Directional fermentation process for improving stability of SOD (superoxide dismutase)
By employing a targeted fermentation process involving strain domestication and multi-parameter synergistic regulation, the problem of poor stability in SOD fermentation production has been solved, achieving efficient extraction and low-energy SOD production, with significantly improved product stability and extraction efficiency.
Patent Information
- Application Number
- CN202511213072.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-28
- Publication Date
- 2025-11-21
AI Technical Summary
Existing SOD fermentation production suffers from poor stability, significant extraction losses, and weak process robustness, resulting in unsatisfactory product stability, high production costs, and large batch-to-batch quality fluctuations.
The method employs targeted strain domestication, multi-parameter synergistic regulation, and mild extraction techniques, including steps such as domesticating strains with gradient concentrations of reactive oxygen species inducers, multi-stage fermentation, low-temperature enzyme release, aqueous two-phase extraction, and freeze spray drying, to improve the thermal and storage stability of SOD.
Significantly improves the thermal and storage stability of SOD. After treatment at 60℃, the residual enzyme activity of the enzyme powder is ≥85%, and after storage at 4℃ for 6 months, the enzyme activity retention rate is ≥90%. The extraction efficiency and activity recovery rate are improved, the process energy consumption is reduced, and the product quality consistency is improved.
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Figure CN120989022A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of bioengineering technology, in particular to a directional fermentation process for improving the stability of superoxide dismutase (SOD), and especially to a method for synergistically improving the thermal stability, storage stability and operational stability of SOD through strain breeding, precise fermentation process control and mild extraction technology. BACKGROUND
[0002] Superoxide dismutase (SOD) is a key antioxidant enzyme in living organisms that can effectively scavenge superoxide anion radicals, and has important application value in the pharmaceutical, health product, cosmetic and food industries. The market demand for high-activity and high-stability SOD products continues to grow.
[0003] However, the inherent molecular structure of SOD makes it sensitive to heat, shear force and changes in chemical environment, leading to severe stability challenges in its industrial production. SOD produced by conventional microbial fermentation processes generally exhibits poor thermal stability and storage stability. At temperatures above 50°C or under long-term storage conditions at room temperature, enzyme activity can quickly decrease, which severely limits the application scenarios and shelf life of the product.
[0004] Further analysis of existing processes shows that common strategies aimed at increasing yield often conflict with stability goals. For example, the accumulation of metabolic byproducts and limited dissolved oxygen conditions during high-density fermentation can exacerbate the oxidative stress environment within the cell, causing damage to SOD molecules. When using strong promoters for high-efficiency induction expression, the rapid protein synthesis rate can cause some enzyme molecules to fold incompletely or incorrectly, affecting their structural stability. In addition, the high-intensity mechanical stirring required to maintain high-density bacterial bodies in the fermenter can also destroy the natural conformation of enzyme proteins.
[0005] In the downstream processing stage after fermentation, stability problems are more prominent. To extract intracellular SOD, cell disruption must be performed. Traditional methods such as ultrasonic disruption or high-pressure homogenization, with their intense mechanical action and the generation of local high temperatures, can easily cause SOD denaturation and inactivation. Subsequent purification steps, such as high-concentration salting-out (ammonium sulfate fractionation precipitation) or organic solvent precipitation, can easily cause unfavorable changes in the conformation of SOD under high ionic strength or organic phase environments, accompanied by significant loss of activity. These factors together result in a SOD activity recovery rate of generally less than 60% throughout the production process.
[0006] Current methods to improve SOD stability focus on post-fermentation modifications, such as PEG chemical modification or formulation optimization with protective agents. However, these methods increase process complexity and cost, introduce exogenous substances that may raise safety concerns, and do not fundamentally address the structural fragility of SOD molecules.
[0007] In summary, the core difficulty of existing SOD production processes lies in the fact that operations aimed at high yields often compromise the intrinsic stability of enzyme molecules, and the harsh conditions of downstream extraction processes further exacerbate activity loss. The end result is that product stability is difficult to meet requirements, production costs are high, and batch-to-batch quality fluctuations are large. Therefore, developing a new integrated process that can improve the intrinsic stability of SOD from the fermentation source and throughout the mild and efficient extraction process is crucial for promoting the large-scale and economic application of SOD. SUMMARY
[0008] The purpose of the present application is to overcome the defects of poor stability, large extraction loss, and weak process robustness in existing SOD fermentation production, and to provide a directional fermentation process for improving SOD stability. This process significantly improves the thermal stability, storage stability, and operational stability of SOD through strain directional domestication, multi-parameter coordinated regulation during fermentation, and integration of mild extraction technology.
[0009] To achieve the above-mentioned purpose, the present application provides the following technical solution: a directional fermentation process for improving SOD stability, comprising the following steps: S1: Strain selection and activation: Taking SOD-producing strains as the object, gradient concentrations of 0.1-1.0 mM active oxygen inducers and temperatures 2-5°C higher than the optimal growth temperature are used for plate subculture domestication for ≥5 rounds to select resistant strains; seed culture medium is inoculated and cultured to OD600=6.0±0.5 to obtain seed liquid; S2: Multi-stage fermentation: the seed liquid obtained in S1 is inoculated into the fermentation system and the following operations are performed: I. Growth phase: 30±0.5°C, DO 30±5%, residual sugar ≤5 g / L, when the exponential feeding liquid is added to OD600=40±5; II. Induction phase: temperature is reduced to 25±0.5°C, DO 50±5%, inducers and glycerol (maintaining 1-3% w / v) are added at a constant flow rate to control residual sugar at 0.5-2.0 g / L; III. Stationary phase: maintain 25±0.5°C, DO 40±5%, add glycerol-containing nutrient solution until fermentation is complete; S3: Enzyme extraction: the fermentation broth is transferred into an enzyme release system at 10±2℃, 0.5-1.5% w / v lysozyme and 5-10 mM EDTA are added, and the mixture is treated for 60±15 min; the treated broth is pumped into a two-water-phase extraction system, PEG4000 and ammonium sulfate are added to a final concentration of 12-16% w / w and 10-14% w / w, respectively, and the upper phase is separated at 4±2℃; S4: Post-treatment: the upper phase obtained in S3 is pumped into a membrane separation system using a freeze spray drying tower, ultrafiltration concentration and buffer replacement are performed on the upper phase using a 10 kDa ultrafiltration membrane, and the buffer is replaced with a phosphate buffer containing 5% w / v trehalose, pH 7.4; after adding 0.5-1.0% w / v gelatin, the mixture is transferred into a drying system, and freeze-spray drying is performed at a cold trap <-50℃ and a vacuum of 10-30 Pa.
[0010] As a further scheme of the present application: the active oxygen inducer is menadione; the inducer added in the induction stage is 0.1-0.5 mM IPTG or 0.05-0.2 mM Cu 2+ / Zn 2+ .
[0011] As a further scheme of the present application: the lysozyme concentration in step S3 is 1.0% w / v, and the two-water-phase system is 14% w / w PEG4000 and 12% w / w ammonium sulfate.
[0012] As a further scheme of the present application: the SOD enzyme powder has an enzyme activity retention rate of ≥85% after being treated at 60℃ for 30 min, and an enzyme activity retention rate of ≥90% after being stored in the dark at 4℃ for 6 months.
[0013] As a further scheme of the present application: the freeze spray drying tower comprises a tower body, a top plate is arranged at the top end of the tower body, a discharge port is fixedly connected to the bottom end of the tower body, a valve is mounted on the outer wall of the discharge port, a vacuum pipe is fixedly connected to one side of the tower body, a vacuum pump is mounted on the outer wall of the vacuum pipe, an inlet pipe is fixedly connected to the top end of the top plate, an atomizing nozzle is mounted at the bottom end of the inlet pipe, a heating jacket is sleeved and mounted on the outer wall of the tower body, the inner wall of the tower body is scraped by a scraping mechanism, and the top plate is automatically opened and closed by a connecting mechanism.
[0014] As a further further scheme of the present application: the scraping mechanism comprises a movable groove, the movable groove is symmetrically opened at the top end of the top plate, the inner wall of the movable groove is slidably connected with a movable frame, the bottom end of the movable frame is fixedly connected with a movable ring, the inner wall of the movable ring is rotatably connected with a rotating cylinder, the bottom end of the rotating cylinder is fixedly connected with a scraping ring, the bottom end of the scraping ring is symmetrically fixedly connected with a scraping rod, the inner wall of the movable groove is fixedly installed with a first sealing ring, the top end of the movable frame is installed with a first motor, the output end of the first motor is connected with a connecting shaft, the bottom end of the connecting shaft is fixedly connected with a spur gear, the spur gear is in contact with the rotating cylinder, one side of the tower body is provided with a supporting seat, the outer wall of the supporting seat is provided with a displacement groove, the inner wall of the displacement groove is slidably connected with a displacement plate, the displacement plate is fixedly connected with the movable frame, the top end of the supporting seat is installed with a second motor, the output end of the second motor is connected with a first threaded rod, and the first threaded rod penetrates the displacement plate.
[0015] As a further further scheme of the present application: the connecting mechanism comprises a first connecting seat, the first connecting seat is symmetrically fixedly connected on both sides of the top plate, the outer wall of the first connecting seat is provided with a slot, the outer wall top end of the tower body is symmetrically fixedly connected with a second connecting seat, the top end of the second connecting seat is fixedly connected with a plug rod, the outer wall of the plug rod is provided with a fixed groove, the top end of the rotating cylinder is fixedly connected with a clamping block, the bottom end of the top plate is rotatably connected with a rotating ring, the outer wall of the rotating ring in the interior of the top plate is rotatably connected with a bevel gear, one end of the bevel gear is fixedly connected with a second threaded rod, the interior of the top plate is slidably connected with a fixed block extending into the inner cavity of the slot, the second threaded rod penetrates the fixed block, the outer wall of the fixed block is rotatably connected with a connecting rod, one end of the connecting rod is rotatably connected with a positioning block, the positioning block is slidably connected in the interior of the top plate, the top end of the tower body is installed with a second sealing ring, and the inner wall of the movable frame is symmetrically provided with a positioning groove.
[0016] As a further further scheme of the present application: the inner wall of the displacement groove is matched with the outer wall of the displacement plate, the outer wall of the displacement plate is provided with a first threaded hole, the first threaded hole is matched with the first threaded rod, the inner wall of the movable groove is matched with the outer wall of the movable frame, and the outer wall of the rotating cylinder is provided with a gear slot, the gear slot is engaged with the spur gear.
[0017] As a further further scheme of the present application: the outer wall of the plug rod is matched with the inner wall of the slot, and the inner wall of the fixed groove is matched with the outer wall of the fixed block.
[0018] As a further scheme of the present application: the bottom end of the rotating ring is provided with a clamping groove matched with the clamping block, the outer wall of the rotating ring is provided with a gear, the gear is engaged with the bevel gear, the outer wall of the fixed block is provided with a second threaded hole matched with the second threaded rod, and the outer wall of the positioning block is matched with the inner wall of the positioning groove.
[0019] Compared with the prior art, the present application has the following advantages: 1. The process can significantly improve the intrinsic stability of SOD: high stability strains are obtained through strain directional tolerance domestication screening; low temperature (25℃) induction combined with heat shock protein inducer (glycerol) feeding is used in the induction stage, which effectively protects the correct folding of SOD and enhances its heat resistance; double water phase extraction (PEG / ammonium sulfate) and freeze-spray drying process maximally maintain the natural conformation of the enzyme. The residual enzyme activity of the obtained SOD enzyme powder is ≥85% after 60℃ treatment for 30 min, and the enzyme activity retention rate is ≥90% after 4℃ storage for 6 months, which is much higher than that of the conventional process (usually <50%); 2. The process can improve the extraction efficiency and activity recovery rate: mild enzyme release (low temperature lysozyme treatment) reduces mechanical damage; double water phase extraction has high selectivity and mild operating conditions (low temperature, normal pressure), which effectively separates SOD from impure proteins and cell fragments; combined with ultrafiltration buffer replacement, trehalose protection and gelatin addition, the whole process has an activity recovery rate of ≥75%, which is significantly improved compared with the traditional centrifugation-salting-out process (≤60%); 3. The process has strong robustness and low energy consumption: multi-stage precise feeding strategy (exponential feeding in the growth stage, constant low sugar feeding in the induction stage, and nutrient maintenance in the stable stage) effectively balances the growth of the bacteria, the expression of SOD and the stability, reduces the accumulation of metabolic by-products and oxidative stress; the whole process (fermentation, extraction, and drying) is carried out at normal or low temperature, avoiding the high energy consumption links such as high temperature sterilization and high temperature drying in the traditional process, and the comprehensive energy consumption is reduced by about 20-25% compared with the conventional fermentation-freeze drying process; 4. The product quality is uniform and stable: based on the precise control of process parameters (temperature, DO, pH, and feeding rate) and the reasonable use of protective agents, the difference CV value of the stability (thermal stability and storage stability) of different batches of products is ≤5%, which meets the strict requirements of high-end applications for quality consistency; 5. The present invention also incorporates a freeze spray drying tower with process improvements. The freeze spray drying tower is equipped with a scraping mechanism. The second motor drives the movable frame to move, and the movable frame moves, causing the scraping ring to move. The scraping ring moves downward to scrape the inner wall of the tower body, scraping off the material adhering to the inner wall of the tower body. At the same time, when the scraping ring moves to the bottom of the tower body, the first motor drives the spur gear to rotate. The rotation of the spur gear drives the scraping ring and the scraping rod to rotate synchronously. The rotation of the scraping rod scrapes the bottom of the tower body, scraping off the adhering material and sliding it into the discharge port, which facilitates the scraping operation of the material adhering to the tower body. 6. The freeze spray drying tower is equipped with a connecting mechanism. The movable ring moves upward to perform a reset operation until the locking block engages with the rotating ring. At this time, the rotating cylinder rotates, and the rotation of the rotating cylinder drives the rotating ring to rotate through the locking block. The rotation of the rotating ring causes the fixed block to move out of the fixing groove, thus canceling the fixation between the top plate and the tower body. At the same time, the displacement of the fixed block drives the positioning block to move through the connecting rod. The positioning block moves into the positioning groove, thereby fixing the movable frame and the top plate. The displacement of the movable frame causes the top plate to move upward, thus automatically opening the tower body. Attached Figure Description
[0020] Figure 1 This is a schematic diagram of the structure of the freeze spray drying tower described in this invention; Figure 2 This is a cross-sectional view of the tower body of the freeze spray drying tower described in this invention; Figure 3 This is a schematic diagram of the movable frame of the freeze spray drying tower described in this invention; Figure 4 The freeze spray drying tower of the present invention Figure 3 Enlarged view of point A in the middle; Figure 5 This is a cross-sectional view of the rotating ring of the freeze spray drying tower described in this invention; Figure 6 The freeze spray drying tower of the present invention Figure 5 Enlarged view of point B in the middle; Figure 7 This is a cross-sectional view of the support base of the freeze spray drying tower according to the present invention; Figure 8 This is a schematic diagram of the installation of the top plate of the freeze spray drying tower described in this invention; Figure 9 The freeze spray drying tower of the present invention Figure 8 Enlarged view of point C in the middle; Figure 10 This is a schematic diagram of the installation of the positioning block of the freeze spray drying tower described in this invention.
[0021] In the figure: 1, tower body; 2, top plate; 3, discharge port; 4, valve; 5, vacuum pipe; 6, vacuum pump; 7, feeding pipe; 8, scraping mechanism; 801, movable groove; 802, movable frame; 803, movable ring; 804, rotating cylinder; 805, scraping ring; 806, scraping rod; 807, first motor; 808, connecting shaft; 809, straight gear; 810, support seat; 811, displacement groove; 812, displacement plate; 813, second motor; 814, first threaded rod; 815, first sealing ring; 9, connecting mechanism; 901, first connecting seat; 902, insertion slot; 903, second connecting seat; 904, insertion rod; 905, fixed groove; 906, clamping block; 907, rotating ring; 908, bevel gear; 909, second threaded rod; 910, fixed block; 911, connecting rod; 912, positioning block; 913, positioning groove; 914, second sealing ring; 10, atomizing nozzle; 11, heating jacket. DETAILED DESCRIPTION
[0022] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.
[0023] In the description of the present application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application. In addition, the terms "first", "second", "third" are only for descriptive purposes and cannot be understood as indicating or implying relative importance. In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connecting", "connecting", "setting" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances. The embodiments of the present application will be described below according to the overall structure of the present application.
[0024] Example 1: A directional fermentation process for improving the stability of SOD Step one, selection and activation of stability-enhanced strain: the SOD-Stab-01 strain was obtained by screening a strain overexpressing human Cu / Zn-SOD gene, BL21 (DE3) as the starting strain, and 6 rounds of passage acclimation on LB plates containing 0.1-1.0 mM menadione and 34°C (optimum 37°C). E. coli BL21 (DE3) as the starting strain, and 6 rounds of passage acclimation on LB plates containing 0.1-1.0 mM menadione and 34°C (optimum 37°C).
[0025] Step two, multi-stage precise feeding fermentation: the seed liquid was inoculated into a 5L fermenter (basic medium: corn syrup dry powder 20g / L, (NH4)2SO4 8g / L, KH2PO4 3g / L, MgSO4·7H2O 1.5g / L, trace element solution 1.5mL / L, glucose 18g / L, pH 6.5) at an inoculation amount of 8%. The trace element solution used in the fermentation medium is composed of the following: FeCl3·6H2O 5 g / L, ZnSO4·7H2O 2 g / L, CuSO4·5H2O 1 g / L, MnSO4·H2O 0.5 g / L, dissolved in 0.1 M HCl solution. When used, add 1.0-2.0 mL / L to the medium.
[0026] I, growth phase (0-12h): 30°C, DO 30%, pH 6.5 (ammonia water adjustment), stirring 400 rpm, aeration 1.2 vvm. When the residual sugar decreases to 5g / L, exponentially add high-nitrogen-carbon ratio feeding liquid (glucose 400g / L, yeast extract 60g / L, (NH4)2SO4 30g / L), μ=0.15 h -1 , to OD600=42.
[0027] II, induction / stability enhancement phase (12-36h): reduce the temperature to 25°C, increase the DO to 50%, adjust the pH to 6.8. Add IPTG (0.3mM final concentration) and glycerol concentrate (maintain a final concentration of 2% w / v). Switch to constant-speed feeding liquid, maintain residual sugar ~1.5g / L.
[0028] III, maintenance phase in the stability period (36-54h): maintain 25°C, DO 40%, pH 6.8. Stop feeding sugar, add maintenance liquid (yeast extract 20g / L, (NH4)2SO4 10g / L, glycerol 2% w / v), harvest at 54h (SOD specific activity reaches peak value).
[0029] Step three, mild enzymatic release and aqueous two-phase extraction: the fermentation broth was cooled to 10℃. Lysozyme was added to 1.0% (w / v) and EDTA to 8 mM, and the solution was treated with mild stirring (100 rpm) for 60 min. PEG4000 and ammonium sulfate were added to a final concentration of 14% (w / w) and 12% (w / w), respectively, at pH 7.0. The mixture was stirred at 4℃ for 10 min, and the phases were allowed to separate for 30 min. The PEG phase containing SOD was collected.
[0030] Step four, membrane separation concentration and freeze-spray drying: the crude enzyme solution was concentrated 10-fold by ultrafiltration through a 10 kDa membrane at 4℃ and 0.2 MPa, and replaced with 10 mM sodium phosphate buffer (pH 7.4) containing 5% trehalose. Gelatin was added to the concentrated solution to a concentration of 0.8% (w / v). After freezing and solidification at -40℃, the solution was dried in a freeze-spray drying tower (cold trap -55℃, vacuum 20 Pa, heating plate 38℃) to obtain SOD enzyme powder.
[0031] The specific activity of the SOD enzyme powder was 4500 U / mg protein. The residual enzyme activity was 88.5% after treatment at 60℃ for 30 min. The enzyme activity retention rate was 93.2% after storage at 4℃ for 6 months. The total activity recovery rate was 78.4%.
[0032] See Figures 1 to 10 In an embodiment of the present application, a targeted fermentation process for improving the stability of SOD includes the following steps: S1: strain selection and activation: using a SOD-producing strain as the object, the strain was subcultured on a plate containing a gradient concentration of 0.1-1.0 mM active oxygen inducer and a temperature 2-5℃ higher than the optimal growth temperature for ≥5 generations to screen for a resistant strain; the seed medium was inoculated and cultured until the OD600=6.0±0.5 to obtain a seed solution; S2: multi-stage fermentation: the seed solution obtained in S1 was inoculated into a fermentation system and the following operations were performed: I. growth phase: 30±0.5℃, DO 30±5%, when the residual sugar was ≤5 g / L, the exponential feeding solution was added until the OD600=40±5; II. induction phase: the temperature was reduced to 25±0.5℃, DO 50±5%, and the inducer and glycerol (maintaining 1-3% w / v) were added at a constant flow rate to control the residual sugar to 0.5-2.0 g / L; III. stationary phase: the temperature was maintained at 25±0.5℃, DO 40±5%, and the nutrient solution containing glycerol was added until the fermentation was complete; S3: Enzyme extraction: the fermentation broth was transferred into the enzyme release system at 10±2℃, 0.5-1.5% w / v lysozyme and 5-10 mM EDTA were added and treated for 60±15 min; the treated broth was pumped into the aqueous two-phase extraction system, PEG4000 and ammonium sulfate were added to the final concentration of 12-16% w / w and 10-14% w / w, respectively, and the upper phase was separated at 4±2℃; S4: Post-treatment: the upper phase obtained in S3 was pumped into the membrane separation system using a freeze spray drying tower, ultrafiltration concentration and buffer replacement were performed using a 10 kDa ultrafiltration membrane, and the buffer was replaced with a phosphate buffer containing 5% w / v trehalose, pH 7.4, 0.5-1.0% w / v gelatin was added, and then transferred into the drying system, and freeze-spray dried under the conditions of a cold trap <-50℃, vacuum 10-30 Pa.
[0033] The active oxygen inducer was menadione; the inducer added during the induction stage was 0.1-0.5 mM IPTG or 0.05-0.2 mM Cu 2+ / Zn 2+ The lysozyme concentration in step S3 was 1.0% w / v, the aqueous two-phase system was 14% w / w PEG4000 and 12% w / w ammonium sulfate; the SOD enzyme powder had an enzyme activity retention rate of ≥85% after being treated at 60℃ for 30 min, and an enzyme activity retention rate of ≥90% after being stored at 4℃ in the dark for 6 months.
[0034] In this embodiment: the intrinsic stability of SOD was significantly improved: a high-stability strain was obtained through strain targeted resistance domestication screening; low temperature (25℃) induction combined with heat shock protein inducer (glycerol) feeding was used in the induction stage, which effectively protected the correct folding of SOD and enhanced its heat resistance; the aqueous two-phase extraction (PEG / ammonium sulfate) and freeze-spray drying process maximized the maintenance of the natural conformation of the enzyme. The residual enzyme activity of the obtained SOD enzyme powder was ≥85% after being treated at 60℃ for 30 min, and the enzyme activity retention rate was ≥90% after being stored at 4℃ for 6 months, which was much higher than that of the conventional process (usually <50%); The extraction efficiency and activity recovery rate were improved: mild enzyme release (lysozyme treatment at low temperature) reduced mechanical damage; the aqueous two-phase extraction was selective and the operating conditions were mild (low temperature, normal pressure), which effectively separated SOD from impurities and cell debris; combined with the addition of trehalose in the buffer replacement and the addition of gelatin, the overall activity recovery rate was ≥75%, which was significantly higher than that of the traditional centrifugation-salting-out process (≤60%); Strong process robustness, reduced energy consumption: multi-stage precise feeding strategy (exponential feeding in growth phase, constant low sugar feeding in induction phase, nutrient maintenance in stationary phase) effectively balances cell growth, SOD expression and stability, reduces accumulation of metabolic by-products and oxidative stress; the entire process (fermentation, extraction, drying) is carried out at room temperature or low temperature, avoiding high energy consumption links such as high temperature sterilization and high temperature drying in traditional process, the comprehensive energy consumption is reduced by about 20-25% compared with conventional fermentation-lyophilization process; Uniform and stable product quality: precise control based on process parameters (temperature, DO, pH, feeding rate) and reasonable use of protective agents, the difference CV value of stability (thermal stability, storage stability) of different batches of products is ≤5%, meeting the stringent requirements of high-end applications for quality consistency.
[0035] Please refer to Figures 1 to 10 , the frozen spray drying tower, including the tower body 1, the top end of the tower body 1 is provided with a top plate 2, the bottom end of the tower body 1 is fixedly connected with a discharge port 3, the outer wall of the discharge port 3 is installed with a valve 4, one side of the tower body 1 is fixedly connected with a vacuum pipe 5, the outer wall of the vacuum pipe 5 is installed with a vacuum pump 6, the top end of the top plate 2 is fixedly connected with a feeding pipe 7, the bottom end of the feeding pipe 7 is installed with an atomizing nozzle 10, the outer wall of the tower body 1 is sleeved with a heating jacket 11, the inner wall of the tower body 1 is scraped by a scraping mechanism 8, and the top plate 2 is automatically opened and closed by a connecting mechanism 9.
[0036] In this embodiment: open the top plate 2, directly inject low-temperature medium (such as liquid nitrogen) into the tower body 1, cool the tower body 1, the feeding pipe 7 transports the material to the atomizing nozzle 10, the atomizing nozzle 10 atomizes the material, the atomized particles are rapidly cooled to form particles, until the low-temperature medium absorbs heat and vaporizes, the vacuum pump 6 is started, the vacuum pipe 5 extracts the gas in the tower body 1, a vacuum environment is formed in the tower body 1, the tower body 1 is heated by the heating jacket 11, and the ice crystals are directly sublimated into water vapor under the vacuum environment, realizing dehydration.
[0037] Please refer to Figures 1 to 7The scraping mechanism 8 comprises a movable groove 801 symmetrically arranged at the top end of the top plate 2, the inner wall of the movable groove 801 being slidably connected with a movable frame 802, the bottom end of the movable frame 802 being fixedly connected with a movable ring 803, the inner wall of the movable ring 803 being rotatably connected with a rotating cylinder 804, the bottom end of the rotating cylinder 804 being fixedly connected with a scraping ring 805, the bottom end of the scraping ring 805 being symmetrically fixedly connected with scraping rods 806, the inner wall of the movable groove 801 being fixedly installed with a first sealing ring 815, the top end of the movable frame 802 being installed with a first motor 807, the output end of the first motor 807 being connected with a connecting shaft 808, the bottom end of the connecting shaft 808 being fixedly connected with a spur gear 809, the spur gear 809 being in contact with the rotating cylinder 804, one side of the tower body 1 being provided with a supporting seat 810, the outer wall of the supporting seat 810 being provided with a displacement groove 811, the inner wall of the displacement groove 811 being slidably connected with a displacement plate 812, the displacement plate 812 being fixedly connected with the movable frame 802, the top end of the supporting seat 810 being installed with a second motor 813, the output end of the second motor 813 being connected with a first threaded rod 814, the first threaded rod 814 penetrating through the displacement plate 812.
[0038] In the embodiment, when the material is discharged, the discharge port 3 is opened by the valve 4, the material is discharged through the discharge port 3, and the second motor 813 can be started to drive the first threaded rod 814 to rotate, the first threaded rod 814 drives the displacement plate 812 to slide in the displacement groove 811, the displacement plate 812 drives the movable frame 802 to displace, the movable frame 802 slides in the movable groove 801, the movable frame 802 drives the movable ring 803 to displace, the movable ring 803 drives the rotating cylinder 804 to displace, the rotating cylinder 804 drives the scraping ring 805 to displace, the scraping ring 805 moves downward to scrape the inner wall of the tower body 1, and the material adhered to the inner wall of the tower body 1 is scraped off, and when the scraping ring 805 moves to the bottom of the tower body 1, the first motor 807 is started, the first motor 807 drives the connecting shaft 808 to rotate, the connecting shaft 808 drives the spur gear 809 to rotate, the spur gear 809 drives the rotating cylinder 804 to rotate, the rotating cylinder 804 drives the scraping ring 805 and the scraping rods 806 to synchronously rotate, and the scraping rods 806 rotate to scrape the bottom of the tower body 1, so that the adhered material is scraped off and slides into the discharge port 3, thereby facilitating the scraping operation of the material adhered to the inner wall of the tower body 1.
[0039] Please refer to Figures 5 to 10The connecting mechanism 9 comprises a first connecting seat 901 fixedly connected to the two sides of the top plate 2, a slot 902 is formed in the outer wall of the first connecting seat 901, a second connecting seat 903 is fixedly connected to the top end of the outer wall of the tower body 1, a plug rod 904 is fixedly connected to the top end of the second connecting seat 903, a fixing groove 905 is formed in the outer wall of the plug rod 904, a clamping block 906 is fixedly connected to the top end of the rotating cylinder 804, a rotating ring 907 is rotatably connected to the bottom end of the top plate 2, a bevel gear 908 is rotatably connected to the inner wall of the rotating ring 907, one end of the bevel gear 908 is fixedly connected to a second threaded rod 909, a fixing block 910 is slidably connected to the inner part of the top plate 2 and extends into the inner cavity of the slot 902, the second threaded rod 909 penetrates through the fixing block 910, a connecting rod 911 is rotatably connected to the outer wall of the fixing block 910, one end of the connecting rod 911 is rotatably connected to a positioning block 912, the positioning block 912 is slidably connected to the inner part of the top plate 2, a second sealing ring 914 is mounted on the top end of the tower body 1, and a positioning groove 913 is symmetrically formed in the inner wall of the movable frame 802.
[0040] In the embodiment, when the tower body 1 is in the closed state, the plug rod 904 is inserted into the slot 902, the fixing block 910 moves into the fixing groove 905, the plug rod 904 is fixed in the slot 902, and the fixing operation of the top plate 2 is completed. When the tower body 1 is opened, the movable ring 803 is upwardly moved for resetting, the movable ring 803 drives the rotating cylinder 804 to move upwardly until the clamping block 906 is clamped with the rotating ring 907, at this time, the rotating cylinder 804 is driven to rotate by the first motor 807, the rotating cylinder 804 drives the rotating ring 907 to rotate through the clamping block 906, the rotating ring 907 drives the bevel gear 908 to rotate, the bevel gear 908 drives the second threaded rod 909 to rotate, the second threaded rod 909 drives the fixing block 910 to displace, the fixing block 910 is displaced out of the fixing groove 905, and the fixing between the top plate 2 and the tower body 1 is cancelled; meanwhile, the fixing block 910 drives the positioning block 912 to displace through the connecting rod 911, the positioning block 912 is inserted into the positioning groove 913, and the fixing operation between the movable frame 802 and the top plate 2 is completed, at this time, the second motor 813 can be started to drive the movable frame 802 to displace, the movable frame 802 drives the top plate 2 to displace upwardly, and the automatic opening operation of the tower body 1 is completed. When the tower body 1 is closed, the movable frame 802 is downwardly moved so that the top plate 2 is pressed against the top end of the tower body 1, then the first motor 807 is started to drive the fixing block 910 to move into the fixing groove 905, the fixing operation between the tower body 1 and the top plate 2 is completed, and meanwhile, the positioning block 912 is moved out of the positioning groove 913, the separation operation between the movable frame 802 and the top plate 2 is completed.
[0041] Please pay attention toFigures 1 to 7 The inner wall of the displacement groove 811 is attached to the outer wall of the displacement plate 812, the outer wall of the displacement plate 812 is provided with a first threaded hole, the first threaded hole is matched with the first threaded rod 814, the inner wall of the movable groove 801 is attached to the outer wall of the movable frame 802, the outer wall of the rotating cylinder 804 is provided with a gear slot, the gear slot is engaged with the spur gear 809.
[0042] In this embodiment: the second motor 813 operates to drive the first threaded rod 814 to rotate, the first threaded rod 814 rotates to drive the displacement plate 812 to slide in the displacement groove 811, the displacement plate 812 is displaced to drive the movable frame 802 to displace, the movable frame 802 slides in the movable groove 801; the first motor 807 operates to drive the connecting shaft 808 to rotate, the connecting shaft 808 rotates to drive the spur gear 809 to rotate, the spur gear 809 rotates to drive the rotating cylinder 804 to rotate, the rotating cylinder 804 rotates to drive the scraping ring 805 and the scraping rod 806 to synchronously rotate.
[0043] Please refer to Figures 5 to 10 The outer wall of the insertion rod 904 is attached to the inner wall of the insertion groove 902, and the inner wall of the fixed groove 905 is attached to the outer wall of the fixed block 910.
[0044] In this embodiment: the insertion rod 904 is inserted into the insertion groove 902, the fixed block 910 is moved into the fixed groove 905, and the insertion rod 904 is fixed in the insertion groove 902, thereby completing the fixing operation of the top plate 2.
[0045] Please refer to Figures 5 to 10 The bottom end of the rotating ring 907 is provided with a clamping groove matched with the clamping block 906, the outer wall of the rotating ring 907 is provided with a gear, the gear is engaged with the bevel gear 908, the outer wall of the fixed block 910 is provided with a second threaded hole, the second threaded hole is matched with the second threaded rod 909, and the outer wall of the positioning block 912 is attached to the inner wall of the positioning groove 913.
[0046] In this embodiment: the clamping block 906 is clamped with the rotating ring 907, the rotating cylinder 804 rotates to drive the rotating ring 907 to rotate through the clamping block 906, the rotating ring 907 rotates to drive the bevel gear 908 to rotate, the bevel gear 908 rotates to drive the second threaded rod 909 to rotate, the second threaded rod 909 rotates to drive the fixed block 910 to displace, the fixed block 910 displaces out of the fixed groove 905 to cancel the fixing between the top plate 2 and the tower body 1; at the same time, the fixed block 910 displaces to drive the positioning block 912 to displace through the connecting rod 911, the positioning block 912 displaces to insert into the positioning groove 913, thereby performing the fixing operation between the movable frame 802 and the top plate 2. Embodiment 2: A directional fermentation process for improving the stability of SOD
[0047] Step one: Acclimation of strain SOD-Stab-01, seed culture same as example 1.
[0048] Step two: Base medium same as example 1.
[0049] I, Growth phase: same as example 1, to OD600=38.
[0050] II, Induction / stabilization phase: temperature 25°C, DO 50%, pH 6.8. Fed-batch CuSO4 / ZnSO4 (final concentration Cu 2+ 0.1 mM, Zn 2+ 0.15 mM) and glycerol (maintained at 3% w / v). Constant feed rate to maintain residual sugar ~1.0 g / L.
[0051] III, Stabilization phase: same as example 1, harvest time 52h.
[0052] Step three: Lysozyme 1.2% (w / v), EDTA 10 mM, 10°C for 50 min. Aqueous two-phase system: PEG4000 15% (w / w) / ammonium sulfate 11% (w / w), pH 7.0, 4°C phase separation.
[0053] Step four: Ultrafiltration same as example 1. Protective agent: gelatin 1.0% (w / v). Freeze spray drying: cold trap -52°C, vacuum 25 Pa, hot plate 40°C.
[0054] The rest same as example 1, not repeated here.
[0055] SOD enzyme powder specific activity 4200 U / mg. Residual activity after 60°C / 30 min 86.2%. Retention rate at 4°C / 6 months 91.5%. Recovery rate 76.1%. Example 3 A directed fermentation process for improving SOD stability
[0056] Step one: Acclimation of strain same as example 1. Seed culture same as example 1.
[0057] Step two: Base medium: corn steep liquor dry powder 25 g / L, the rest same as example 1.
[0058] I, Growth phase: same as example 1, to OD600=45.
[0059] II, Induction / stabilization phase: temperature 25°C, DO 45%, pH 6.9. Fed-batch IPTG (0.5 mM final concentration) and glycerol (maintained at 1.5% w / v). Constant feed rate to maintain residual sugar ~0.8 g / L.
[0060] III, Stabilization phase: same as example 1, harvest time 50h.
[0061] Step three: Lysozyme 0.8% (w / v), EDTA 6mM, 10°C for 70 min. Aqueous two-phase system: PEG4000 13% (w / w) / ammonium sulfate 13% (w / w), pH 7.2, 4°C phase separation.
[0062] Step four: Ultrafiltration as in Example 1. Protective agent: gelatin 0.6% (w / v). Freeze spray drying: cold trap -58°C, vacuum 15 Pa, heating plate 36°C.
[0063] The rest is the same as Example 1, which will not be repeated here.
[0064] SOD enzyme powder specific activity 4800 U / mg. 60°C / 30min residual activity 90.1%. 4°C / 6 months retention rate 94.8%. Recovery rate 80.2%.
[0065] Comparative Example 1 Conventional fermentation and freeze-drying Step two replacement: Single temperature fermentation at 30°C, no glycerol feeding, DO maintained at 30% during induction phase, residual sugar controlled at ~5g / L. Harvest time 40h.
[0066] Step three replacement: Centrifugal collection of bacterial cells, ultrasonic crushing (ice bath, power 400W, work 5s, intermittent 5s, total time extended to 15min or until the cell crushing rate under microscope >90%), ammonium sulfate fractionation precipitation (40%-80% saturation).
[0067] Step four replacement: After dialysis, freeze at -80°C, then transfer to a conventional freeze dryer for drying.
[0068] The rest is the same as Example 1, which will not be repeated here.
[0069] Results: Specific activity 3800 U / mg. 60°C / 30min residual activity 42.3%. 4°C / 6 months retention rate 68.7%. Recovery rate 52.0%.
[0070] Comparative Example 2 No aqueous two-phase extraction Step three replacement: After lysozyme treatment, direct centrifugation (12000 g, 20 min, 4°C) to take the supernatant as the crude enzyme solution.
[0071] Step four: Ultrafiltration concentration (with trehalose buffer) and freeze spray drying as in Example 1.
[0072] Results: Specific activity 4000 U / mg. 60°C / 30min residual activity 75.6%. 4°C / 6 months retention rate 85.1%. Recovery rate 60.8% (due to high impurities, low ultrafiltration flux and increased loss).
[0073] Performance test The SOD enzyme powders obtained in Examples 1-3 and Comparative Examples 1-2 were subjected to key performance index tests: 1. Specific activity (U / mg protein): enzyme activity was determined according to the modified Marklund method, and protein was determined by the Bradford method.
[0074] 2. Thermal stability: enzyme solution (1 mg / mL in pH 7.4 PBS) was treated in a 60℃ water bath for 30 min, immediately ice-bathed, and residual enzyme activity (%) was determined.
[0075] 3. Storage stability: enzyme powder was sealed and stored in the dark at 4℃, and enzyme activity was determined every month, and enzyme activity retention rate (%) after 6 months was calculated.
[0076] 4. Total activity recovery rate (%): total enzyme activity of the final product / total enzyme activity of the fermentation broth) x 100%.
[0077] The test results are shown in Table 1 below: Table 1 Comparison of key performance indexes of SOD enzyme powders
[0078] In summary, the present application provides a directed fermentation process for improving the stability of SOD. Through the combination of core technologies such as stability-enhanced strain breeding, multi-stage precise feeding fermentation (low-temperature induction combined with heat shock protein induction), mild enzyme release and aqueous two-phase extraction, and membrane separation combined with freeze-spray drying, the key problems of poor stability, high extraction loss, and high energy consumption in SOD production are successfully solved. This process significantly improves the intrinsic thermal stability (60℃ / 30min residual activity≥85%) and long-term storage stability (4℃ / 6 months retention rate≥90%) of SOD, greatly improves the activity recovery rate (≥75%), and reduces the comprehensive energy consumption (about 20-25%). The product stability is uniform and reliable, providing an efficient, economical, and reliable technical solution for the production of high-value SOD enzyme preparations.
[0079] The above description is only a preferred embodiment of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can make equivalent substitutions or changes to the technical solutions and inventive concepts of the present application within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.
Claims
1. A directional fermentation process for improving SOD stability, characterized in that, Includes the following steps: S1: Strain selection and activation: Using SOD-producing strains as the target, the strains were subjected to ≥5 rounds of upbringing on plates containing gradient concentrations of 0.1-1.0 mM reactive oxygen species inducers and at temperatures 2-5°C above the optimum growth temperature. Resistant strains were then screened. Seed culture was obtained by inoculating the culture medium with seed culture until OD600 = 6.0 ± 0.
5. S2: Multi-stage fermentation: The seed culture obtained in S1 is introduced into the fermentation system for the following operations: I. Growth stage: At 30±0.5℃, DO 30±5%, and residual sugar ≤5 g / L, add feed solution in an exponential flow until OD600=40±5; II. Induction phase: Cool to 25±0.5℃, DO 50±5%, add inducer and glycerol (maintain 1-3% w / v), and add at a constant rate to control residual sugar at 0.5-2.0 g / L; III. Stabilization period: Maintain 25±0.5℃ and DO 40±5%, and add glycerol-containing nutrient solution until fermentation ends; S3: Enzyme extraction: The fermentation broth was transferred to an enzyme release system at 10±2℃, and 0.5-1.5% w / v lysozyme and 5-10 mMEDTA were added for 60±15 min. The treated solution was then pumped into an aqueous two-phase extraction system, and PEG4000 and ammonium sulfate were added to a final concentration of 12-16% w / w and 10-14% w / w, respectively. The upper phase was separated at 4℃. S4: Post-processing: Using a freeze spray drying tower, the upper phase obtained in S3 was pumped into the membrane separation system and concentrated by ultrafiltration through a 10 kDa ultrafiltration membrane. The buffer solution was replaced with phosphate buffer containing 5% w / v trehalose, pH 7.
4. After adding 0.5-1.0% w / v gelatin, it was transferred to the drying system and freeze-spray dried under cold trap conditions of <-50℃ and vacuum conditions of 10-30 Pa.
2. The directional fermentation process for improving SOD stability according to claim 1, characterized in that, The reactive oxygen species inducer is menadione; the inducer added during the induction phase is 0.1-0.5 mM IPTG or 0.05-0.2 mM Cu. 2+ / Zn 2+ .
3. The directional fermentation process for improving SOD stability according to claim 1, characterized in that, In step S3, the lysozyme concentration is 1.0% w / v, and the aqueous two-phase system consists of 14% w / w PEG4000 and 12% w / w ammonium sulfate.
4. The directional fermentation process for improving SOD stability according to claim 1, characterized in that, The SOD enzyme powder has an enzyme activity retention rate of ≥85% after treatment at 60℃ for 30 min and an enzyme activity retention rate of ≥90% after storage at 4℃ in the dark for 6 months.
5. The directional fermentation process for improving SOD stability according to claim 1, characterized in that, The freeze spray drying tower includes a tower body (1), a top plate (2) is provided at the top of the tower body (1), a discharge port (3) is fixedly connected to the bottom of the tower body (1), a valve (4) is installed on the outer wall of the discharge port (3), a vacuum tube (5) is fixedly connected to one side of the tower body (1), a vacuum pump (6) is installed on the outer wall of the vacuum tube (5), a feed pipe (7) is fixedly connected to the top of the top plate (2), an atomizing nozzle (10) is installed at the bottom of the feed pipe (7), a heating jacket (11) is sleeved on the outer wall of the tower body (1), the inner wall of the tower body (1) is scraped by a scraping mechanism (8), and the top plate (2) is automatically opened and closed by a connecting mechanism (9).
6. The directional fermentation process for improving SOD stability according to claim 5, characterized in that, The scraping mechanism (8) includes a movable groove (801), which is symmetrically located at the top of the top plate (2). A movable frame (802) is slidably connected to the inner wall of the movable groove (801). A movable ring (803) is fixedly connected to the bottom end of the movable frame (802). A rotating cylinder (804) is rotatably connected to the inner wall of the movable ring (803). A scraping ring (805) is fixedly connected to the bottom end of the rotating cylinder (804). Scraping rods (806) are symmetrically fixedly connected to the bottom end of the scraping ring (805). A first sealing ring (815) is fixedly installed on the inner wall of the movable groove (801). A first motor (807) is installed at the top of the movable frame (802). The output end of the tower body (1) is connected to a connecting shaft (808), and a spur gear (809) is fixedly connected to the bottom end of the connecting shaft (808). The spur gear (809) is in contact with the rotating cylinder (804). A support base (810) is provided on one side of the tower body (1). A displacement groove (811) is opened on the outer wall of the support base (810). A displacement plate (812) is slidably connected to the inner wall of the displacement groove (811). The displacement plate (812) is fixedly connected to the movable frame (802). A second motor (813) is installed at the top of the support base (810). A first threaded rod (814) is connected to the output end of the second motor (813). The first threaded rod (814) passes through the displacement plate (812).
7. The directional fermentation process for improving SOD stability according to claim 6, characterized in that, The connecting mechanism (9) includes a first connecting seat (901), which is symmetrically fixedly connected to both sides of the top plate (2). The outer wall of the first connecting seat (901) is provided with a slot (902). The top of the outer wall of the tower body (1) is symmetrically fixedly connected to a second connecting seat (903). The top of the second connecting seat (903) is fixedly connected to a plug rod (904). The outer wall of the plug rod (904) is provided with a fixing groove (905). The top of the rotating cylinder (804) is fixedly connected to a locking block (906). The bottom of the top plate (2) is rotatably connected to a rotating ring (907). The interior of the top plate (2) is located on the outer wall of the rotating ring (907). A bevel gear (908) is rotatably connected, and a second threaded rod (909) is fixedly connected to one end of the bevel gear (908). A fixed block (910) extending into the cavity of the slot (902) is slidably connected inside the top plate (2). The second threaded rod (909) passes through the fixed block (910). A connecting rod (911) is rotatably connected to the outer wall of the fixed block (910). A positioning block (912) is rotatably connected to one end of the connecting rod (911). The positioning block (912) is slidably connected to the inside of the top plate (2). A second sealing ring (914) is installed at the top of the tower body (1). Positioning grooves (913) are symmetrically opened on the inner wall of the movable frame (802).
8. The directional fermentation process for improving SOD stability according to claim 6, characterized in that, The inner wall of the displacement groove (811) is in contact with the outer wall of the displacement plate (812). The outer wall of the displacement plate (812) is provided with a first threaded hole, which matches the first threaded rod (814). The inner wall of the movable groove (801) is in contact with the outer wall of the movable frame (802). The outer wall of the rotating cylinder (804) is provided with a toothed groove, which meshes with the spur gear (809).
9. The directional fermentation process for improving SOD stability according to claim 7, characterized in that, The outer wall of the insertion rod (904) is in contact with the inner wall of the slot (902), and the inner wall of the fixing groove (905) is in contact with the outer wall of the fixing block (910).
10. The directional fermentation process for improving SOD stability according to claim 7, characterized in that, The bottom end of the rotating ring (907) is provided with a slot that engages with the locking block (906). The outer wall of the rotating ring (907) is provided with gear teeth that mesh with the bevel gear (908). The outer wall of the fixing block (910) is provided with a second threaded hole that matches the second threaded rod (909). The outer wall of the positioning block (912) is in contact with the inner wall of the positioning groove (913).