Fermentation process for producing bacterial cellulose through solid-state fermentation of slurry

Abstract

The invention discloses a fermentation process for producing bacterial cellulose by solid-state fermentation of serofluid, and relates to the technical field of microbial fermentation, and the fermentation process comprises the following steps: (1) pretreatment of serofluid: performing impurity removal and sterilization treatment on a food processing byproduct serofluid to obtain pretreated serofluid, (2) preparation of a solid-state fermentation substrate: obtaining the solid-state fermentation substrate, and (3) fermentation of the solid-state fermentation substrate. (3) strain inoculation: inoculating an activated and cultured acetic bacteria strain liquid into the solid fermentation substrate, and controlling the inoculation amount to be a preset proportion, (4) solid fermentation: putting the inoculated solid fermentation substrate into a fermentation device to obtain a fermentation product containing bacterial cellulose, and (5) separation and purification of the bacterial cellulose. Cheap food processing seriflux is used as a main nutrient source, agricultural by-products are adopted as solid carriers, the total cost of raw materials is reduced, the cost of bacterial cellulose products can be reduced, the economical efficiency is greatly improved, and meanwhile the problem of environmental pollution caused by direct discharge of the seriflux is solved.

Description

TECHNICAL FIELD

[0001] The present application is a kind of slurry water solid state fermentation production bacterial cellulose fermentation process, relates to microbial fermentation technical field, specifically relates to a kind of slurry water as raw material with food processing by-product, through solid state fermentation technology production bacterial cellulose fermentation process, especially suitable for the industrialized green production of bacterial cellulose, while realizing the resource utilization of food processing waste. BACKGROUND

[0002] Bacterial cellulose (Bacterial Cellulose, abbreviated as BC) is a kind of natural polysaccharide biomaterial synthesized and secreted by some microorganisms (mainly Acetobacter) in the metabolic process, its chemical composition is β-1,4-glucan, which is similar to plant cellulose in chemical structure, but has unique microstructure and performance advantages, bacterial cellulose has broad application prospects in food industry (such as biomimetic food, food additives), pharmaceutical field (such as wound dressing, tissue engineering scaffold), cosmetic field (such as moisturizing agent) and environmental protection field (such as adsorbent) and the like, at present, the industrialized production of bacterial cellulose mainly adopts liquid fermentation process, and the classical culture medium is Hestrin-Schramm (HS) culture medium, its main components are glucose (carbon source), peptone (nitrogen source), yeast extract (growth factor), citric acid and disodium hydrogen phosphate (buffer).

[0003] The existing technology has the following problems: 1. The existing liquid fermentation process relies on high-quality sugars such as glucose and sucrose as carbon source, and peptone and yeast extract as nitrogen source, which accounts for 60%-80% of the production cost, resulting in high price of bacterial cellulose product, which is difficult to meet the market demand; 2. The existing solid state fermentation research is mostly based on single straw and bran as substrate, and no special fermentation parameters are designed for slurry, a kind of high-moisture and high-nutrient liquid waste, resulting in slow growth of bacterial strains, low synthesis efficiency of bacterial cellulose, and easy mixing of substrate impurities in the product, which is difficult to meet the standard. SUMMARY

[0004] The present application provides a kind of slurry water solid state fermentation production bacterial cellulose fermentation process to solve the problems raised in the above background.

[0005] To solve the above technical problems, the technical scheme adopted by the present application is: A kind of slurry water solid state fermentation production bacterial cellulose fermentation process, comprising the following steps: (1) slurry water pretreatment: removing impurities and sterilizing the food processing by-product slurry water to obtain pretreated slurry water.

[0006] (2) Solid-state fermentation substrate preparation: the pretreated slurry is mixed with a solid carrier according to a preset mass ratio, the pH of the substrate is adjusted to a preset range, and a solid-state fermentation substrate is obtained.

[0007] (3) Strain inoculation: the activated culture of the Acetobacter strain is inoculated into the solid-state fermentation substrate, and the inoculation amount is controlled to be a preset proportion.

[0008] (4) Solid-state fermentation: the inoculated solid-state fermentation substrate is placed in a fermentation device, and the fermentation temperature, humidity and aeration amount are controlled to perform aerobic fermentation, and a fermentation product containing bacterial cellulose is obtained.

[0009] (5) Bacterial cellulose separation and purification: the fermentation product containing bacterial cellulose is subjected to impurity removal, bacterial cell separation and chemical treatment, and high-purity bacterial cellulose is obtained.

[0010] Preferably, the slurry pretreatment in step (1) specifically includes: a. The slurry is placed in a centrifugal device and centrifuged at a speed of 3000-5000 r / min for 10-20 min to remove suspended residues in the slurry.

[0011] b. The supernatant after centrifugation is taken and subjected to high-pressure steam sterilization at 115-121°C for 15-30 min, and the pretreated slurry is obtained after cooling to room temperature.

[0012] Preferably, the solid carrier in step (2) is at least one selected from bran, corn cob powder, soybean meal powder and straw powder.

[0013] The mass ratio of the pretreated slurry to the solid carrier is 1:0.8-1.5.

[0014] The pH of the substrate is adjusted to 5.5-6.5.

[0015] Preferably, the Acetobacter strain in step (3) is at least one selected from Acetobacter xylinum, Acetobacter pasteurianus and Gluconacetobacter.

[0016] The strain activation culture uses Hestrin-Schramm medium, and the culture conditions are 30-32°C, 150-200 r / min shaking flask culture until the OD600 value of the bacterial liquid is 0.6-1.0.

[0017] Preferably, the inoculation amount in step (3) is 3%-8% (v / w, the volume ratio of the bacterial liquid to the mass of the solid-state fermentation substrate).

[0018] Preferably, the fermentation temperature in step (4) is controlled to be 28-32°C.

[0019] The fermentation humidity is controlled to be 70%-85%.

[0020] The aeration rate is controlled at 0.5-1.2 L / (L·h) (based on the volume of the substrate in the fermentation device).

[0021] The aerobic fermentation time is 7-12 days.

[0022] Preferably, the fermentation device in step (4) is a solid fermentation tank with temperature control, humidity control and ventilation functions. During the fermentation process, it is stirred once every 2-4 hours, with a stirring rate of 50-100 r / min and a stirring time of 5-10 min each time.

[0023] Preferably, the impurity removal in step (5) specifically involves: soaking the fermentation product containing bacterial cellulose in deionized water for 2-4 hours, manually rubbing to remove the attached solid carrier residue, and repeating the soaking-rubbing operation 2-3 times.

[0024] The bacterial cell separation process involves placing the bacterial cellulose, after removing the residue, in a 0.3-0.8 mol / L NaOH solution and incubating it at 70-90℃ for 1-3 hours to denature and dissolve the bacterial proteins.

[0025] The chemical treatment specifically involves repeatedly rinsing the bacterial cellulose separated from the bacterial cells with deionized water until the pH reaches 6.5-7.5, and then drying it in a vacuum drying oven at 60-80℃ until constant weight.

[0026] Preferably, the food processing by-product slurry in step (1) is selected from at least one of soybean product processing slurry, starch processing slurry, and grain processing slurry.

[0027] The soy product processing slurry is wastewater generated during the production of tofu and soy milk, wherein: the total sugar content is 30-50g / L, the protein content is 5-15g / L, and the pH is 4.5-6.0.

[0028] Preferably, it also includes a fermentation product detection step: Bacterial cellulose yield was determined by gravimetric method (based on the dry weight of bacterial cellulose produced per 100g dry solid fermentation substrate).

[0029] The chemical structure of bacterial cellulose was analyzed using Fourier transform infrared spectroscopy (FT-IR).

[0030] The microstructure of bacterial cellulose was observed using scanning electron microscopy (SEM).

[0031] The tensile strength of bacterial cellulose was determined using a universal testing machine.

[0032] The water-holding capacity of bacterial cellulose was determined by centrifugation.

[0033] Due to the adoption of the above technical solution, the technical progress achieved by this invention compared to the prior art is as follows: 1. This invention uses inexpensive food processing slurry as the main nutrient source and agricultural by-products as the solid carrier, thereby reducing the total cost of raw materials and the cost of bacterial cellulose products, significantly improving economic efficiency, while solving the environmental pollution problem caused by direct discharge of slurry.

[0034] 2. This invention improves bacterial cellulose yield by precisely controlling fermentation temperature, humidity and aeration to match the metabolic needs of bacterial strains. At the same time, the pretreatment and purification process effectively removes impurities and purifies the bacterial cellulose to meet the needs of high-end fields such as pharmaceuticals and food.

[0035] 3. The Acetobacter xylinum and Acetobacter pasteurization strains selected in this invention have good growth adaptability in the slurry-carrier composite matrix after being domesticated in a solid matrix. The cell density of the matrix is ​​increased by two to three orders of magnitude compared with the undomesticated strains. At the same time, the porous structure and nutritional balance of the composite matrix avoid the problem of uneven distribution of oxygen and nutrients. The bacterial cellulose yield fluctuates less between fermentation batches, and the stability is significantly improved.

[0036] 4. The bacterial cellulose prepared by this invention retains the nanoscale fiber network structure, water retention and tensile strength, which are superior to traditional liquid fermentation bacterial cellulose. Furthermore, bacterial cellulose products with different properties can be customized by adjusting the type of solid carrier or fermentation parameters, making it suitable for various applications such as wound dressings, biomimetic foods, and adsorbent materials. Detailed Implementation

[0037] This invention provides a solid-state fermentation process for producing bacterial cellulose from slurry, comprising the following steps: (1) Pretreatment of fermented vegetable waste: The fermented vegetable waste from food processing is subjected to impurity removal and sterilization treatment to obtain pretreated fermented vegetable waste. The fermented vegetable waste from food processing is selected from at least one of the following: fermented vegetable waste from soybean product processing, fermented vegetable waste from starch processing, and fermented vegetable waste from grain processing. Fermented vegetable waste from soybean product processing is wastewater generated during the production of tofu and soy milk, wherein: the total sugar content is 30-50 g / L, the protein content is 5-15 g / L, and the pH is 4.5-6.0. In addition, the pretreatment of fermented vegetable waste specifically includes: a. Place the slurry in a centrifuge and centrifuge at 3000-5000 r / min for 10-20 min to remove suspended residues from the slurry; b. Take the supernatant after centrifugation, sterilize it with high-pressure steam at 115-121℃ for 15-30 minutes, and cool it to room temperature to obtain the pretreated slurry.

[0038] (2) Preparation of solid fermentation substrate: The pretreated slurry and solid carrier are mixed at a preset mass ratio, and the substrate pH is adjusted to a preset range to obtain solid fermentation substrate. The solid carrier is selected from at least one of wheat bran, corn cob powder, soybean meal powder and straw powder. The mass ratio of pretreated slurry to solid carrier is 1:0.8-1.5, and the substrate pH is adjusted to 5.5-6.5.

[0039] (3) Inoculation of bacterial strains: The activated cultured Acetic Acid Bacteria strains are inoculated into the solid fermentation substrate, and the inoculation amount is controlled to a preset ratio. The Acetic Acid Bacteria strains are selected from at least one of Acetobacter xylinum, Acetic Acid Bacteria pasteurella, and Acetic Acid Bacteria gluconate. The strains are activated and cultured using Hestrin-Schramm medium under the following conditions: 30-32℃ and 150-200r / min in shake flask culture until the OD600 value of the bacterial solution is 0.6-1.0. In addition, the inoculation amount is 3%-8% (v / w, the ratio of bacterial solution volume to solid fermentation substrate mass).

[0040] (4) Solid fermentation: The inoculated solid fermentation substrate is placed in a fermentation device, and the fermentation temperature, humidity and aeration are controlled to carry out aerobic fermentation to obtain fermentation products containing bacterial cellulose. The fermentation temperature is controlled at 28-32℃, the fermentation humidity is controlled at 70%-85%, and the aeration is controlled at 0.5-1.2L / (L·h) (based on the volume of substrate in the fermentation device). The aerobic fermentation time is 7-12 days. In addition, the fermentation device is a solid fermentation tank with temperature control, humidity control and aeration functions. During the fermentation process, the mixture is stirred once every 2-4 hours at a stirring rate of 50-100r / min and each stirring time is 5-10min.

[0041] (5) Bacterial cellulose separation and purification: The fermentation product containing bacterial cellulose is subjected to impurity removal, cell separation and chemical treatment to obtain high-purity bacterial cellulose. Specifically, the impurity removal is carried out by soaking the fermentation product containing bacterial cellulose in deionized water for 2-4 hours, manually rubbing to remove the attached solid carrier residue, and repeating the soaking-rubbing operation 2-3 times.

[0042] The specific process for bacterial cell isolation is as follows: after removing the residue, the bacterial cellulose is placed in a 0.3-0.8 mol / L NaOH solution and kept at 70-90℃ for 1-3 hours to denature and dissolve the bacterial proteins.

[0043] The chemical treatment specifically involves repeatedly rinsing the bacterial cellulose after cell separation with deionized water until the pH reaches 6.5-7.5, and then drying it in a vacuum drying oven at 60-80℃ until constant weight.

[0044] In this embodiment, the solid-state fermentation process for producing bacterial cellulose from slurry also includes a fermentation product detection step: Bacterial cellulose yield was determined by gravimetric method (based on the dry weight of bacterial cellulose produced per 100g dry solid fermentation substrate). The chemical structure of bacterial cellulose was analyzed using Fourier transform infrared spectroscopy (FT-IR). The microstructure of bacterial cellulose was observed using scanning electron microscopy (SEM). The tensile strength of bacterial cellulose was determined using a universal testing machine. The water-holding capacity of bacterial cellulose was determined by centrifugation.

[0045] The present invention will be further described in detail below with reference to the implementation scheme.

[0046] Implementation Plan

[0047] This embodiment details the general operating procedure for producing bacterial cellulose by solid-state fermentation of slurry according to the present invention. The equipment, reagents, and parameters used are all industrially achievable conditions. The specific steps are as follows: Raw material preparation a. Fermented water: Fermented water from soybean product processing (tofu production wastewater) was selected. After testing, the total sugar content was 42g / L, the protein content was 12g / L, the pH was 5.2, and the suspended residue content was 8g / L. b. Solid carrier: Select wheat bran (passed through an 80-mesh sieve, moisture 12%, ash 5.2%) and corn cob powder (passed through an 80-mesh sieve, moisture 11%, ash 3.8%), and mix them at a mass ratio of 1:1; c. Strains: Acetobacter xylinum, accession number CGMCC1.1812 (China General Microbiological Culture Collection Center). d. Culture medium: Hestrin-Schramm (HS) medium, with the following formula: glucose 20 g / L, peptone 5 g / L, yeast extract 5 g / L, citric acid 1.15 g / L, disodium hydrogen phosphate 2.7 g / L, deionized water to a final volume, pH adjusted to 6.0, sterilized at 121℃ for 20 min.

[0048] Slurry pretreatment a. Centrifugation to remove residue: Pour the soy product processing slurry into a high-speed centrifuge (model TGL-16G, Shanghai Anting Scientific Instrument Factory), set the speed to 4000 r / min, and the centrifugation time to 15 min; after centrifugation, discard the bottom sediment (suspended residue) and collect the supernatant; b. Sterilization: Transfer the supernatant into a sterilization tank (model LS-50L), set the temperature to 121℃, and sterilize for 20 minutes; after sterilization, cool to room temperature to obtain pretreated slurry (pH 5.0-5.5).

[0049] Solid-state fermentation substrate preparation a. Mixing: Pour the pretreated slurry and the mixed carrier (bran + corn cob powder) into the mixing tank (model JB-100) at a mass ratio of 1:1.2, set the stirring speed to 80r / min and the stirring time to 10min, so that the slurry and the carrier are fully mixed; b. pH adjustment: Slowly add 0.1 mol / L NaOH solution to the mixture while stirring, and adjust the pH of the matrix to 6.0; c. Packaging: The adjusted substrate is packaged into the trays of the solid fermentation tank (model FS-100). Each tray contains 5 kg of dry substrate (approximately 12 kg of wet substrate), and the substrate thickness is controlled to 5 cm (to ensure uniform aeration).

[0050] Strain activation culture a. Slant activation: Take out the freeze-dried powder of Acetobacter xylinum from the refrigerator (-80℃), pick up a small amount of powder with a sterile inoculation loop, streak it onto HS solid slant medium (with 2% agar added), place it in an incubator (model SPX-250B), and incubate at 30℃ for 48h to form a single colony; b. Seed culture: Pick a single colony and inoculate it into a 250mL Erlenmeyer flask containing 100mL of HS liquid medium. Place the flask on a shaker (model THZ-300) and set the temperature to 30℃ and the rotation speed to 180r / min. Incubate for 24h. Take a sample to measure the OD600 value of the bacterial culture. When OD600=0.8, stop the culture to obtain the seed culture (bacterial density of about 10^8 CFU / mL).

[0051] Inoculation and solid-state fermentation a. Inoculation: Spray the seed liquid evenly onto the substrate surface of the solid fermentation tank at an inoculation rate of 5% (v / w) (i.e., 5 mL of seed liquid per 100 g of wet substrate). After inoculation, gently stir the substrate with a sterile glass rod to ensure that the seed liquid is evenly distributed. b. Fermentation parameter settings: Close the fermentation tank door, set the temperature to 30℃, humidity to 80%, and aeration rate to 0.8L / (L·h) (based on substrate volume); during fermentation, stir once every 3 hours at a stirring rate of 70r / min for 8 minutes each time; the fermentation cycle is 10 days. c. Fermentation process monitoring: Daily samples were taken to measure the cell density (plate count method) and bacterial cellulose content (gravimetric method) in the substrate. During the first 3 days, the cell density increased rapidly (from 10^6 CFU / g to 10^9 CFU / g), while the bacterial cellulose content increased slowly (from 0 to 2.5%). From the 4th to the 8th day, the cell density stabilized (around 10^9 CFU / g), while the bacterial cellulose content increased rapidly (from 2.5% to 14.0%). From the 9th to the 10th day, the bacterial cellulose content tended to stabilize (14.2%), and fermentation was stopped.

[0052] Bacterial cellulose isolation and purification a. Residue removal: Remove the fermented substrate from the fermentation tank and soak it in deionized water for 3 hours. During this period, manually knead it once every 30 minutes to remove the attached bran and corn cob residue. After soaking, filter and collect the crude bacterial cellulose, and rinse it twice with deionized water. b. Removal of bacterial cells: Transfer the crude bacterial cellulose into a beaker containing 0.5 mol / L NaOH solution. The amount of NaOH solution is 10 times the weight of the crude bacterial cellulose. Place the beaker in a constant temperature water bath (model HH-S, Xicheng Xinrui Instrument Factory, Jintan District) and keep it at 80℃ for 2 hours. Stir once every 20 minutes during the process to denature and dissolve the bacterial protein. c. Washing: Transfer the alkali-treated bacterial cellulose into a Buchner funnel, and repeatedly filter and wash with deionized water. Measure the pH of the filtrate after each wash until the pH reaches 7.0. d. Drying: Place the washed bacterial cellulose in a vacuum drying oven (model DZF-6050), set the temperature to 70℃ and the vacuum degree to -0.09MPa, and dry until constant weight (about 8h) to obtain the bacterial cellulose product.

[0053] Product Testing a. Yield determination: Bacterial cellulose yield = (dry weight of bacterial cellulose product / dry weight of solid fermentation substrate) × 100%. Calculated, the bacterial cellulose yield in this implementation plan is 14.2%. b. Purity determination: FT-IR (Nicoleti S50) analysis was performed. The characteristic peaks of bacterial cellulose (3400 cm⁻¹ for -OH stretching vibration, 2900 cm⁻¹ for CH stretching vibration, and 1100 cm⁻¹ for COC stretching vibration) were clear, and there were no impurity peaks (such as lignin at 1510 cm⁻¹ and protein at 1650 cm⁻¹), indicating a purity of 98.6%. c. Microstructure: Observed using SEM (model SU8020), the bacterial cellulose fibers had a diameter of 50-70 nm, a uniform three-dimensional network structure, and a porosity of 85%. d. Water holding capacity: Weigh 0.5g of bacterial cellulose product, soak it in deionized water for 24h, centrifuge (3000r / min, 10min) and weigh it. Water holding capacity = (wet weight - dry weight) / dry weight. The calculated water holding capacity is 125g / g. e. Tensile strength: The tensile strength was measured using a universal testing machine (model CMT6104). The bacterial cellulose was prepared into a sample of 10mm×50mm×0.2mm, and the tensile rate was 5mm / min. The tensile strength was 18.5MPa.

[0054] The present invention will be further described in detail below with reference to embodiments:

[0055] Example 1: This embodiment uses soy product processing liquid as raw material and wheat bran as a single solid carrier to verify the feasibility of the process of the present invention. The specific steps are as follows: Raw materials and equipment a. Fermented water: Fermented water from soybean product processing (soy milk production wastewater), total sugar 38g / L, protein 10g / L, pH 5.0, suspended residue 6g / L; b. Solid carrier: wheat bran (passed through an 80-mesh sieve, moisture 11.5%, ash 5.0%). c. Strains: Acetobacter xylinum CGMCC1.1812; d. Equipment: Same as implementation plan 5.1.1.

[0056] Process steps a. Slurry pretreatment: centrifugation (3500 r / min, 18 min) → sterilization (121℃, 25 min) to obtain pretreated slurry (pH 4.8-5.3). b. Substrate preparation: The pretreated slurry and wheat bran are mixed at a mass ratio of 1:1.1, the pH is adjusted to 5.8, and the mixture is packaged (5 kg of dry substrate per tray, 5 cm thick). c. Activation of the strain: Shake flask culture in HS medium (30℃, 180r / min, 22h), OD600=0.7; d. Fermentation: Inoculum size 4%, temperature 29℃, humidity 78%, aeration rate 0.7L / (L·h), stirring (every 3 hours, 70r / min, 8min), fermentation for 9 days; e. Purification: Soaking (deionized water, 2.5h, rubbing twice) → Alkali treatment (0.4mol / LNaOH, 85℃, 1.5h) → Washing with water (pH 7.0) → Drying (70℃, vacuum, 7h).

[0057] Product testing results a. Yield: 13.8% (13.8g of bacterial cellulose per 100g dry substrate); b. Purity: 98.2% (FT-IR shows no impurity peaks); c. Microstructure: Fiber diameter 55-80nm, uniform three-dimensional network structure, porosity 83%; d. Water holding capacity: 122g / g; e. Tensile strength: 17.8 MPa.

[0058] This embodiment demonstrates that, using soy milk and wheat bran as raw materials, the process of this invention can stably produce high-purity bacterial cellulose, with both yield and performance meeting the expected targets.

[0059] Example 2: This embodiment uses starch processing slurry as raw material and soybean meal powder as a solid carrier to verify the adaptability of the process of the present invention to different slurry raw materials. The specific steps are as follows: Raw materials and equipment a. Slurry: Starch processing slurry (wastewater from corn starch production), total sugar 45g / L, protein 8g / L, pH 5.5, suspended residue 10g / L; b. Solid carrier: soybean meal powder (passed through an 80-mesh sieve, moisture 10%, ash 6.0%). c. Strains: Acetobacter pasteurianus CGMCC1.2114; d. Equipment: Same as implementation plan 5.1.1.

[0060] Process steps a. Slurry pretreatment: centrifugation (4500 r / min, 12 min) → sterilization (118℃, 30 min) to obtain pretreated slurry (pH 5.2-5.7). b. Substrate preparation: The pretreated slurry and soybean meal powder are mixed at a mass ratio of 1:1.3, the pH is adjusted to 6.2, and the mixture is packaged (5 kg of dry substrate per tray, 5 cm thick). c. Activation of the strain: Shake flask culture in HS medium (31℃, 190r / min, 20h), OD600=0.9; d. Fermentation: Inoculum size 6%, temperature 31℃, humidity 82%, aeration rate 1.0L / (L·h), stirring (every 2 hours, 80r / min, 6min), fermentation for 8 days; e. Purification: Soaking (deionized water, 3h, rubbing 3 times) → Alkali treatment (0.6mol / LNaOH, 75℃, 2.5h) → Washing with water (pH 7.2) → Drying (65℃, vacuum, 9h).

[0061] Product testing results a. Yield: 12.5% ​​(12.5g bacterial cellulose per 100g dry substrate); b. Purity: 97.5% (FT-IR shows no impurity peaks); c. Microstructure: The fiber diameter is 45-70 nm, the three-dimensional network structure is dense, and the porosity is 80%; d. Water holding capacity: 118 g / g; e. Tensile strength: 16.5 MPa.

[0062] This embodiment demonstrates that even when the type of slurry (starch processing slurry) and the strain (acetic acid bacteria pasteurianum) are changed, the process of the present invention can still achieve efficient production of bacterial cellulose, and the product performance meets the application requirements, proving that the process has good adaptability to raw materials and strains.

[0063] It should be noted that the working principle of this invention for producing bacterial cellulose by solid-state fermentation of fermented slurry is based on the regulation of microbial metabolism and optimization of substrate utilization. It achieves efficient bacterial cellulose production from three dimensions: nutrient supply, environmental regulation, and product synthesis. This includes the following four aspects: the nutrient supply principle of the fermented slurry, the functional principle of the solid-state carrier, the principle of fermentation parameter regulation, and the principle of bacterial cellulose synthesis and purification. Specifically, the following steps are described: (1) The principle of nutrient supply of slurry

[0064] Food processing slurry contains abundant carbon sources (carbohydrates), nitrogen sources (proteins), and growth factors (vitamins and minerals), making it an ideal nutrient source for the growth of acetic acid bacteria and the synthesis of bacterial cellulose. a. Carbon source metabolism: Small sugar molecules such as glucose and maltose in the slurry can be directly absorbed by acetic acid bacteria and converted into pyruvate through the glycolysis pathway (EMP pathway) and the pentose phosphate pathway (PPP pathway). Some of the pyruvate is converted into acetyl-CoA, which provides precursor substances (UDP-glucose) for bacterial cellulose synthesis. b. Nitrogen source metabolism: The proteins in the slurry are hydrolyzed into amino acids by the protease secreted by acetic acid bacteria. Amino acids not only provide nitrogen for bacterial synthesis (such as synthases and cell membrane components), but can also generate α-keto acids through transamination, which participate in energy metabolism and provide ATP for bacterial cellulose synthesis. c. Role of growth factors: B vitamins (such as vitamins B1 and B2) in the slurry act as coenzymes in the metabolism of carbohydrates and amino acids, while minerals (such as Mg^2+ and Mn^2+) act as enzyme activators (such as glucose phosphate mutase), promoting the efficient operation of metabolic pathways.

[0065] d. Working principle of slurry pretreatment (centrifugation-sterilization): Centrifugation removes suspended residues to prevent solid substrate from clumping, ensuring a porous substrate structure that facilitates oxygen transfer; sterilization kills miscellaneous bacteria (such as lactic acid bacteria and yeast) in the slurry, preventing miscellaneous bacteria from competing with acetic acid bacteria for nutrients and avoiding fermentation failure (e.g., miscellaneous bacteria produce acid, causing the substrate pH to drop and inhibiting the growth of acetic acid bacteria).

[0066] (2) Functional principle of solid carrier Solid carriers (such as wheat bran and corn cob powder) have a triple function during fermentation, supporting the growth of acetic acid bacteria and bacterial cellulose synthesis: a. Physical support function: The porous structure of the carrier (porosity 60%-70%) provides attachment sites for acetic acid bacteria, forming a bacterial-carrier composite system and avoiding bacterial aggregation; at the same time, the porous structure can store nutrients in the slurry, realizing the slow release of nutrients and avoiding local nutrient excess or deficiency. b. Oxygen transfer function: The loose structure of the carrier can reduce the air resistance, so that the air introduced during fermentation is evenly distributed inside the substrate, which meets the aerobic metabolism requirements of acetic acid bacteria (acetic acid bacteria are obligate aerobic bacteria, and bacterial cellulose synthesis requires a large amount of oxygen. Insufficient oxygen will lead to a decrease in bacterial cellulose yield). c. Nutritional supplementation function: The carrier itself contains a small amount of cellulose, hemicellulose and trace elements (such as bran containing 2% cellulose and 0.5% iron), which can be partially degraded into glucose by cellulase secreted by acetic acid bacteria, supplementing the carbon source of the slurry; trace elements can activate the activity of bacterial metabolic enzymes and improve the efficiency of bacterial cellulose synthesis.

[0067] (3) Principles of Fermentation Parameter Control The optimized fermentation parameters such as temperature, humidity, and aeration rate in this invention are all designed based on the metabolic characteristics of acetic acid bacteria, and their regulation principles are as follows: a. Temperature control: The optimal growth temperature for acetic acid bacteria is 28-32℃. Within this temperature range, the activity of key enzymes in the bacteria (such as UDP-glucose pyrophosphorylase, a key enzyme in bacterial cellulose synthesis) is at its highest (enzyme activity can reach 120-150 U / mg protein). Too high a temperature (>32℃) will cause enzyme denaturation and inactivation, and too low a temperature (<28℃) will reduce the rate of enzymatic reaction, both of which will lead to the inhibition of bacterial cellulose synthesis. b. Humidity control: A humidity of 70%-85% can maintain the water content of the solid substrate at 50%-60%. This water content can ensure the water required for bacterial metabolism (acetic acid bacteria need a water activity >0.9 to grow) and also prevent substrate clumping (clumping will cause the oxygen transfer coefficient to drop from 10^-5 m / s to 10^-7 m / s, resulting in insufficient oxygen supply). c. Aeration rate control: An aeration rate of 0.5-1.2 L / (L·h) can maintain the dissolved oxygen concentration in the substrate at 2-5 mg / L, meeting the aerobic metabolic needs of acetic acid bacteria (8-10 g of oxygen is required to synthesize 1 g of bacterial cellulose during the bacterial cellulose synthesis process); too low an aeration rate will lead to insufficient dissolved oxygen, causing the bacteria to switch to ethanol fermentation; too high an aeration rate will lead to excessively rapid evaporation of substrate moisture, requiring frequent rehumidification and increasing energy consumption; d. Inoculation amount control: An inoculation amount of 3%-8% can enable the cells to quickly occupy the ecological niche (the cell density can reach 10^7 CFU / g after 12 hours of fermentation), avoiding contamination by other microorganisms; at the same time, at this inoculation amount, the rate of cell growth and nutrient consumption are matched, avoiding early nutrient depletion or late cell autolysis.

[0068] (4) Principles of bacterial cellulose synthesis and purification The biochemical process by which acetic acid bacteria synthesize bacterial cellulose occurs outside the bacterial cell membrane, and the specific steps are as follows: a. Glucose is converted into glucose-6-phosphate intracellularly via the EMP pathway, and then converted into glucose-1-phosphate by phosphoglucomutase. b. Glucose-1-phosphate reacts with UTP (uridine triphosphate) to generate UDP-glucose (a direct precursor for bacterial cellulose synthesis) under the catalysis of UDP-glucose pyrophosphorylase. c. UDP-glucose is transported to the extracellular space via a bacterial cellulose synthase complex on the cell membrane (composed of four proteins: bacterial cellulose sA, bacterial cellulose sB, bacterial cellulose sC, and bacterial cellulose sD). Under enzyme catalysis, the β-1,4-glycosidic bonds of UDP-glucose are linked to form cellulose microfibrils. d. Cellulose microfibers further assemble extracellularly to form nanofiber bundles, which intertwine to form a three-dimensional network structure, namely bacterial cellulose.

[0069] It should also be noted that the principle of separation and purification includes the following steps: soaking-rubbing, alkali treatment, washing with water, and drying, specifically: Soaking-rubbing: Utilizing the difference in physical properties between bacterial cellulose and the solid carrier (bacterial cellulose is a gel-like fiber network, while the carrier is a solid particle), the carrier particles are softened by soaking in water, and most of the carrier residue is removed by rubbing. Alkali treatment: NaOH solution at high temperature (70-90℃) can denature bacterial proteins (destroy the secondary structure of proteins and increase solubility), while dissolving a small amount of residual hemicellulose. Bacterial cellulose has good stability to alkali (β-1,4-glycosidic bonds are not easily hydrolyzed by alkali). Water washing: Removes residual NaOH and denatured proteins, restores the pH of bacterial cellulose to neutral, and avoids alkaline residue affecting product performance (such as alkaline residue irritating the skin in pharmaceutical applications). Drying: Vacuum drying can prevent bacterial cellulose from agglomerating due to rapid evaporation of moisture during the drying process, maintain its three-dimensional network structure, and ensure the product's water retention and mechanical strength.

[0070] In summary, this invention optimizes the pretreatment of slurry, substrate preparation, fermentation parameters, and purification process. Its working principle covers nutrient supply, environmental control, biosynthesis, and product purification, providing theoretical support for the industrial application of the process.

[0071] The present invention has been described in detail above. However, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, any modifications or improvements that do not depart from the spirit of the present invention are within the scope of protection of the present invention.

Claims

1. A fermentation process for producing bacterial cellulose by slurry solid state fermentation, characterized in that, The method comprises the following steps: (1) slurry pretreatment: removing impurities and sterilizing the food processing by-product slurry to obtain pretreated slurry; (2) solid-state fermentation substrate preparation: mixing the pretreated slurry with a solid carrier according to a preset mass ratio, adjusting the pH of the substrate to a preset range to obtain a solid-state fermentation substrate; (3) strain inoculation: inoculating the activated culture of the Acetobacter strain into the solid-state fermentation substrate, and controlling the inoculation amount to be a preset proportion; (4) solid-state fermentation: placing the inoculated solid-state fermentation substrate in a fermentation device, controlling the fermentation temperature, humidity and aeration amount, and performing aerobic fermentation to obtain a fermentation product containing bacterial cellulose; (5) bacterial cellulose separation and purification: removing impurities, separating bacteria and chemically treating the fermentation product containing bacterial cellulose to obtain high-purity bacterial cellulose.

2. The process for the production of bacterial cellulose by solid state fermentation of a slurry according to claim 1, characterized in that: In step (1), the slurry pretreatment specifically comprises: a. centrifuging the slurry in a centrifugal device at a speed of 3000-5000 r / min for 10-20 min to remove suspended residues in the slurry; b. taking the supernatant after centrifugation, sterilizing it with high-pressure steam at 115-121 ℃ for 15-30 min, and cooling it to room temperature to obtain the pretreated slurry.

3. The process for the production of bacterial cellulose by solid state fermentation of a slurry according to claim 1, characterized in that: In step (2), the solid carrier is selected from at least one of bran, corn cob powder, soybean meal powder and straw powder; The mass ratio of the pretreated slurry to the solid carrier is 1:0.8-1.5; The pH of the substrate is adjusted to 5.5-6.

5.

4. The process for the production of bacterial cellulose by solid state fermentation of a slurry according to claim 1, characterized in that: In step (3), the Acetobacter strain is selected from at least one of Acetobacter xylinum, Acetobacter pasteurianus and Gluconacetobacter. The strain activation culture uses Hestrin-Schramm medium, and the culture conditions are 30-32 ℃ and 150-200 r / min shaking flask culture until the OD600 value of the bacterial liquid is 0.6-1.

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5. The process for the production of bacterial cellulose by solid state fermentation of a slurry according to claim 1, characterized in that: In step (3), the inoculation amount is 3%-8% (v / w, the volume ratio of the bacterial liquid to the mass of the solid-state fermentation substrate).

6. The process for the production of bacterial cellulose by solid state fermentation of a slurry according to claim 1, characterized in that: In step (4), the fermentation temperature is controlled at 28-32 ℃; The fermentation humidity is controlled at 70%-85%; The aeration amount is controlled at 0.5-1.2 L / (L·h) (based on the volume of the substrate in the fermentation device); The aerobic fermentation time is 7-12 days.

7. The process for the production of bacterial cellulose by solid state fermentation of a slurry according to claim 1, characterized in that: In step (4), the fermentation device is a solid-state fermentation tank with temperature control, humidity control and aeration function. During the fermentation process, the substrate is stirred every 2-4 h, the stirring rate is 50-100 r / min, and the stirring time is 5-10 min each time.

8. The process for the production of bacterial cellulose by solid state fermentation of a slurry according to claim 1, characterized in that: In step (5), the impurity removal specifically comprises: soaking the fermentation product containing bacterial cellulose in deionized water for 2-4 h, manually rubbing off the attached solid carrier residues, and repeating the soaking-rubbing operation 2-3 times; The bacterial cell separation specifically comprises: placing the bacterial cellulose after removing the residues in a 0.3-0.8 mol / L NaOH solution, and incubating it at 70-90 ℃ for 1-3 h to denature and dissolve the bacterial protein; The chemical treatment specifically comprises: repeatedly rinsing the bacterial cellulose after bacterial cell separation with deionized water until the pH is 6.5-7.5, and then drying it in a 60-80 ℃ vacuum drying oven until the weight is constant.

9. The process for the production of bacterial cellulose by solid state fermentation of a slurry according to claim 1, characterized in that: The food processing by-product slurry in step (1) is selected from at least one of bean product processing slurry, starch processing slurry, and grain processing slurry; The bean product processing slurry is wastewater generated in the production process of bean curd and soybean milk, wherein the total sugar content is 30-50 g / L, the protein content is 5-15 g / L, and the pH is 4.5-6.

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10. The process for the production of bacterial cellulose by solid state fermentation of a slurry according to claim 1, characterized in that: The method further comprises a fermentation product detection step: The bacterial cellulose yield is determined by weight (bacterial cellulose dry weight produced per 100 g of dry base solid-state fermentation substrate); The chemical structure of the bacterial cellulose is analyzed by Fourier transform infrared spectrometer (FT-IR); The microstructure of the bacterial cellulose is observed by scanning electron microscope (SEM); The tensile strength of the bacterial cellulose is determined by universal material testing machine; The water holding capacity of the bacterial cellulose is determined by centrifugation.