Method for preparing bacterial nanocellulose by using animal slaughtering tail water
By treating and chemically modifying slaughterhouse wastewater, bacterial nanocellulose with excellent swelling resistance was prepared, solving the problems of insufficient resource utilization and swelling resistance of slaughterhouse wastewater, and achieving efficient resource conversion and material performance improvement.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HAINAN BAIKERUI BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-02-09
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies are difficult to effectively utilize slaughter tailwater to prepare bacterial nanocellulose, and their swelling resistance is insufficient, failing to meet practical application requirements.
Bacterial cellulose wet membranes were prepared by treating slaughterhouse wastewater through centrifugation, enzymatic hydrolysis, and membrane filtration. The membranes were then modified with silane coupling agents and nano-TiO2, and combined with fluorinated hydrophobic monomers and copolymerization of styrene and divinylbenzene to construct a dense interpenetrating network structure, thereby enhancing the hydrophobicity and swelling resistance of the material.
This method enables the efficient resource utilization of slaughterhouse wastewater, significantly improves the swelling resistance of bacterial nanocellulose, solves the problems of resource waste and water eutrophication in traditional methods, and enhances the swelling resistance of the material.
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Figure CN122012650A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomanufacturing technology, specifically a method for preparing bacterial nanocellulose using animal slaughter tailwater. Background Technology
[0002] Bacterial nanocellulose (BNC) is a natural nanoscale biomass material synthesized by microbial metabolism. Its molecular chains are linked by β-1,4 glycosidic bonds, exhibiting high crystallinity, excellent mechanical strength, good biocompatibility, and biodegradability. It shows broad application prospects in medical dressings, food packaging, and biosensors. Compared to plant-derived cellulose, bacterial nanocellulose has higher purity and aspect ratio, and possesses a unique three-dimensional network structure, making it an ideal substrate for high-performance functional materials. The slaughtering of animals (livestock such as pigs, cattle, and sheep; poultry such as chickens and ducks; and seafood such as edible fish) generates large amounts of bloody wastewater. This wastewater is rich in biomass nutrients such as proteins, peptides, nitrogen, and phosphorus, and is characterized by high COD and high oil content, belonging to typical high-concentration organic wastewater. Traditional treatment processes often employ biochemical methods or physicochemical-biochemical combined processes, which are not only costly but also waste nitrogen and phosphorus resources and easily lead to eutrophication of water bodies. In recent years, researchers have begun to explore the use of animal slaughter tail water as a culture medium to prepare bacterial cellulose in order to realize the resource utilization of waste. However, due to the fact that the molecular chain surface of natural bacterial nanocellulose is rich in a large number of hydrophilic hydroxyl groups and its own three-dimensional network structure has a strong water adsorption and permeation capacity, its swelling resistance has always been difficult to meet the needs of practical applications. Summary of the Invention
[0003] The purpose of this invention is to provide a method for preparing bacterial nanocellulose using animal slaughter tail water, so as to solve the technical problems mentioned in the background art.
[0004] To achieve the above objectives, the present invention provides the following technical solution:
[0005] A method for preparing bacterial nanocellulose using animal slaughter tail water includes the following steps:
[0006] (1) Centrifuge the animal slaughter tail water, take the supernatant, heat and adjust the pH to alkaline, add alkaline protease for enzymatic hydrolysis, then heat to inactivate the enzyme and denature the impurities, cool and adjust the pH to weak acid and let stand, then perform membrane filtration to obtain refined tail water filtrate.
[0007] (2) Add disodium hydrogen phosphate and magnesium sulfate to the purified tailwater filtrate, adjust the pH and sterilize to obtain solution A; sterilize the glucose solution separately to obtain solution B; select anhydrous ethanol as solution C; mix solution A, solution B and solution C under sterile conditions, inoculate with Acetobacter xylinum and incubate statically to obtain bacterial cellulose wet film.
[0008] (3) The bacterial cellulose wet film was boiled with alkaline solution, washed until neutral, crushed and prepared into a suspension. After high pressure microfluidic homogenization, a bacterial nanocellulose aqueous dispersion was obtained.
[0009] (4) After centrifuging the bacterial nanocellulose aqueous dispersion, it was redispersed in a mixed solvent of ethanol and water. Silane coupling agent KH570 was added, and the mixture was pre-hydrolyzed under acidic conditions and then heated under reflux. After the reaction was completed, the mixture was centrifuged and washed to obtain bacterial nanocellulose with double bonds grafted on the surface.
[0010] (5) Disperse nano-TiO2 in anhydrous ethanol, add silane coupling agent KH570, heat and reflux under acidic conditions, and centrifuge and wash after reaction to obtain surface-modified TiO2.
[0011] (6) Surface-modified TiO2 and surface-grafted double bond bacterial nanocellulose was dispersed in tetrahydrofuran, dodecafluoroheptyl methacrylate monomer and initiator azobisisobutyronitrile were added, and polymerization was carried out under an inert atmosphere. After the reaction was completed, the mixture was centrifuged, washed with tetrahydrofuran, and then solvent-replaced with tert-butanol. Finally, the mixture was freeze-dried to obtain bacterial nanocellulose.
[0012] In this invention, the animal slaughter effluent is first centrifuged to remove bottom sediment and insoluble blood clots. The supernatant is then adjusted to alkaline pH, and alkaline protease is added. Through enzymatic hydrolysis, the large hemoglobin molecules in the effluent are degraded into small polypeptides and free amino acids that are easily utilized by Acetobacter xylinum. The enzymes are then inactivated by high temperature, and the impurities are denatured. After cooling, the pH is adjusted back to weakly acidic to promote the early precipitation of inorganic salts that are prone to precipitate during subsequent fermentation. Membrane filtration is then used to remove bacteria and impurities while retaining soluble nutrients, resulting in a refined effluent filtrate. The refined effluent filtrate containing nitrogen sources and inorganic salts is then separately sterilized to obtain solution A, and a glucose solution is separately sterilized. Alcohol-based sterilization solution B and anhydrous ethanol solution C were used. A carbon-nitrogen source phase-separated sterilization method was adopted to avoid the production of antibacterial toxins by the Maillard reaction. After mixing the three solutions A, B, and C under aseptic conditions, Acetobacter xylinum was inoculated. Acetobacter xylinum utilized the small molecule nutrients in the filtrate and substrates such as glucose and ethanol to metabolize and synthesize, forming a bacterial cellulose wet film. Finally, the bacterial cells and residual protein impurities in the wet film were removed by boiling with alkaline solution. After washing to neutrality, the film was crushed and prepared into a suspension. With the help of the strong shear force, cavitation effect, and high-speed impact force generated by high-pressure microfluidic homogenization, the micron-sized cellulose bundles were efficiently exfoliated into single nanofibers, and bacterial nanocellulose was finally obtained.
[0013] This invention synergistically improves the swelling resistance of bacterial nanocellulose in the following ways: Firstly, KH570 undergoes a condensation reaction with the hydroxyl groups on the surface of bacterial nanocellulose, introducing highly active double bond functional groups on the surface. This partially shields the hydrophilic hydroxyl groups on the surface of bacterial nanocellulose through chemical bonding, reducing their binding sites with water molecules, thereby initially reducing the hydrophilic solubility of bacterial nanocellulose. On the other hand, the fluorinated hydrophobic monomer dodecafluoroheptyl methacrylate (DFMA) undergoes a ternary free radical copolymerization reaction with the double-bonded functional groups on the surface of bacterial nanocellulose and nano-titanium dioxide to form a dense interpenetrating network structure. This network structure can firmly anchor inorganic nano-titanium dioxide to the surface of bacterial nanocellulose through chemical bonding, preventing it from falling off during use. At the same time, the fluorinated groups formed after DFMA polymerization have ultra-low surface energy, which can construct a stable hydrophobic barrier on the material surface. Combined with the micro-nano rough structure of the material itself, it significantly reduces the contact area between the material and water molecules, reducing the adsorption and penetration of water molecules. In addition, solvent replacement with tert-butanol and freeze-drying can form a fluffy aerogel structure without collapsing the nano-skeleton of the material, further enhancing the hydrophobic effect. Finally, through the synergistic effect of hydrophobic group shielding, interpenetrating network binding and micro-nano hydrophobic structure, the water absorption and swelling degree of bacterial nanocellulose is significantly reduced, and the swelling resistance is greatly improved.
[0014] Preferably, in step (1), the animal slaughter tail water is the slaughter tail water of livestock, poultry, seafood or river fish.
[0015] Preferably, in step (1), the amount of alkaline protease added is 0.2 to 0.4 wt% of the effluent mass.
[0016] Preferably, in step (2), the amount of disodium hydrogen phosphate added is 0.4 to 0.6 wt% of the mass of the refined tailwater filtrate; and the amount of magnesium sulfate added is 0.2 to 0.4 wt% of the mass of the refined tailwater filtrate.
[0017] Preferably, in step (4), the mass ratio of bacterial nanocellulose to silane coupling agent KH570 is 10:(1-3).
[0018] Preferably, in step (5), the mass ratio of nano-TiO2 to silane coupling agent KH570 is 5:(0.3-0.6).
[0019] Preferably, in step (6), the mass ratio of bacterial nanocellulose with surface-grafted double bonds to surface-modified TiO2 is 2.5:(0.2-0.5).
[0020] Preferably, in step (6), the mass ratio of bacterial nanocellulose with surface-grafted double bonds to dodecafluoroheptyl methacrylate is 2.5:(6-10).
[0021] Preferably, in step (6), styrene and divinylbenzene are added to the reaction solution before the polymerization reaction is carried out by heating.
[0022] This invention discovered in experiments that the large side chains of the fluorinated monomer (DFMA) used result in significant steric hindrance, leading to grafting blind zones on the surface of BNC fibers that are difficult for macromolecules to cover. Furthermore, the excessive flexibility of the linear polymer chains generated by single polymerization makes them prone to conformational relaxation in aqueous environments, lacking sufficient physical binding force to limit the water absorption and swelling of the fiber matrix, thus affecting the anti-swelling performance of bacterial nanofibers. To address this technical problem, this invention introduces styrene and divinylbenzene to construct a dense, rigid network system. Utilizing the small molecular size and rigid benzene ring of styrene, it is used as a micro-filler, precisely inserted into the grafting gaps that DFMA cannot penetrate through free radical copolymerization, effectively eliminating coverage blind zones and improving the overall rigidity of the coating. Simultaneously, the divinylbenzene's divinyl structure acts as a chemical interlock, weaving the originally loose linear polymer chains in situ into a high-density three-dimensional interpenetrating network structure. This constructs a triple synergistic anti-swelling mechanism of low-energy water repellency from fluorinated groups, rigid and dense benzene ring filling, and mechanical binding of the cross-linked network, blocking water molecule penetration and fiber volume expansion from both physical and mechanical dimensions and surface chemistry.
[0023] Preferably, in step (6), the mass ratio of styrene to divinylbenzene is 10:(0.5-1.0).
[0024] Compared with the prior art, the beneficial effects of the present invention are:
[0025] 1. By using centrifugation, enzymatic hydrolysis and membrane filtration to treat slaughterhouse wastewater in a synergistic manner, impurities are effectively removed while small molecule nutrients are retained. The wastewater is transformed into a sterile culture medium with high bioavailability, realizing the high-value utilization of waste and solving the problems of nutrient loss and sterilization scaling in traditional methods.
[0026] 2. By using silane coupling agent (KH570) to undergo a condensation reaction with the hydroxyl groups on the surface of BNC and nano TiO2, active double bonds are introduced on the surface of both through chemical bonding. This not only partially shields the hydrophilic groups of BNC itself and reduces the initial hydrophilicity, but also provides uniform and strong active sites for subsequent copolymerization reactions.
[0027] 3. By ternary copolymerization of DFMA with the double bonds on the surfaces of bacterial nanocellulose and nano titanium dioxide, a stable composite hydrophobic structure integrating particle anchoring and an ultra-low surface energy fluorine-containing layer is constructed through chemical bonding, thereby achieving long-term barrier to water molecules and significantly improving the swelling resistance of the material.
[0028] 4. Styrene and divinylbenzene are introduced as comonomers. The small-molecule styrene, as a sealant, effectively compensates for the grafting blind spots caused by the steric hindrance of the fluorinated macromonomer (DFMA), improving the density and rigidity of the coating. Divinylbenzene acts as a chemical interlocking agent, crosslinking the linear polymer into a high-strength three-dimensional interpenetrating network. This network, in synergy with the ultra-low surface energy fluorinated layer provided by DFMA, significantly blocks water molecules from both physical and surface chemical dimensions. Attached Figure Description
[0029] Figure 1 This is a low-magnification SEM image of the surface of the bacterial nanocellulose prepared in Example 1 of the present invention after being pressed into a film.
[0030] Figure 2 This is a medium-magnification SEM image of the surface of the bacterial nanocellulose prepared in Example 1 of the present invention after being pressed into a film.
[0031] Figure 3 This is a high-magnification SEM image of the surface of the bacterial nanocellulose prepared in Example 1 of the present invention after being pressed into a film.
[0032] Figure 4 XPS spectra of bacterial nanocellulose prepared in Example 1 of this invention after being compressed into a membrane. Detailed Implementation
[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0034] Example 1
[0035] A method for preparing bacterial nanocellulose using animal slaughter tail water includes the following steps:
[0036] Step 1: Take 10L of fresh bloody pig slaughter tail water and place it in a large-capacity centrifuge. Centrifuge at 4000 rpm for 15 minutes, discarding the bottom sediment and insoluble precipitate, and collect the supernatant. Heat the supernatant to 55℃, adjust the pH to 8.0 using 1mol / L sodium hydroxide solution, add 0.35wt% alkaline protease (by weight of tail water), and stir for 4 hours under constant temperature conditions for enzymatic hydrolysis. After enzymatic hydrolysis, rapidly raise the temperature to 95℃ and maintain it for 15 minutes to inactivate the enzyme and denature and precipitate some heat-labile proteins. After the solution cools to room temperature, adjust the pH to 6.0 using citric acid, let it stand for 30 minutes to further settle inorganic salt impurities, and finally pump the supernatant into a ceramic membrane filter with a pore size of 0.22μm for filtration, collecting a clear and transparent purified tail water filtrate.
[0037] Step 2: Take 9.5 L of purified tailwater filtrate, add 0.55 wt% disodium hydrogen phosphate and 0.35 wt% magnesium sulfate (based on the filtrate mass), adjust the pH to 5.5 with acetic acid, sterilize at 115℃ for 20 min, and cool to obtain solution A; separately, prepare a 50 wt% concentrated solution of 475 g glucose, sterilize it separately at 115℃ for 15 min, and cool to obtain solution B; take 120 mL of anhydrous ethanol as solution C. In a sterile operating table, mix solutions A, B, and C, inoculate with 6 wt% of the mixture with liquid inoculum of Acetobacter xylinum, and incubate at 30℃ for 7 days. Harvest the bacterial cellulose wet film formed on the surface.
[0038] Step 3: The bacterial cellulose wet membrane was repeatedly rinsed with deionized water, then placed in a 0.1 mol / L sodium hydroxide solution and boiled at 90°C for 90 min to remove bacterial cells and residual protein. After removal, it was washed with deionized water until the pH of the washing solution was neutral. The purified wet membrane was pulped using a tissue homogenizer, and deionized water was added to prepare a cellulose suspension with a solid content of 0.5 wt%. This suspension was passed through a high-pressure microfluidic homogenizer and homogenized five times at a pressure of 150 MPa to obtain a uniformly dispersed bacterial nanocellulose aqueous dispersion.
[0039] Step 4: Take the bacterial nanocellulose aqueous dispersion (containing 10g of dry bacterial nanocellulose), centrifuge to remove water, and redisperse it in 2L of a mixed solvent of 95% ethanol and 5% water. Add 2.5g of silane coupling agent KH570, adjust the pH of the system to 4.0 with glacial acetic acid, stir and pre-hydrolyze at 25℃ for 30min, then heat to 60℃ and reflux for 6h. After the reaction is complete, centrifuge the product and wash it three times with anhydrous ethanol to remove unreacted silane, obtaining bacterial nanocellulose with surface-grafted double bonds.
[0040] Step 5: Disperse 5.0 g of nano-TiO2 in 100 mL of anhydrous ethanol, add 0.5 g of silane coupling agent KH570, adjust the pH to 4.0 with glacial acetic acid, and heat under reflux at 75 °C for 4 h. After the reaction is complete, centrifuge the mixture and wash it three times with anhydrous ethanol to obtain surface-modified TiO2.
[0041] Step 6: 2.5 g of bacterial nanocellulose with surface-grafted double bonds and 0.4 g of surface-modified TiO2 were dispersed together in 250 mL of tetrahydrofuran solvent and ultrasonically dispersed for 15 min. 9 g of dodecafluoroheptyl methacrylate monomer and 0.1 g of azobisisobutyronitrile (AIB) initiator were added to the system, followed by 1.6 g of styrene and 0.13 g of divinylbenzene. Nitrogen gas was purged for 30 min to replace air and create an inert atmosphere. The temperature was raised to 75 °C for polymerization reaction for 14 h. After the reaction, the precipitate was centrifuged. The precipitate was washed twice with tetrahydrofuran, then subjected to three solvent replacements with tert-butanol, and finally pre-frozen at -20 °C and dried in a freeze dryer for 24 h to obtain bacterial nanocellulose.
[0042] Example 2
[0043] A method for preparing bacterial nanocellulose using animal slaughter tail water includes the following steps:
[0044] Step 1: Take 10L of fresh, bloody cattle slaughter tail water and place it in a large-capacity centrifuge. Centrifuge at 4000 rpm for 15 minutes, discarding the bottom sediment and insoluble precipitate, and collect the supernatant. Heat the supernatant to 55℃, adjust the pH to 8.0 using 1mol / L sodium hydroxide solution, and add 0.25wt% alkaline protease (by weight of tail water). Stir and hydrolyze under constant temperature for 4 hours. After hydrolysis, rapidly raise the temperature to 95℃ and maintain it for 15 minutes to inactivate the enzyme and denature and precipitate some heat-labile proteins. After cooling the solution to room temperature, adjust the pH to 6.0 using citric acid, and let it stand for 30 minutes to further settle inorganic salt impurities. Finally, pump the supernatant into a 0.22μm ceramic membrane filter for filtration, collecting a clear and transparent purified tail water filtrate.
[0045] Step 2: Take 9.5 L of purified tailwater filtrate, add 0.45 wt% disodium hydrogen phosphate and 0.25 wt% magnesium sulfate (based on the filtrate mass), adjust the pH to 5.5 with acetic acid, sterilize at 115℃ for 20 min, and obtain solution A after cooling; separately, prepare a 50 wt% concentrated solution with 475 g of glucose, sterilize separately at 115℃ for 15 min, and obtain solution B after cooling; take 120 mL of anhydrous ethanol as solution C. In a sterile operating table, mix solutions A, B, and C, inoculate with 6 wt% of Acetobacter xylinum liquid inoculum, and incubate in a 30℃ constant temperature incubator for 7 days, harvesting the bacterial cellulose wet film formed on the surface.
[0046] Step 3: The bacterial cellulose wet membrane was repeatedly rinsed with deionized water, then placed in a 0.1 mol / L sodium hydroxide solution and boiled at 90°C for 90 min to remove bacterial cells and residual protein. After removal, it was washed with deionized water until the pH of the washing solution was neutral. The purified wet membrane was pulped using a tissue homogenizer, and deionized water was added to prepare a cellulose suspension with a solid content of 0.5 wt%. This suspension was passed through a high-pressure microfluidic homogenizer and homogenized five times at a pressure of 150 MPa to obtain a uniformly dispersed bacterial nanocellulose aqueous dispersion.
[0047] Step 4: Take the bacterial nanocellulose aqueous dispersion (containing 10g of dry bacterial nanocellulose), centrifuge to remove water, and redisperse it in 2L of a mixed solvent of 95% ethanol and 5% water. Add 1.5g of silane coupling agent KH570, adjust the pH of the system to 4.0 with glacial acetic acid, stir and pre-hydrolyze at 25℃ for 30min, then heat to 60℃ and reflux for 6h. After the reaction is complete, centrifuge the product and wash it three times with anhydrous ethanol to remove unreacted silane, obtaining bacterial nanocellulose with surface-grafted double bonds.
[0048] Step 5: Disperse 5.0 g of nano-TiO2 in 100 mL of anhydrous ethanol, add 0.4 g of silane coupling agent KH570, adjust the pH to 4.0 with glacial acetic acid, and heat under reflux at 75 °C for 4 h. After the reaction is complete, centrifuge the mixture and wash it three times with anhydrous ethanol to obtain surface-modified TiO2.
[0049] Step 6: 2.5 g of bacterial nanocellulose with surface-grafted double bonds and 0.3 g of surface-modified TiO2 were dispersed together in 250 mL of tetrahydrofuran solvent and ultrasonically dispersed for 15 min. 7 g of dodecafluoroheptyl methacrylate monomer and 0.1 g of azobisisobutyronitrile (AIB) initiator were added to the system, followed by 1.6 g of styrene and 0.09 g of divinylbenzene. Nitrogen gas was purged for 30 min to replace air and create an inert atmosphere. The temperature was raised to 75 °C for polymerization reaction for 12 h. After the reaction, the precipitate was centrifuged. The precipitate was washed twice with tetrahydrofuran, then subjected to three solvent replacements with tert-butanol, and finally pre-frozen at -20 °C and dried in a freeze dryer for 24 h to obtain bacterial nanocellulose.
[0050] Example 3
[0051] A method for preparing bacterial nanocellulose using animal slaughter tail water includes the following steps:
[0052] Step 1: Take 10L of fresh sheep slaughter tail water containing blood and place it in a large-capacity centrifuge. Centrifuge at 4000 rpm for 15 minutes, discarding the bottom sediment and insoluble precipitate, and collect the supernatant. Heat the supernatant to 55℃, adjust the pH to 8.0 using 1mol / L sodium hydroxide solution, add 0.3wt% alkaline protease (by weight of tail water), and stir for 4 hours under constant temperature conditions for enzymatic hydrolysis. After enzymatic hydrolysis, rapidly raise the temperature to 95℃ and maintain it for 15 minutes to inactivate the enzyme and denature and precipitate some heat-labile proteins. After the solution cools to room temperature, adjust the pH to 6.0 using citric acid, let it stand for 30 minutes to further settle inorganic salt impurities, and finally pump the supernatant into a ceramic membrane filter with a pore size of 0.22μm for filtration, collecting a clear and transparent purified tail water filtrate.
[0053] Step 2: Take 9.5 L of purified tailwater filtrate, add 0.5 wt% disodium hydrogen phosphate and 0.3 wt% magnesium sulfate (based on the filtrate mass), adjust the pH to 5.5 with acetic acid, sterilize at 115℃ for 20 min, and cool to obtain solution A; separately, prepare a 50 wt% concentrated solution with 475 g of glucose, sterilize separately at 115℃ for 15 min, and cool to obtain solution B; take 120 mL of anhydrous ethanol as solution C. In a sterile operating table, mix solutions A, B, and C, inoculate with 6 wt% of the mixture with Acetobacter xylinum liquid inoculum, and incubate at 30℃ for 7 days. Harvest the bacterial cellulose wet film formed on the surface.
[0054] Step 3: The bacterial cellulose wet membrane was repeatedly rinsed with deionized water, then placed in a 0.1 mol / L sodium hydroxide solution and boiled at 90°C for 90 min to remove bacterial cells and residual protein. After removal, it was washed with deionized water until the pH of the washing solution was neutral. The purified wet membrane was pulped using a tissue homogenizer, and deionized water was added to prepare a cellulose suspension with a solid content of 0.5 wt%. This suspension was passed through a high-pressure microfluidic homogenizer and homogenized five times at a pressure of 150 MPa to obtain a uniformly dispersed bacterial nanocellulose aqueous dispersion.
[0055] Step 4: Take the bacterial nanocellulose aqueous dispersion (containing 10g of dry bacterial nanocellulose), centrifuge to remove water, and redisperse it in 2L of a mixed solvent of 95% ethanol and 5% water. Add 2g of silane coupling agent KH570, adjust the pH of the system to 4.0 with glacial acetic acid, stir and pre-hydrolyze at 25℃ for 30min, then heat to 60℃ and reflux for 6h. After the reaction is complete, centrifuge the product and wash it three times with anhydrous ethanol to remove unreacted silane, obtaining bacterial nanocellulose with surface-grafted double bonds.
[0056] Step 5: Disperse 5.0 g of nano-TiO2 in 100 mL of anhydrous ethanol, add 0.45 g of silane coupling agent KH570, adjust the pH to 4.0 with glacial acetic acid, and heat under reflux at 75 °C for 4 h. After the reaction is complete, centrifuge the mixture and wash it three times with anhydrous ethanol to obtain surface-modified TiO2.
[0057] Step 6: 2.5 g of bacterial nanocellulose with surface-grafted double bonds and 0.35 g of surface-modified TiO2 were dispersed together in 250 mL of tetrahydrofuran solvent and ultrasonically dispersed for 15 min. 8 g of dodecafluoroheptyl methacrylate monomer and 0.1 g of azobisisobutyronitrile (AIB) initiator were added to the system, followed by 1.6 g of styrene and 0.10 g of divinylbenzene. Nitrogen gas was purged for 30 min to replace air and create an inert atmosphere. The temperature was raised to 75 °C for polymerization reaction for 13 h. After the reaction, the precipitate was centrifuged. The precipitate was washed twice with tetrahydrofuran, then subjected to three solvent replacements with tert-butanol, and finally pre-frozen at -20 °C and dried in a freeze dryer for 24 h to obtain bacterial nanocellulose.
[0058] Example 4
[0059] A method for preparing bacterial nanocellulose using animal slaughter tail water includes the following steps:
[0060] Step 1: Take 10L of fresh chicken and duck slaughter tail water containing blood and place it in a large-capacity centrifuge. Centrifuge at 4000 rpm for 15 minutes, discarding the bottom sediment and insoluble precipitate, and collect the supernatant. Heat the supernatant to 55℃, adjust the pH to 8.0 using 1mol / L sodium hydroxide solution, add 0.4wt% alkaline protease (by weight of tail water), and stir for 4 hours under constant temperature conditions for enzymatic hydrolysis. After enzymatic hydrolysis, rapidly raise the temperature to 95℃ and maintain it for 15 minutes to inactivate the enzyme and denature and precipitate some heat-labile proteins. After the solution cools to room temperature, adjust the pH to 6.0 using citric acid, let it stand for 30 minutes to further settle inorganic salt impurities, and finally pump the supernatant into a ceramic membrane filter with a pore size of 0.22μm for filtration, collecting a clear and transparent purified tail water filtrate.
[0061] Step 2: Take 9.5 L of purified tailwater filtrate, add 0.6 wt% disodium hydrogen phosphate and 0.4 wt% magnesium sulfate (based on the filtrate mass), adjust the pH to 5.5 with acetic acid, sterilize at 115℃ for 20 min, and obtain solution A after cooling; separately, prepare a 50 wt% concentrated solution with 475 g of glucose, sterilize separately at 115℃ for 15 min, and obtain solution B after cooling; take 120 mL of anhydrous ethanol as solution C. In a sterile operating table, mix solutions A, B, and C, inoculate with 6 wt% of Acetobacter xylinum liquid inoculum, and incubate in a 30℃ constant temperature incubator for 7 days, harvesting the bacterial cellulose wet film formed on the surface.
[0062] Step 3: The bacterial cellulose wet membrane was repeatedly rinsed with deionized water, then placed in a 0.1 mol / L sodium hydroxide solution and boiled at 90°C for 90 min to remove bacterial cells and residual protein. After removal, it was washed with deionized water until the pH of the washing solution was neutral. The purified wet membrane was pulped using a tissue homogenizer, and deionized water was added to prepare a cellulose suspension with a solid content of 0.5 wt%. This suspension was passed through a high-pressure microfluidic homogenizer and homogenized five times at a pressure of 150 MPa to obtain a uniformly dispersed bacterial nanocellulose aqueous dispersion.
[0063] Step 4: Take the bacterial nanocellulose aqueous dispersion (containing 10g of dry bacterial nanocellulose), centrifuge to remove water, and redisperse it in 2L of a mixed solvent of 95% ethanol and 5% water. Add 3g of silane coupling agent KH570, adjust the pH of the system to 4.0 with glacial acetic acid, stir and pre-hydrolyze at 25℃ for 30min, then heat to 60℃ and reflux for 6h. After the reaction is complete, centrifuge the product and wash it three times with anhydrous ethanol to remove unreacted silane, obtaining bacterial nanocellulose with surface-grafted double bonds.
[0064] Step 5: Disperse 5.0 g of nano-TiO2 in 100 mL of anhydrous ethanol, add 0.6 g of silane coupling agent KH570, adjust the pH to 4.0 with glacial acetic acid, and heat under reflux at 75 °C for 4 h. After the reaction is complete, centrifuge the mixture and wash it three times with anhydrous ethanol to obtain surface-modified TiO2.
[0065] Step 6: 2.5 g of bacterial nanocellulose with surface-grafted double bonds and 0.5 g of surface-modified TiO2 were dispersed together in 250 mL of tetrahydrofuran solvent and ultrasonically dispersed for 15 min. 10 g of dodecafluoroheptyl methacrylate monomer and 0.1 g of azobisisobutyronitrile (AIB) initiator were added to the system, followed by 1.6 g of styrene and 0.16 g of divinylbenzene. Nitrogen gas was purged for 30 min to replace air and create an inert atmosphere. The temperature was raised to 80 °C for polymerization reaction for 15 h. After the reaction, the precipitate was centrifuged, washed twice with tetrahydrofuran, then subjected to three solvent replacements with tert-butanol, and finally pre-frozen at -20 °C and dried in a freeze dryer for 24 h to obtain bacterial nanocellulose.
[0066] Example 5
[0067] A method for preparing bacterial nanocellulose using animal slaughter tail water includes the following steps:
[0068] Step 1: Take 10L of fresh fish slaughter tail water containing blood and place it in a large-capacity centrifuge. Centrifuge at 4000 rpm for 15 minutes, discarding the bottom sediment and insoluble precipitate, and collect the supernatant. Heat the supernatant to 55℃, adjust the pH to 8.0 using 1mol / L sodium hydroxide solution, add 0.2wt% alkaline protease (by weight of tail water), and stir for 4 hours under constant temperature conditions for enzymatic hydrolysis. After enzymatic hydrolysis, rapidly raise the temperature to 95℃ and maintain it for 15 minutes to inactivate the enzyme and denature and precipitate some heat-labile proteins. After the solution cools to room temperature, adjust the pH to 6.0 using citric acid, let it stand for 30 minutes to further settle inorganic salt impurities, and finally pump the supernatant into a ceramic membrane filter with a pore size of 0.22μm for filtration, collecting a clear and transparent purified tail water filtrate.
[0069] Step 2: Take 9.5 L of purified tailwater filtrate, add 0.4 wt% disodium hydrogen phosphate and 0.2 wt% magnesium sulfate (based on the filtrate mass), adjust the pH to 5.5 with acetic acid, sterilize at 115℃ for 20 min, and obtain solution A after cooling; separately, prepare a 50 wt% concentrated solution with 475 g of glucose, sterilize separately at 115℃ for 15 min, and obtain solution B after cooling; take 120 mL of anhydrous ethanol as solution C. In a sterile operating table, mix solutions A, B, and C, inoculate with 6 wt% of Acetobacter xylinum liquid inoculum, and incubate in a 30℃ constant temperature incubator for 7 days, harvesting the bacterial cellulose wet film formed on the surface.
[0070] Step 3: The bacterial cellulose wet membrane was repeatedly rinsed with deionized water, then placed in a 0.1 mol / L sodium hydroxide solution and boiled at 90°C for 90 min to remove bacterial cells and residual protein. After removal, it was washed with deionized water until the pH of the washing solution was neutral. The purified wet membrane was pulped using a tissue homogenizer, and deionized water was added to prepare a cellulose suspension with a solid content of 0.5 wt%. This suspension was passed through a high-pressure microfluidic homogenizer and homogenized five times at a pressure of 150 MPa to obtain a uniformly dispersed bacterial nanocellulose aqueous dispersion.
[0071] Step 4: Take the bacterial nanocellulose aqueous dispersion (containing 10g of dry bacterial nanocellulose), centrifuge to remove water, and redisperse it in 2L of a mixed solvent of 95% ethanol and 5% water. Add 1g of silane coupling agent KH570, adjust the pH of the system to 4.0 with glacial acetic acid, stir and pre-hydrolyze at 25℃ for 30min, then heat to 60℃ and reflux for 6h. After the reaction is complete, centrifuge the product and wash it three times with anhydrous ethanol to remove unreacted silane, obtaining bacterial nanocellulose with surface-grafted double bonds.
[0072] Step 5: Disperse 5.0 g of nano-TiO2 in 100 mL of anhydrous ethanol, add 0.3 g of silane coupling agent KH570, adjust the pH to 4.0 with glacial acetic acid, and heat under reflux at 75 °C for 4 h. After the reaction is complete, centrifuge the mixture and wash it three times with anhydrous ethanol to obtain surface-modified TiO2.
[0073] Step 6: 2.5 g of bacterial nanocellulose with surface-grafted double bonds and 0.2 g of surface-modified TiO2 were dispersed together in 250 mL of tetrahydrofuran solvent and ultrasonically dispersed for 15 min. 6 g of dodecafluoroheptyl methacrylate monomer and 0.1 g of azobisisobutyronitrile (AIB) initiator were added to the system, followed by 1.6 g of styrene and 0.08 g of divinylbenzene. Nitrogen gas was purged for 30 min to replace air and create an inert atmosphere. The temperature was raised to 70 °C for polymerization reaction for 10 h. After the reaction, the precipitate was centrifuged. The precipitate was washed twice with tetrahydrofuran, then subjected to three solvent replacements with tert-butanol, and finally pre-frozen at -20 °C and dried in a freeze dryer for 24 h to obtain bacterial nanocellulose.
[0074] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that in step 4, the silane coupling agent KH570 is not used to graft and modify the bacterial nanocellulose.
[0075] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that no surface-modified TiO2 is added in step 6.
[0076] Comparative Example 3: The difference between Comparative Example 3 and Example 1 is that dodecafluoroheptyl methacrylate monomer is not added in step 6.
[0077] Comparative Example 4: The difference between Comparative Example 4 and Example 1 is that styrene and divinylbenzene are not added in step 6.
[0078] Performance testing:
[0079] 1. Bacterial cellulose yield test: After fermentation, the wet bacterial cellulose film was purified by alkaline boiling, washed with water until neutral, and then dried in a 60℃ oven to constant weight. The mass of the dried film was then measured. The yield calculation formula is: Yield (g / L) = Mass of dried bacterial cellulose (g) / Volume of fermentation medium (L). This indicator is used to evaluate the fermentation efficiency of using slaughter tail water as a culture medium. The test results are shown in Table 1.
[0080] 2. Swelling test: Weigh 1.0 g of bacterial nanocellulose sample dried to constant weight and record its dry weight m1. Place the sample in a beaker containing 50 mL of deionized water and soak it at a constant temperature of 25℃ for 24 h. After removal, quickly absorb the free water on the sample surface with filter paper and immediately weigh and record the wet weight m2. The swelling degree is calculated using the formula: Swelling degree (%) = [(m2-m1) / m1] × 100%. Each group of samples was tested in parallel three times, and the average value was taken as the final result. The test results are shown in Table 1.
[0081] 3. Static Contact Angle Test: Bacterial nanocellulose samples were pressed into uniformly thick dry films and placed on the sample stage of a contact angle measuring instrument. 5 μL of deionized water was drawn using a microsyringe and dropped onto the surface of the dry film. After the droplet stabilized, its morphology was photographed, and the static contact angle was calculated using the instrument's built-in software. Five different test points were selected for each sample group, and the average value was taken as the final result. A larger static contact angle indicates better hydrophobic properties. The test results are shown in Table 1.
[0082] 4. Coating Bond Strength Test: To verify the bonding strength between the hydrophobic modified layer and the BNC substrate, the sample with the initial contact angle tested was immersed in a beaker containing anhydrous ethanol and ultrasonically cleaned at a frequency of 40 kHz for 30 minutes. After removal, it was dried at 60℃, and its water contact angle was measured again using the above method. If the change in contact angle before and after cleaning is small, it indicates that the organic-inorganic composite modified layer is firmly anchored by chemical bonding; otherwise, it indicates that the modified layer is prone to detachment. The test results are shown in Table 1.
[0083] 5. Mechanical Property Testing: Bacterial nanocellulose samples were prepared into standard tensile specimens with dimensions of 20mm × 5mm × 0.2mm. A universal testing machine was used for testing, with a tensile rate of 1mm / min. The tensile strength at fracture was recorded. Each group of samples was tested in parallel five times, and the average value was taken as the final result. This indicator reflects the effect of the rigid interpenetrating network constructed from styrene and divinylbenzene on improving the mechanical strength of the material. The test results are shown in Table 1.
[0084] Table 1
[0085] Yield (g / L) Swelling degree (%) Static contact angle (°) Contact angle (°) after cleaning Tensile strength (MPa) Example 1 2.81 127 156.3 154.6 67.5 Example 2 2.70 140 153.7 152.3 65.2 Example 3 2.78 136 155.0 153.7 66.8 Example 4 2.85 123 158.6 156.2 68.4 Example 5 2.65 152 150.2 149.6 63.9 Comparative Example 1 2.83 853 75.6 70.3 28.1 Comparative Example 2 2.84 321 110.4 108.5 62.3 Comparative Example 3 2.82 482 88.3 86.3 58.7 Comparative Example 4 2.86 216 142.7 139.7 45.6
[0086] Analysis of the test data above shows that the BNC yield of all examples and comparative examples is stable in the range of 2.65 to 2.86 g / L, which strongly confirms the stability and high-value utilization potential of the centrifugation-enzymatic hydrolysis-membrane filtration slaughter tailwater pretreatment process adopted in this invention.
[0087] In terms of material properties, Examples 1-5 all exhibited excellent overall performance, with static water contact angles greater than 150° (up to 158.6°), water absorption swelling rates effectively controlled at a low level of 123%-152%, and tensile strength maintained at a high level of 63.9-68.4 MPa. In contrast, the performance of the comparative examples decreased significantly: the contact angle data of Comparative Example 1 (without KH570) and Comparative Example 2 (without TiO2) indicate that without chemical bonding anchoring or micro / nano rough structures, the materials cannot achieve a stable superhydrophobic effect.
[0088] Comparative Example 4 (without styrene and divinylbenzene) had a water absorption swelling rate as high as 216% and a tensile strength that dropped to 45.6 MPa, which was in stark contrast to Example 1 (swelling rate of 127% and strength of 67.5 MPa). This directly verifies the decisive role of introducing styrene as a sealant and constructing a rigid interpenetrating network with divinylbenzene in physically binding fiber swelling and improving the mechanical strength of the material.
[0089] In summary, this invention successfully prepared high-performance bacterial nanocellulose with high strength, superhydrophobicity, and low swelling properties through multi-component synergistic modification.
[0090] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for preparing bacterial nanocellulose using animal slaughter tail water, characterized in that, Includes the following steps: (1) Centrifuge the animal slaughter tail water, take the supernatant, heat and adjust the pH to alkaline, add alkaline protease for enzymatic hydrolysis, then heat to inactivate the enzyme and denature the impurities, cool and adjust the pH to weak acid and let stand, then perform membrane filtration to obtain refined tail water filtrate. (2) Add disodium hydrogen phosphate and magnesium sulfate to the purified tailwater filtrate, adjust the pH and sterilize to obtain solution A; sterilize the glucose solution separately to obtain solution B; select anhydrous ethanol as solution C; mix solution A, solution B and solution C under sterile conditions, inoculate with Acetobacter xylinum and incubate statically to obtain bacterial cellulose wet film. (3) The bacterial cellulose wet film was boiled with alkaline solution, washed until neutral, crushed and prepared into a suspension. After high pressure microfluidic homogenization, a bacterial nanocellulose aqueous dispersion was obtained. (4) After centrifuging the bacterial nanocellulose aqueous dispersion, it was redispersed in a mixed solvent of ethanol and water. Silane coupling agent KH570 was added, and the mixture was pre-hydrolyzed under acidic conditions and then heated under reflux. After the reaction was completed, the mixture was centrifuged and washed to obtain bacterial nanocellulose with double bonds grafted on the surface. (5) Disperse nano-TiO2 in anhydrous ethanol, add silane coupling agent KH570, heat and reflux under acidic conditions, and centrifuge and wash after reaction to obtain surface-modified TiO2. (6) Surface-modified TiO2 and surface-grafted double bond bacterial nanocellulose was dispersed in tetrahydrofuran, dodecafluoroheptyl methacrylate monomer and initiator azobisisobutyronitrile were added, and polymerization was carried out under an inert atmosphere. After the reaction was completed, the mixture was centrifuged, washed with tetrahydrofuran, and then solvent-replaced with tert-butanol. Finally, the mixture was freeze-dried to obtain bacterial nanocellulose.
2. The method for preparing bacterial nanocellulose using animal slaughter tail water according to claim 1, characterized in that, In step (1), the animal slaughter tail water is the slaughter tail water of livestock, poultry, seafood or river fish.
3. The method for preparing bacterial nanocellulose using animal slaughter tail water according to claim 1, characterized in that, In step (1), the amount of alkaline protease added is 0.2 to 0.4 wt% of the effluent mass.
4. The method for preparing bacterial nanocellulose using animal slaughter tail water according to claim 1, characterized in that, In step (2), the amount of disodium hydrogen phosphate added is 0.4 to 0.6 wt% of the mass of the purified tailwater filtrate; The amount of magnesium sulfate added is 0.2 to 0.4 wt% of the mass of the refined tailwater filtrate.
5. The method for preparing bacterial nanocellulose using animal slaughter tail water according to claim 1, characterized in that, In step (4), the mass ratio of bacterial nanocellulose to silane coupling agent KH570 is 10:(1-3).
6. The method for preparing bacterial nanocellulose using animal slaughter tail water according to claim 1, characterized in that, In step (5), the mass ratio of nano-TiO2 to silane coupling agent KH570 is 5:(0.3-0.6).
7. The method for preparing bacterial nanocellulose using animal slaughter tail water according to claim 1, characterized in that, In step (6), the mass ratio of bacterial nanocellulose with surface-grafted double bonds to surface-modified TiO2 is 2.5:(0.2-0.5).
8. A method for preparing bacterial nanocellulose using animal slaughter tail water according to claim 1, characterized in that, In step (6), the mass ratio of bacterial nanocellulose with surface-grafted double bonds to dodecafluoroheptyl methacrylate is 2.5:(6-10).
9. A method for preparing bacterial nanocellulose using animal slaughter tail water according to claim 1, characterized in that, In step (6), styrene and divinylbenzene are added to the reaction solution before the polymerization reaction is carried out by heating.
10. A method for preparing bacterial nanocellulose using animal slaughter tail water according to claim 1, characterized in that, In step (6), the mass ratio of styrene to divinylbenzene is 10:(0.5-1.0).