose is further improved.

Description

Technical Field

[0001] The present application relates to the field of microbial fermentation technology, and in particular to a method for increasing bacterial cellulose production by utilizing low-dose ultraviolet irradiation. Background Art

[0002] Bacterial cellulose (BC) is secreted by some Gram-negative bacteria and is produced as white gel-like BC particles at the air-liquid interface. It has attracted great interest from materials scientists and polymer chemists due to its unique properties, such as high purity (free of pectin, lignin and hemicellulose) and crystallinity (>80%).

[0003] Due to the unique properties of bacterial cellulose, BC has been widely explored as an important biomaterial for applications in many fields, such as food, cosmetics, biomedicine, textiles, and high-end acoustic diaphragms. Although BC producers use various substrates to produce BC, their conversion efficiency is low, resulting in low yields and high production costs, which limits their widespread application. In order to improve the substrate conversion efficiency of BC producers and minimize the production cost of BC, hydrolysates of agro-industrial wastes, such as wheat straw, sunflower seed meal, and colored rice, which are rich sources of different sugars, have been converted into BC. During the de novo synthesis of BC, some by-products are also produced, such as α-glucuronic acid-based organic acids, which can reduce the overall yield of BC. Therefore, how to increase the yield of bacterial cellulose is a technical problem that needs to be solved urgently. Summary of the invention

[0004] The present application provides a method for increasing the yield of bacterial cellulose by using low-dose ultraviolet irradiation to solve the following technical problem: how to increase the yield of bacterial cellulose.

[0005] The present application provides a method for increasing bacterial cellulose production by low-dose ultraviolet irradiation, the method comprising:

[0006] inoculating Taonella mepensis seed liquid into a fermentation medium for fermentation treatment, and performing ultraviolet irradiation under set conditions during the fermentation process to obtain a fermentation liquid containing a bacterial cellulose membrane; and

[0007] The bacterial cellulose membrane in the fermentation liquid is taken out, and then the bacterial cellulose membrane is purified to obtain bacterial cellulose.

[0008] Optionally, the setting conditions include at least one of the following conditions: ultraviolet irradiation power is 3W to 5W, ultraviolet irradiation wavelength is 254nm, ultraviolet irradiation distance is 45cm to 55cm, and ultraviolet irradiation time is 15s / h to 45s / h.

[0009] Optionally, the chemical components of the fermentation medium include, in terms of mass concentration: 5g / L to 6g / L yeast extract, 4.5g / L to 5.5g / L peptone, 15g / L to 20g / L glucose, 1g / L to 1.15g / L citric acid monohydrate, 2.5g / L to 2.7g / L disodium hydrogen phosphate and 15g / L to 18g / L agar.

[0010] Optionally, the pH value of the fermentation medium is 6.5-7.0.

[0011] Optionally, the viable count of the Taonella mepensis seed solution is 10 6 CFU / mL~10 7 CFU / mL.

[0012] Optionally, the inoculation amount is 5% to 15%.

[0013] Optionally, the fermentation temperature is 28°C to 30°C, and the fermentation time is 7 days.

[0014] Optionally, the purifying treatment of the bacterial cellulose membrane comprises:

[0015] The bacterial cellulose membrane is sequentially subjected to a first heating and boiling, water washing, a second heating and boiling, and water washing until neutrality to obtain bacterial cellulose; wherein the first heating and boiling uses a sodium carbonate solution.

[0016] Optionally, the yield of bacterial cellulose is ≥2.78 g / L.

[0017] Optionally, the bacterial cellulose satisfies at least one of the following properties: a crystallinity index of 75% to 80%, a tensile strength of 10.5 MPa to 12 MPa, a Young's modulus of 6 GPa to 7 GPa, an elongation at break of 2.6% to 3.2%, a DP of 1920 to 2020, and a specific surface area of ​​27 m 2 / g~34m 2 / g.

[0018] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0019] The present application provides a method for increasing the yield of bacterial cellulose by low-dose ultraviolet irradiation, the method comprising: inoculating Taonella mepensis seed liquid into a fermentation medium for fermentation treatment, and performing ultraviolet irradiation under set conditions during the fermentation process to obtain a fermentation liquid containing a bacterial cellulose film; taking out the bacterial cellulose film in the fermentation liquid, and then purifying the bacterial cellulose film to obtain bacterial cellulose. Based on the HS medium formula and adjusting the ultraviolet irradiation time at the same time, the activity of enzymes related to cellulose synthesis, such as UDP-glucose pyrophosphorylase (UGP) and phosphoglucomutase (PGM) is increased, and the activity of enzymes related to glucose decomposition pathway, such as pyruvate kinase (PK) and phosphofructokinase (PFK) is inhibited, thereby increasing the yield of bacterial cellulose. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0021] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0022] Figure 1 A schematic diagram of a process for increasing bacterial cellulose production by low-dose ultraviolet irradiation provided in an embodiment of the present application;

[0023] Figure 2 A trend chart showing the effect of ultraviolet irradiation on bacterial cellulose production provided in Examples 1 to 3 and Comparative Examples 1 to 2 of the present application;

[0024] Figure 3 A graph showing a trend of glucose concentration changes in Bacillus xylosus provided in Example 2 and Comparative Example 1 of the present application;

[0025] Figure 4 A graph showing the variation trend of bacterial cellulose production of Bacillus xylosus provided in Example 2 and Comparative Example 1 of the present application;

[0026] Figure 5 This is a comparison chart of infrared spectra of bacterial cellulose provided in Example 2 and Comparative Example 1 of the present application;

[0027] Figure 6 A comparison chart of the X-ray diffraction curves of bacterial cellulose provided in Example 2 and Comparative Example 1 of the present application;

[0028] Figure 7 This is a TGA thermal analysis comparison diagram of bacterial cellulose provided in Example 2 and Comparative Example 1 of the present application;

[0029] Figure 8 This is a comparison chart of DTG thermal analysis of bacterial cellulose provided in Example 2 and Comparative Example 1 of the present application;

[0030] Fig. 9 This is a graph showing the effect of the culture methods provided in Example 2 and Comparative Example 1 of the present application on the activity of phosphofructokinase (PFK) involved in BC anabolism and glucose catabolism;

[0031] Fig.10 This is a graph showing the effect of the culture methods provided in Example 2 and Comparative Example 1 of the present application on the activity of pyruvate kinase (PK) involved in BC anabolism and glucose catabolism;

[0032] Fig.11 This is a graph showing the effect of the culture methods provided in Example 2 and Comparative Example 1 of the present application on the activity of phosphoglucomutase (PGM) involved in BC anabolism and glucose catabolism;

[0033] Fig.12 This is a graph showing the effect of the culture methods provided in Example 2 and Comparative Example 1 of the present application on the activity of UDP-glucose pyrophosphorylase (UGP) involved in BC anabolism and glucose catabolism. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0035] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0036] In addition, in the description of the specification of the present application, the terms "including", "comprising", etc. mean "including but not limited to". In this article, relational terms such as "first" and "second", etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. In this article, "and / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A and B can be singular or plural. In this article, "at least one" means one or more, and "plurality" means two or more. "At least one", "the following at least one item (items)" or similar expressions refer to any combination of these items, including any combination of single items (items) or plural items (items). For example, "at least one of a, b, or c", or "at least one of a, b, and c", can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple. "Parts" such as parts by weight and parts by mass indicate the proportional relationship between the components. In the proportional relationship involved in this article, the parameters that need to be described by proportion should be understood as the first term of the proportional formula in the order of description, and the proportional numbers should be understood as the second term of the proportional formula. For example, the mass ratio of substance A, substance B, and substance C is 1:2:3, then substance A, substance B, and substance C should correspond one-to-one with the proportional numbers in the proportional formula in the order of description, that is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

[0037] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0038] Figure 1A schematic flow chart of a method for increasing bacterial cellulose production by low-dose ultraviolet irradiation is provided in an embodiment of the present application.

[0039] See also Figure 1 The present application provides a method for increasing the yield of bacterial cellulose by low-dose ultraviolet irradiation, the method comprising:

[0040] S1, inoculating Taonella mepensis seed liquid into a fermentation medium for fermentation treatment, and performing ultraviolet irradiation under set conditions during the fermentation process to obtain a fermentation liquid containing a bacterial cellulose membrane;

[0041] Ultraviolet (UV) is an electromagnetic wave with a wavelength between 10nm and 400nm, which is shorter than visible light but longer than X-rays. Sunlight contains some ultraviolet rays, and electric arcs, mercury lamps, and black lights also emit ultraviolet rays. Ultraviolet rays are not ionizing radiation, but can trigger chemical reactions and make some substances fluoresce. In the field of synthetic biology, ultraviolet radiation is used to activate certain biosynthetic pathways. For example, UV-B irradiation can induce plants to produce more flavonoids or polyphenols, which have a "sunscreen" effect. In addition, ultraviolet radiation, especially UV-B, has an important effect on the biosynthesis of secondary metabolites in plants. Studies have shown that UV-B irradiation can induce the expression of genes related to anthocyanin synthesis in plants, such as the UVR8 gene, thereby promoting the synthesis of anthocyanins. In summary, ultraviolet radiation plays an important role in promoting biosynthesis, especially in the synthesis of secondary metabolites in plants. These research results provide a scientific basis and potential application prospects for regulating the synthesis of bacterial cellulose by ultraviolet radiation.

[0042] Taonella mepensis, also known as a type of xylose acetobacter, belongs to the genus Taonella. The colony morphology of Taonella mepensis is small, round, smooth and light yellow. The colony diameter is usually between 1 and 3 mm, with round and neat edges, moist surface, brittle texture, and difficult to pick. The cell size of Taonella mepensis is about (1.5-3.0) × (0.8-1.0) μm. Taonella mepensis has an efficient metabolic pathway and can make full use of external carbon sources (such as glucose, xylose, etc.) for fermentation. During the fermentation process, the strain converts the carbon source into a precursor of bacterial cellulose through a series of enzymatic reactions, and then synthesizes bacterial cellulose. Therefore, Taonella mepensis can produce a large amount of bacterial cellulose.

[0043] Adjusting the irradiation time of ultraviolet light during the bacterial cellulose production process is an effective method to increase the yield of bacterial cellulose. At present, there is no report on the use of regulating the ultraviolet irradiation time to promote the synthesis of bacterial cellulose.

[0044] Therefore, the present application creatively uses the HS culture medium formula as the basis and regulates the ultraviolet irradiation time at the same time, thereby increasing the activity of enzymes related to cellulose synthesis, such as UDP-glucose pyrophosphorylase (UGP) and phosphoglucomutase (PGM), and inhibiting the activity of enzymes related to the glucose decomposition pathway, such as pyruvate kinase (PK) and phosphofructokinase (PFK), thereby increasing the bacterial cellulose yield.

[0045] In some embodiments, the set conditions include at least one of the following conditions: ultraviolet irradiation power is 3W to 5W, ultraviolet irradiation wavelength is 254nm, ultraviolet irradiation distance is 45cm to 55cm, and ultraviolet irradiation time is 15s / h to 45s / h.

[0046] The ultraviolet irradiation time is 15s / h to 45s / h, which means that the ultraviolet lamp will irradiate for 15s to 45s per hour. By adjusting the ultraviolet irradiation time, the activity of enzymes related to cellulose synthesis is increased, and the activity of enzymes related to the glucose decomposition pathway is inhibited, thereby optimizing the synthesis process of bacterial cellulose. The ultraviolet irradiation power is limited to 3W to 5W, the ultraviolet irradiation wavelength is 254nm, the ultraviolet irradiation distance is 45cm to 55cm, and the ultraviolet irradiation time is 15s / h to 45s / h, so as to induce the required enzyme activity changes while avoiding excessive damage, or even cell death or growth inhibition. Exemplarily, the UV irradiation power can be 3W, 3.2W, 3.5W, 3.8W, 4W, 4.5W, 5W, etc., the UV irradiation distance can be 45cm, 46cm, 47cm, 48cm, 49cm, 50cm, 52cm, 54cm, 55cm, etc., and the UV irradiation time can be 15s / h, 20s / h, 25s / h, 30s / h, 35s / h, 40s / h, 45s / h, etc.

[0047] In some embodiments, the chemical components of the fermentation medium include, by mass concentration, 5 g / L to 6 g / L yeast extract, 4.5 g / L to 5.5 g / L peptone, 15 g / L to 20 g / L glucose, 1 g / L to 1.15 g / L citric acid monohydrate, 2.5 g / L to 2.7 g / L disodium hydrogen phosphate, and 15 g / L to 18 g / L agar.

[0048] In some embodiments, the pH value of the fermentation medium is 6.5-7.0.

[0049] The chemical components of the fermentation medium provide the carbon source, nitrogen source, inorganic salts and other nutrients required for bacterial growth, and are the basis for the synthesis of bacterial cellulose. Limiting the pH value of the fermentation medium to 6.5-7.0 helps maintain the normal metabolic activities of bacteria and promotes the synthesis of bacterial cellulose. Exemplarily, the mass concentration of yeast extract can be 5g / L, 5.2g / L, 5.4g / L, 5.6g / L, 5.8g / L, 6g / L, etc., the mass concentration of peptone can be 4.5g / L, 4.7g / L, 4.9g / L, 5.0g / L, 5.2g / L, 5.4g / L, 5.5g / L, etc., and the mass concentration of glucose can be 15g / L, 16g / L, 17g / L, 18g / The mass concentration of the fermentation medium can be 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, etc. The mass concentration of the fermentation medium can be 1 g / L, 1.05 g / L, 1.10 g / L, 1.12 g / L, 1.15 g / L, etc. The mass concentration of the disodium hydrogen phosphate can be 2.5 g / L, 2.55 g / L, 2.6 g / L, 2.65 g / L, 2.7 g / L, etc. The mass concentration of the agar can be 15 g / L, 16 g / L, 17 g / L, 18 g / L, etc. The pH value of the fermentation medium can be 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, etc.

[0050] In some embodiments, the viable count of the Taonella mepensis seed solution is 10 6 CFU / mL~10 7 CFU / mL.

[0051] In some embodiments, the inoculation amount is 5% to 15%.

[0052] The viable count of the seed solution of Bacillus xylosus refers to the number of viable bacteria contained in a unit volume of the seed solution. The inoculation amount refers to the ratio of the seed solution to the fermentation medium. The viable count of the seed solution of Bacillus xylosus is limited to 10 6 CFU / mL~10 7 CFU / mL and the inoculation amount are 5% to 15%, so that the bacteria can multiply rapidly in the fermentation broth and reach a high cell density, which is conducive to the synthesis of bacterial cellulose. 6 CFU / mL, 2×10 6 CFU / mL, 4×10 6 CFU / mL, 6×10 6 CFU / mL, 8×10 6 CFU / mL, 107 CFU / mL and the inoculum size of the inoculation are 5%, 7%, 9%, 10%, 12%, 14%, 15%, etc.

[0053] In some embodiments, the fermentation temperature is 28° C. to 30° C., and the fermentation time is 7 days.

[0054] The fermentation temperature is limited to 28°C to 30°C, and the fermentation time is 7 days. Xylosaccharomyces cerevisiae can maintain a good growth state, and the cell metabolism is active, which is conducive to the synthesis of bacterial cellulose. By optimizing the fermentation time to 7 days, the quality of bacterial cellulose can be guaranteed while maximizing its yield.

[0055] S2, taking out the bacterial cellulose membrane in the fermentation liquid, and then purifying the bacterial cellulose membrane to obtain bacterial cellulose.

[0056] In some embodiments, the purifying the bacterial cellulose membrane comprises:

[0057] The bacterial cellulose membrane is sequentially subjected to a first heating and boiling, water washing, a second heating and boiling, and water washing until neutrality to obtain bacterial cellulose; wherein the first heating and boiling uses a sodium carbonate solution.

[0058] In some embodiments, the yield of bacterial cellulose is ≥2.78 g / L.

[0059] In some embodiments, the bacterial cellulose satisfies at least one of the following properties: a crystallinity index of 75% to 80%, a tensile strength of 10.5 MPa to 12 MPa, a Young's modulus of 6 GPa to 7 GPa, an elongation at break of 2.6% to 3.2%, a DP of 1920 to 2020, and a specific surface area of ​​27 m 2 / g~34m 2 / g.

[0060] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to industry standards. If there is no corresponding industry standard, then the conditions recommended by the manufacturer are followed.

[0061] The strain used in this application is Taonella mepensis, with the biological deposit number being CICC10529.

[0062] Example 1

[0063] This embodiment provides a method for increasing bacterial cellulose yield by low-dose ultraviolet irradiation, which may specifically include the following steps:

[0064] Step 1: Activation of Bacillus xylosus

[0065] Pour 0.5mL of sterile water into the freeze-dried tube and gently blow to fully dissolve into a suspension. Pipette the bacterial suspension and inject 200μL into the surface of the culture medium. Culture at 30℃. After two days, new colonies will grow on the solid culture medium of Xylosaccharomyces cerevisiae. The solid culture medium of Xylosaccharomyces cerevisiae: 5g yeast extract, 5g peptone, 20g glucose, 1.15g citric acid monohydrate, 2.7g Na2HPO4, 18.0g agar, 1.0L distilled water.

[0066] Step 2: Expansion of Bacillus xylosus

[0067] A liquid culture medium of Bacillus xylosus was prepared, wherein the formula included 5 g / L yeast extract, 5 g / L peptone, 20 g / L glucose, 1.15 g / L citric acid monohydrate, and 2.7 g / L Na2HPO4, and the pH value was adjusted to 7.0. Activated Bacillus xylosus was inoculated into the culture medium, and the culture was shaken at 30° C. for 24 hours to obtain a seed liquid of Bacillus xylosus. The concentration of Bacillus xylosus in the seed liquid of Bacillus xylosus was 10 6 CFU / mL~10 7 CFU / mL.

[0068] Step 3: Preparation of bacterial cellulose fermentation medium

[0069] Take 1 L of distilled water, 5 g of yeast extract, 5 g of peptone, 20 g of glucose, 1.15 g of citric acid monohydrate and 2.7 g of Na2HPO4, adjust the pH value to 7.0, and obtain a bacterial fiber fermentation medium.

[0070] Step 4: Preparation of bacterial cellulose fermentation broth

[0071] Take the seed solution of Bacillus xylosus (concentration 10 6 CFU / mL~10 7 CFU / mL), inoculate the xylose calcite seed liquid into the bacterial cellulose fermentation medium at an inoculation rate of 10 mL per 90 mL, and irradiate with ultraviolet light Xin Yat T5-4W type, wavelength 254nm, 4W, distance 50cm, irradiation for 15 seconds / hour under static fermentation at 28°C, and terminate the fermentation after 7 days to obtain bacterial cellulose fermentation liquid.

[0072] Step 5: Preparation of bacterial cellulose

[0073] Take the bacterial cellulose film in the bacterial cellulose fermentation broth, put the bacterial cellulose film into 200mL, 2% sodium carbonate solution at 100℃ water bath for 30min, rinse with deionized water several times, put the bacterial cellulose film into 200mL deionized water at 100℃ water bath for 30min, and then rinse with deionized water several times until neutral to obtain a pure bacterial cellulose film. The yield is calculated according to the dry weight of bacterial cellulose (BC) / total volume of culture medium, and its yield is 2.97±0.04g / L.

[0074] Example 2

[0075] This embodiment provides a method for increasing bacterial cellulose yield by low-dose ultraviolet irradiation, which may specifically include the following steps:

[0076] Step 1: Activation of Bacillus xylosus

[0077] Same as Example 1.

[0078] Step 2: Expansion of Bacillus xylosus

[0079] Same as Example 1.

[0080] Step 3: Preparation of bacterial cellulose fermentation medium

[0081] Take 1 L of distilled water, add 5 g of yeast extract, 5 g of peptone, 20 g of glucose, 1.15 g of citric acid monohydrate and 2.7 g of Na2HPO4, adjust the pH value to 7.0, and obtain a bacterial fiber fermentation medium.

[0082] Step 4: Preparation of bacterial cellulose fermentation broth

[0083] Take the seed solution of Bacillus xylosus (concentration 10 6 CFU / mL~10 7 The seed solution of Bacillus xylosus was inoculated into the bacterial cellulose fermentation medium at an inoculation rate of 10 mL per 90 mL. Under static fermentation at 28°C, ultraviolet rays of the New Yat T5-4W type were irradiated with a wavelength of 254 nm, 4 W, a distance of 50 cm, and 30 seconds / hour. The fermentation was terminated after 7 days to obtain the bacterial cellulose fermentation liquid.

[0084] Step 5: Preparation of bacterial cellulose:

[0085] Take the bacterial cellulose film in the bacterial cellulose fermentation broth, put the bacterial cellulose film into 200mL, 2% sodium carbonate solution at 100℃ water bath for 30min, rinse with deionized water several times, put the bacterial cellulose film into 200mL deionized water at 100℃ water bath for 30min, and then rinse with deionized water several times to obtain a pure bacterial cellulose film. The yield is calculated according to the dry weight of bacterial cellulose (BC) / total volume of culture medium, and its yield is 3.41±0.06g / L.

[0086] Example 3

[0087] This embodiment provides a method for increasing bacterial cellulose yield by low-dose ultraviolet irradiation, which may specifically include the following steps:

[0088] Step 1: Activation of Bacillus xylosus

[0089] Same as Example 1.

[0090] Step 2: Expansion of Bacillus xylosus

[0091] Same as Example 1.

[0092] Step 3: Preparation of bacterial cellulose fermentation medium

[0093] Take 1 L of distilled water, add 5 g of yeast extract, 5 g of peptone, 20 g of glucose, 1.15 g of citric acid monohydrate and 2.7 g of Na2HPO4, adjust the pH value to 7.0, and obtain a bacterial fiber fermentation medium.

[0094] Step 4: Preparation of bacterial cellulose fermentation broth

[0095] Take the seed solution of Bacillus xylosus (concentration 10 6 CFU / mL~10 7 CFU / mL), and the seed liquid of Bacillus xylosus was inoculated into the bacterial cellulose fermentation medium at an inoculation rate of 10 mL per 90 mL. Under static fermentation at 28°C, ultraviolet rays of Xinyate T5-4W type, wavelength of 254 nm, 4 W, distance of 50 cm, irradiation for 45 seconds / hour were applied. The fermentation was terminated after 7 days to obtain bacterial cellulose fermentation liquid.

[0096] Step 5: Preparation of bacterial cellulose

[0097] Take the bacterial cellulose film in the bacterial cellulose fermentation broth, put the bacterial cellulose film into 200mL, 2% sodium carbonate solution at 100℃ water bath for 30min, rinse with deionized water several times, put the bacterial cellulose film into 200mL deionized water at 100℃ water bath for 30min, and then rinse with deionized water several times to obtain a pure bacterial cellulose film. The yield is calculated according to the dry weight of bacterial cellulose (BC) / total volume of culture medium, and the yield is 2.78±0.05g / L.

[0098] Comparative Example 1

[0099] This comparative example provides a method for increasing the yield of bacterial cellulose by low-dose ultraviolet irradiation, which may specifically include the following steps:

[0100] Step 1: Activation of Bacillus xylosus

[0101] Same as Example 1.

[0102] Step 2: Expansion of Bacillus xylosus

[0103] Same as Example 1.

[0104] Step 3: Preparation of bacterial cellulose fermentation medium

[0105] Take 1L of distilled water, add 5g of yeast extract, 5g of peptone, 20g of glucose, 1.15g of citric acid monohydrate and 2.7g of Na2HPO4, adjust the pH value to 7.0, and obtain bacterial fiber fermentation medium.

[0106] Step 4: Preparation of bacterial cellulose fermentation broth:

[0107] Take the seed solution of Bacillus xylosus (concentration 10 6 CFU / mL~10 7 CFU / mL), and the seed liquid of Bacillus xylosus was inoculated into the bacterial cellulose fermentation medium at an inoculation rate of 10 mL per 90 mL. The fermentation was terminated after static fermentation at 28°C without ultraviolet radiation for 7 days to obtain the bacterial cellulose fermentation liquid.

[0108] Step 5: Preparation of bacterial cellulose

[0109] Take the bacterial cellulose film in the bacterial cellulose fermentation broth, put the bacterial cellulose film into 200 mL, 2% sodium carbonate solution in a water bath at 100°C for 30 minutes, rinse it with deionized water several times, put the bacterial cellulose (film into 200 mL deionized water in a water bath at 100°C for 30 minutes, and then rinse it with deionized water several times to obtain a pure bacterial cellulose film. The yield is calculated according to the dry weight of bacterial cellulose (BC) / total volume of culture medium, and the yield is 2.56±0.06g / L.

[0110] Comparative Example 2

[0111] This comparative example provides a method for increasing the yield of bacterial cellulose by low-dose ultraviolet irradiation, which may specifically include the following steps:

[0112] Step 1: Activation of Bacillus xylosus

[0113] Same as Example 1.

[0114] Step 2: Expansion of Bacillus xylosus

[0115] Same as Example 1.

[0116] Step 3: Preparation of bacterial cellulose fermentation medium

[0117] Take 1L of distilled water, add 5g of yeast extract, 5g of peptone, 20g of glucose, 1.15g of citric acid monohydrate and 2.7g of Na2HPO4, adjust the pH value to 6.5, and obtain a bacterial fiber fermentation medium.

[0118] Step 4: Preparation of bacterial cellulose fermentation broth

[0119] Take the seed solution of Bacillus xylosus (concentration 10 6 CFU / mL~10 7 CFU / mL), inoculate the xylose bacillus seed liquid into the bacterial cellulose fermentation medium at an inoculation rate of 15 mL per 85 mL, and irradiate with ultraviolet light Xin Yat T5-4W type, wavelength 254nm, 4W, distance 50cm, irradiation for 60 seconds / hour under static fermentation at 28°C, and terminate the fermentation after 7 days to obtain bacterial cellulose fermentation liquid.

[0120] Step 5: Preparation of bacterial cellulose

[0121] Take the bacterial cellulose film in the bacterial cellulose fermentation broth, put the bacterial cellulose film into 200mL, 2% sodium carbonate solution at 100℃ water bath for 30min, rinse with deionized water several times, put the bacterial cellulose film into 200mL deionized water at 100℃ water bath for 30min, and then rinse with deionized water several times to obtain a pure bacterial cellulose film. The yield is calculated according to the dry weight of bacterial cellulose (BC) / total volume of culture medium, and the yield is 1.74±0.03g / L.

[0122] In order to better illustrate that ultraviolet irradiation of a certain time and an appropriate dose has a promoting effect on the yield of bacterial cellulose, the applicant studied the yield of bacterial cellulose (BC) prepared by Examples 1 to 3 and Comparative Examples 1 to 2.

[0123] Figure 2The trend diagram of the effect of ultraviolet irradiation on bacterial cellulose production provided in Examples 1 to 3 and Comparative Examples 1 to 2 of the present application; Figure 2 It can be seen that the BC yield in the culture increases as the ultraviolet irradiation time increases to 30s / h, and then the yield drops sharply. The ultraviolet irradiation time is 30 seconds / hour for 7 days, at which time the BC yield reaches the highest 3.41g / L. On the contrary, only 2.56g / L BC was produced in Comparative Example 1, indicating that the BC yield fermented under ultraviolet irradiation conditions is much higher. In Comparative Example 2, the ultraviolet irradiation time is 60 seconds / hour, the ultraviolet irradiation time is too long, and the activity of Bacillus xylosus is inhibited. The BC yield under the optimal ultraviolet irradiation time fermentation in this application is 133.2% of that under normal conditions.

[0124] In order to better illustrate the reason why the bacterial cellulose fermentation medium of the present invention is used to ferment Bacillus xylosus to improve the yield of bacterial cellulose, the time for Bacillus xylosus to secrete bacterial cellulose and the structural characteristics of bacterial cellulose were fermented with the bacterial cellulose fermentation medium (optimal fermentation group under ultraviolet irradiation) prepared in Example 2. The bacterial cellulose fermentation liquid prepared in Example 2 is referred to as the optimal fermentation group under ultraviolet irradiation, and the bacterial cellulose liquid prepared in Comparative Example 1 is referred to as the control fermentation group.

[0125] Figure 3 A graph showing a trend of glucose concentration changes in Bacillus xylosus provided in Example 2 and Comparative Example 1 of the present application; Figure 4 The variation trend diagram of bacterial cellulose production of Bacillus xylosus provided in Example 2 and Comparative Example 1 of the present application; Figure 3 and Figure 4 It can be seen that by comparing the growth curves of the cultures grown in the fermentation group under control conditions and the optimal fermentation group under UV irradiation, during the entire fermentation period, when growing under UV irradiation conditions of 30s / h, Bacillus xylosus absorbed more glucose than under the control conditions; at the same time, the maximum BC yield of 3.41g / L obtained from the optimal UV irradiation group was significantly higher than the BC yield obtained in the control group (2.56g / L).

[0126] Figure 5 This is a comparison chart of the infrared spectra of bacterial cellulose provided in Example 2 and Comparative Example 1 of the present application; Figure 5 It can be seen that the BC spectrum from the UV irradiation group (Example 2) is almost similar to the BC spectrum obtained from the control group (Comparative Example 1), with the peaks at 1020, 1410, 2894 and 3340 cm -1 The characteristic peaks of cellulose are shown at , and there is no obvious difference in the FTIR spectra of the two samples.

[0127] Figure 6 This is a comparison chart of the X-ray diffraction curves of bacterial cellulose provided in Example 2 and Comparative Example 1 of the present application; Figure 6 It can be seen that the two BC films obtained by the two different preparation methods showed similar patterns, with three diffraction peaks at 2θ = 14.6°, 16.7° and 22.9°, corresponding to the (100), (010) and (110) lattice planes, respectively. This is because the BC sample has all the characteristic bands of classical cellulose (type I). The CI of the BC-UV irradiation group (76.92%) is slightly lower than that of the BC-control group (80.83%), which may be due to the fact that UV irradiation may destroy the crystallization of BC during the culture process. In addition, UV irradiation changes the cross-linked hydrogen bond network of BC and increases the distance between cellulose chains, thereby destroying the process of linear β-glucan chains arranging into an ordered crystal structure.

[0128] Figure 7 This is a TGA thermal analysis comparison diagram of bacterial cellulose provided in Example 2 and Comparative Example 1 of the present application; Figure 7 It can be seen that the TGA thermal analysis diagrams of different BC films show that the thermal depolymerization of the BC-UV irradiation group (red curve) starts at 330°C, and the thermal depolymerization of the BC-control group (black curve) starts at 335°C.

[0129] Figure 8 This is a DTG thermal analysis comparison diagram of bacterial cellulose provided in Example 2 and Comparative Example 1 of the present application; Figure 8 It can be seen that the maximum degradation temperatures of the BC-ultraviolet irradiation group (Example 2) and the BC-control group (Comparative Example 1) are 369°C and 373°C, respectively, indicating that the thermal stability of the BC-ultraviolet irradiation group is slightly lower than that of the BC-control group. The maximum degradation of BC occurring at this temperature may lead to the depolymerization of glucan.

[0130] The average degree of polymerization (DP) values ​​of the BC-control group and the BC-UV irradiation group are shown in Table 1. After UV irradiation, the DP of BC decreased slightly. The DP value of the BC-UV irradiation group was 1978, which was significantly lower than that of the BC-control group (2031), which was consistent with the CI determined by XRD. BC has commendable mechanical properties, making it suitable for various uses, such as biomedicine, food additives, and sewage purification. The effect of additives on the mechanical behavior of BC was studied by measuring Young's modulus, tensile strength, and elongation at break, and the data are shown in Table 1. The applicant found that the elongation at break of the BC-UV irradiation group was slightly longer than that of the BC-control group (p>0.05), while the Young's modulus and tensile strength of the BC-UV irradiation group were slightly decreased compared with the BC-control group (p>0.05). The slight decrease in mechanical properties may be related to the decrease in DP and CI.

[0131] Table 1 Performance of bacterial cellulose provided in Example 2 and Comparative Example 1

[0132] performance Control group (Comparative example 1) Optimal UV irradiation (Example 2) Crystallinity index (%) 80.30±2.34 76.92±2.17 Tensile strength(MPa) 12.85±0.31 11.24±0.28 Young's modulus(GPa) 6.35±0.24 6.27±0.27 Elongation at break (%) 2.78±0.17 2.89±0.18 DP 2031±33 1978±25 Specific surface area 21.069±1.375 30.284±2.237

[0133] In order to explore the molecular mechanism of PETAH promoting cellulose biosynthesis, key enzymes of sugar metabolism were also studied. The results showed that the activities of glucose catabolism-related enzymes: pyruvate kinase (PK) and phosphofructokinase (PFK) were reduced, and sugar decomposition was inhibited. At the same time, the activities of key enzymes for cellulose synthesis: UDP-glucose pyrophosphorylase (UGP) and glucose phosphomutase (PGM) were enhanced, accelerating the synthesis of bacterial cellulose.

[0134] Fig. 9 This is a graph showing the effect of the culture methods provided in Example 2 and Comparative Example 1 of the present application on the activity of phosphofructokinase (PFK) involved in BC anabolism and glucose catabolism; Fig.10 This is a graph showing the effect of the culture methods provided in Example 2 and Comparative Example 1 of the present application on the activity of pyruvate kinase (PK) involved in BC anabolism and glucose catabolism; Fig.11 This is a graph showing the effect of the culture methods provided in Example 2 and Comparative Example 1 of the present application on the activity of phosphoglucomutase (PGM) involved in BC anabolism and glucose catabolism; Fig.12 This is a graph showing the effect of the culture methods provided in Example 2 and Comparative Example 1 on the activity of UDP-glucose pyrophosphorylase (UGP) involved in BC anabolism and glucose catabolism. Figures 9 to 12 It can be seen that the samples irradiated with UV for 30 seconds / hour increased the activities of UDP-glucose pyrophosphorylase (UGP) and phosphoglucomutase (PGM) by 60.25% and 187.08%, respectively, and decreased the activities of pyruvate kinase (PK) and phosphofructokinase (PFK) by 41.56% and 50.41%.

[0135] In summary, giving an appropriate dose of UV irradiation for an appropriate time under traditional fermentation conditions is a very simple and effective method to increase BC production without significantly changing its characteristics. The significant increase in BC production is due to the regulation of the activity of four key glucose metabolic enzymes (PFK, PK, PGM and UGP). The method provided by the present invention can increase BC production using simple UV irradiation as a regulatory factor.

[0136] In addition, one or more technical solutions in the embodiments of the present application have at least the following technical effects or advantages:

[0137] In the examples of the present application, the prepared bacterial cellulose (BC) exhibits lower crystallinity and better surface area. Compared with the traditional HS medium fermentation culture of Bacillus xylosus, the specific surface area of ​​the bacterial cellulose prepared by the present invention is increased by about 1.43 times, and the crystallinity is reduced by 5.26%.

[0138] In the examples of the present application, the bacterial cellulose culture medium of the present invention is obtained by adjusting the appropriate dose of ultraviolet radiation to irradiate the traditional HS culture medium, which effectively improves the yield of bacterial cellulose on the basis of low cost and low operating difficulty, and is suitable for large-scale production of bacterial cellulose.

[0139] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A method for increasing bacterial cellulose production by low-dose ultraviolet irradiation, the method comprising: inoculating Taonella mepensis seed liquid into a fermentation medium for fermentation treatment, and performing ultraviolet irradiation under set conditions during the fermentation process to obtain a fermentation liquid containing a bacterial cellulose membrane; as well as The bacterial cellulose membrane in the fermentation liquid is taken out, and then the bacterial cellulose membrane is purified to obtain bacterial cellulose.

2. The method according to claim 1, characterized in that: The setting conditions include at least one of the following conditions: ultraviolet irradiation power is 3W to 5W, ultraviolet irradiation wavelength is 254nm, ultraviolet irradiation distance is 45cm to 55cm, and ultraviolet irradiation time is 15s / h to 45s / h.

3. The method according to claim 1, characterized in that In terms of mass concentration, the chemical components of the fermentation medium include: 5g / L-6g / L yeast extract, 4.5g / L-5.5g / L peptone, 15g / L-20g / L glucose, 1g / L-1.15g / L citric acid monohydrate, 2.5g / L-2.7g / L disodium hydrogen phosphate and 15g / L-18g / L agar.

4. The method according to claim 3, characterized in that The pH value of the fermentation medium is 6.5-7.

0.

5. The method according to claim 1, characterized in that The viable count of the seed solution of Taonella mepensis xylosus was 10 6 CFU / mL~10 7 CFU / mL.

6. The method according to claim 1, characterized in that The inoculation amount of the inoculation is 5% to 15%.

7. The method according to claim 1, characterized in that The temperature of the fermentation treatment is 28° C. to 30° C., and the time of the fermentation treatment is 7 days.

8. The method according to claim 1, characterized in that: The purification process of the bacterial cellulose membrane comprises: The bacterial cellulose membrane is sequentially subjected to a first heating and boiling, water washing, a second heating and boiling, and water washing until neutrality to obtain bacterial cellulose; wherein the first heating and boiling uses a sodium carbonate solution.

9. The method according to claim 1, characterized in that: The yield of the bacterial cellulose is ≥2.78 g / L.

10. The method according to claim 1, characterized in that The bacterial cellulose meets at least one of the following properties: a crystallinity index of 75% to 80%, a tensile strength of 10.5 MPa to 12 MPa, a Young's modulus of 6 GPa to 7 GPa, an elongation at break of 2.6% to 3.2%, a DP of 1920 to 2020, and a specific surface area of ​​27 m 2 / g~34m 2 / g.

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