Oliganase fermentation process control system and method based on pH and dissolved oxygen electrode feedback

Abstract

The invention discloses an oligosaccharide enzyme fermentation process control system and method based on pH and dissolved oxygen electrode feedback, relates to the technical field of oligosaccharide enzyme fermentation, and aims to improve the yield and activity of oligosaccharide enzyme. The system comprises a pH electrode, a dissolved oxygen electrode, a stirrer, a ventilation system and a temperature control system, and accurate control is realized through a data acquisition and processing unit, a logic control unit and a human-computer interface. The control method comprises the steps of seed culture, shake flask fermentation, fermentation tank culture, monitoring and evaluation as well as optimization and adjustment. Through a segmented oxygen control strategy and dissolved oxygen and stirring linkage control, the dissolved oxygen level is accurately controlled, the yield and activity of the oligosaccharide enzyme are remarkably improved, the fermentation process conditions are optimized, and the monitoring and evaluation capability is enhanced. The method is suitable for industrial production of the oligosaccharide enzyme, and is beneficial for improving the product quality and the economic benefit.

Description

Technical Field

[0001] The invention relates to the technical field of oligosaccharidase fermentation, in particular to an oligosaccharidase fermentation process control system and method based on pH and dissolved oxygen electrode feedback. Background Art

[0002] Oligosaccharidases are a class of enzymes that can degrade oligosaccharides; oligosaccharides, also known as oligosaccharides or oligosaccharides, refer to small straight-chain or branched polymers formed by 2-10 monosaccharides connected by glycosidic bonds. Oligosaccharidases can act on these oligosaccharide molecules and degrade them into smaller sugar molecules. Taking alginate oligosaccharidase as an example, this enzyme can degrade alginate polysaccharides and oligosaccharides into monosaccharides. The number of this enzyme is relatively small in nature, and its activity may not be ideal. Therefore, researchers may clone the gene of this enzyme from specific microorganisms and recombinantly express it to improve its enzyme activity and stability, and then apply it to the fields of monosaccharide preparation of oligosaccharides.

[0003] At present, the research on oligosaccharidase at home and abroad mainly focuses on Bacillus subtilis, Bacillus licheniformis, alkaliphilic Bacillus and Pseudomonas in bacteria; Aspergillus niger and Trichoderma in fungi; and Streptomyces in actinomycetes. Oligosaccharidase is mainly limited by its low enzyme activity level in actual production and application. Therefore, trying to improve the fermentation yield of oligosaccharidase is the current research focus in the industry. The most direct way to increase enzyme yield is to optimize the culture medium and culture conditions. Fermentation conditions not only affect the growth and reproduction of bacteria, but also affect the synthesis of enzymes; in addition, the optimal reaction pH, isoelectric point and optimal reaction temperature for oligosaccharidase produced by different types of microorganisms also have different degrees of differences. Therefore, the present invention proposes an oligosaccharidase fermentation process control system and method based on pH and dissolved oxygen electrode feedback. Summary of the invention

[0004] To achieve the above object, the present invention provides the following technical solution: an oligosaccharidase fermentation process control system based on pH and dissolved oxygen electrode feedback, which includes a hardware system and a control system, wherein the hardware system includes:

[0005] pH electrode: used to measure the pH value in the fermentation broth online;

[0006] Dissolved oxygen electrode: real-time monitoring of dissolved oxygen content in the fermentation liquid, providing feedback for the control system to adjust the stirring speed and ventilation volume;

[0007] Agitator: Affects the dissolved oxygen level in the fermentation liquid by adjusting the stirring speed, and works in conjunction with the dissolved oxygen electrode to achieve precise control of the dissolved oxygen content;

[0008] Ventilation system: including air pump and gas flow meter equipment, used to introduce sterile air into the fermentation tank and control the dissolved oxygen content in the fermentation liquid by adjusting the ventilation volume;

[0009] Temperature control system: Although not directly mentioned in the document, it is necessary to maintain a constant temperature during the fermentation process, so a temperature probe and heating / cooling device should be included to maintain the set fermentation temperature;

[0010] The control system includes:

[0011] The data acquisition and processing unit receives signals from the pH electrode and dissolved oxygen electrode, processes and analyzes the data, and sends the processed data to the logic control unit as a basis for formulating the control strategy;

[0012] The logic control unit formulates and executes control strategies based on the data provided by the data acquisition and processing unit; sends control signals to the agitator, ventilation system and acid-base adjustment equipment to achieve precise control of pH value and dissolved oxygen content;

[0013] Human-machine interface: provides a platform for operators to interact with the control system and displays the parameters of the current fermentation process;

[0014] The pH electrode and the pH electrode of the control system measure the pH value in the fermentation liquid in real time and transmit the signal to the data acquisition and processing unit in the control system; the control system determines whether the pH value of the fermentation liquid needs to be adjusted according to the received pH value signal, and adjusts the pH value to the set range by controlling the device for adding acid and alkali solution;

[0015] The dissolved oxygen electrode and the dissolved oxygen electrode of the control system measure the dissolved oxygen content in the fermentation liquid in real time and transmit the signal to the control system; the control system adjusts the speed of the agitator and the ventilation volume of the ventilation system through the logic control unit according to the dissolved oxygen signal to keep the dissolved oxygen content within the set range;

[0016] The agitator of the agitator and ventilation system affects the dissolved oxygen content in the fermentation liquid by adjusting the rotation speed, and is linked to the feedback signal of the dissolved oxygen electrode; the ventilation system further controls the dissolved oxygen content by adjusting the ventilation volume, and works together with the agitator to ensure accurate control of the dissolved oxygen content.

[0017] The oligosaccharide enzyme fermentation process control method based on pH and dissolved oxygen electrode feedback is carried out in the following steps:

[0018] S1, seed culture;

[0019] Slant seed activation: Activate the slant seeds under suitable conditions for 4 hours to prepare for subsequent inoculation;

[0020] Seed culture medium inoculation: inoculate the activated slant seeds into the seed culture medium and culture for 20 hours; during this stage, the shaking speed is controlled at 200 rpm and the temperature is 30°C;

[0021] S2, shake flask fermentation;

[0022] Shake flask preparation: Fill 50 ml of fermentation medium into a 500 ml Erlenmeyer flask;

[0023] Inoculation and cultivation: Inoculate the seeds into the fermentation medium at a 10% inoculation rate, maintain the shaker speed at 200 rpm, set the temperature at 30°C, and cultivate for 30 hours;

[0024] S3, fermentation tank culture;

[0025] S31, fermentation tank preparation: the fermentation tank is filled with 4L of liquid, the inoculation amount is 10% of the fermentation liquid, the ventilation volume is set to 5.0L / min, and the temperature is controlled at 30°C;

[0026] S32, pH value control: Use a pH electrode to measure the pH value of the fermentation liquid online, and adjust the pH value to 5.5 by automatically adding 3 mol / L H2SO4 and NaOH solution;

[0027] S33, dissolved oxygen control:

[0028] Initial control: The initial stirring speed was set at 300 rpm, and the stirring speed was adjusted in real time to maintain a constant dissolved oxygen concentration;

[0029] Early stage: dissolved oxygen levels drop naturally, and no additional control measures are taken;

[0030] Logarithmic growth period: When the dissolved oxygen drops to 15%, the dissolved oxygen level is maintained at 15% by linking the stirring speed with the dissolved oxygen;

[0031] Later stage: Continue to control the dissolved oxygen level at 45% by linking the stirring speed with the dissolved oxygen until the fermentation is completed;

[0032] S4. Monitoring and evaluation;

[0033] S41, determination of bacterial concentration: regularly determine the bacterial concentration in the fermentation broth by dry weight method; the specific operation is to take 50 mL of fermentation broth and filter to obtain bacterial cells, dry to constant weight and weigh, and subtract the weight of the filter paper to obtain the actual bacterial concentration;

[0034] S42, enzyme activity determination: oligosaccharidase activity was determined by spectrophotometry; an appropriate amount of diluted enzyme solution was added to the salicin solution and kept warm, and then the DNS reagent was added to perform a color reaction, and finally the absorbance was measured and the enzyme activity was calculated;

[0035] S5. Optimization and adjustment;

[0036] S51, ventilation ratio optimization: optimize biomass and enzyme activity by changing the ventilation ratio to find the best ventilation conditions;

[0037] S52, linkage optimization of dissolved oxygen and stirring: in the early stage, logarithmic growth stage and late stage of the fermentation process, precise regulation of dissolved oxygen is achieved through linkage control of stirring speed and dissolved oxygen level, thereby increasing biomass and enzyme activity;

[0038] S53. Product synthesis kinetics analysis: Optimize fermentation control by analyzing the relationship between product synthesis rate qP and cell growth rate μ.

[0039] In a preferred solution, the specific process of step S41 includes:

[0040] S411, sampling: regularly take out 50 mL of fermentation liquid from the fermentation tank as a sample;

[0041] S412, filtration: filtering the sampled fermentation liquid through filter paper to separate the bacteria in the fermentation liquid;

[0042] S413, drying: placing the bacteria on the filter paper in an oven at 100° C. for drying until the bacteria reach a constant weight; maintaining a constant temperature during the drying process to prevent the bacteria from being burned;

[0043] S414. Weighing: weigh the dried filter paper and the bacteria together, and then subtract the weight of the filter paper to obtain the actual dry weight of the bacteria; this dry weight is the concentration of the bacteria in the fermentation liquid.

[0044] In a preferred solution, the specific process of step S42 includes:

[0045] S421, preparing enzyme solution: taking out an appropriate amount of enzyme solution from the fermentation solution and diluting it appropriately to ensure that the concentration of the enzyme solution is within the measurement range of the spectrophotometer;

[0046] S422, adding substrate: taking a certain amount of 0.5 mL of diluted enzyme solution, adding 1 mL of 1.0% salicin solution; salicin is a common substrate of oligosaccharidase and is used to detect enzyme activity;

[0047] S423, heat preservation reaction: place the mixed solution in a 50°C water bath for a certain period of time of 30 minutes to allow the enzyme and substrate to fully react;

[0048] S424, color reaction: add 3 mL of DNS reagent to the reaction solution, wherein the DNS reagent is 3,5-dinitrosalicylic acid, and then place the solution in a boiling water bath and heat for 10 minutes to color the reaction product; the DNS reagent can react with reducing sugar to generate a colored compound, which is convenient for subsequent absorbance measurement;

[0049] S425, cooling and diluting: cooling the reaction solution after color development to room temperature, and diluting it to 25 mL with water for measurement on a spectrophotometer;

[0050] S426, measuring absorbance: setting the wavelength to 540 nm on the spectrophotometer, and measuring the absorbance of the diluted reaction solution; the absorbance is proportional to the enzyme activity, and the actual enzyme activity is calculated by calibration using a standard curve or a sample with known enzyme activity;

[0051] S427. Calculate enzyme activity: Calculate the activity unit of oligosaccharidase based on the measured absorbance value and the known standard curve or correction factor; the activity unit of oligosaccharidase is the amount of enzyme required to release 1 μmol of reducing sugar from a 1.0% salicin solution per minute under the conditions of 50°C and pH 4.8.

[0052] In a preferred solution, the specific process of step S51 includes:

[0053] S511, selecting a series of different ventilation ratios as experimental conditions, and performing multiple repeated experiments under each ventilation ratio condition to reduce random errors; and gradually adjusting the ventilation ratio to a predetermined value at different stages during the fermentation process;

[0054] S512, using a dissolved oxygen electrode to monitor the dissolved oxygen content in the fermentation liquid in real time, and simultaneously observing and recording key parameters such as pH value, temperature, and stirring speed during the fermentation process;

[0055] S513. After the fermentation is completed, the bacterial concentration, i.e., biomass, of each experimental group is determined by the dry weight method, and the biomass and enzyme activity data under different ventilation ratios are compared. The influence of ventilation ratio on the fermentation effect is analyzed by statistical methods; it is determined under which ventilation ratio the biomass and enzyme activity are optimal; and repeated experiments are carried out under the selected optimal ventilation ratio to verify.

[0056] In a preferred solution, the specific process of step S52 includes:

[0057] S521, dissolved oxygen monitoring: when the dissolved oxygen content drops to 15% of the preset value, the linkage control of the stirring speed and the dissolved oxygen level is started;

[0058] S522, adjusting the stirring speed: according to the feedback signal of the dissolved oxygen electrode, automatically adjust the stirring speed to maintain the dissolved oxygen content at 15%; if the dissolved oxygen has an upward trend, appropriately reduce the stirring speed; if the dissolved oxygen has a downward trend, increase the stirring speed;

[0059] S523, maintain stable ventilation: At this stage, the ventilation volume can be kept relatively stable, or fine-tuned as needed to assist the stirring speed in maintaining the dissolved oxygen level;

[0060] S524, dissolved oxygen staged control: according to the fermentation process requirements, the dissolved oxygen level is gradually increased to 45% in the late fermentation period; before each increase in the dissolved oxygen level, the stirring speed is quickly adjusted to make the dissolved oxygen close to the target value, and then gradually stabilized at this level;

[0061] S525, Dynamic Adjustment: In the process of increasing the dissolved oxygen level, continuously monitor the changes in dissolved oxygen, and dynamically adjust the stirring speed and ventilation volume as needed to ensure that the dissolved oxygen is stable within the target range;

[0062] S526. Monitor biomass and enzyme activity: During the process of staged dissolved oxygen increase, take samples regularly to measure biomass and enzyme activity, and evaluate the impact of changes in dissolved oxygen levels on fermentation results.

[0063] In a preferred solution, the specific process of step S53 includes:

[0064] Samples were taken at different time points of the fermentation, and the product concentration was determined by spectrophotometry; the product synthesis rate qP was calculated based on the change in product concentration; qP was defined as the increase in product concentration per unit time divided by the biomass;

[0065] Samples were taken at different time points of the fermentation to measure the biomass, and the cell growth rate μ was calculated based on the change in biomass; μ is usually defined as the natural logarithmic rate of change of biomass per unit time;

[0066] Construct a mathematical relationship model between product synthesis rate qP and cell growth rate μqP=aμ+β;

[0067] Among them, a represents the size of the product synthesis capacity under the condition of constant cell growth capacity;

[0068] β represents the ability to synthesize products when the cell stops growing;

[0069] The nonlinear regression method is used to estimate the parameters a and β in the model, and the model prediction results are compared with the experimental data to verify the accuracy and reliability of the model;

[0070] The model is used to analyze the changing trends and influencing factors of the product synthesis capacity qp under different fermentation conditions; the fermentation control strategy is adjusted according to the analysis results, and the dissolved oxygen content, pH value, stirring speed, and ventilation volume parameters are adjusted to optimize the product synthesis rate and biomass accumulation; if the model shows that the product synthesis capacity is highest at a certain dissolved oxygen content, then this dissolved oxygen level can be maintained during the fermentation process.

[0071] Compared with the prior art, the present invention has the following beneficial effects:

[0072] The present invention achieves precise control and significant optimization of the oligosaccharidase fermentation process by introducing an advanced pH and dissolved oxygen electrode feedback control system and combining it with a scientific fermentation process optimization strategy, thereby increasing the yield and activity of the oligosaccharidase. Specifically,

[0073] Significantly improved oligosaccharidase production and activity: By introducing a pH and dissolved oxygen electrode feedback control system and combining it with a segmented oxygen control strategy, the optimized oligosaccharidase fermentation process can significantly improve the production and activity of oligosaccharidase. Specifically, the maximum enzyme activity increased from about 1600U / mL before optimization to 2406.175U / mL, an increase of about 50%.

[0074] Achieve precise control of dissolved oxygen levels: The system can achieve precise control of dissolved oxygen levels at different stages of the fermentation process through linkage control of the stirring speed and dissolved oxygen electrode, as well as fine adjustment of the ventilation ratio. This control method is more effective than adjusting the stirring speed or ventilation ratio alone, and can better meet the demand for dissolved oxygen at different fermentation stages.

[0075] Improve the stability and repeatability of the fermentation process: Due to the use of automated pH and dissolved oxygen control systems, the uncertainty of manual operation is reduced, making the fermentation process more stable and improving the repeatability of the experiment. This is particularly important for industrial production and helps to ensure the uniformity of product quality.

[0076] Optimized fermentation process conditions: The best combination of fermentation conditions was determined by optimizing process parameters such as different ventilation ratios, stirring speeds, and pH values. These optimized conditions not only increased the yield and activity of oligosaccharidase, but also helped reduce production costs and improve economic benefits.

[0077] Enhanced monitoring and evaluation capabilities for the fermentation process: The system is equipped with a complete monitoring and evaluation mechanism, including regular testing of key indicators such as bacterial concentration and enzyme activity. These data provide strong support for further optimization of the fermentation process and also provide an important basis for product quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 The oligosaccharidase fermentation flow chart of the present invention. DETAILED DESCRIPTION

[0079] See also Figure 1 The present invention provides an oligosaccharidase fermentation process control system based on pH and dissolved oxygen electrode feedback, which includes a hardware system and a control system, wherein the hardware system includes:

[0080] pH electrode: used to measure the pH value in the fermentation broth online;

[0081] Dissolved oxygen electrode: real-time monitoring of dissolved oxygen content in the fermentation liquid, providing feedback for the control system to adjust the stirring speed and ventilation volume;

[0082] Agitator: Affects the dissolved oxygen level in the fermentation liquid by adjusting the stirring speed, and works in conjunction with the dissolved oxygen electrode to achieve precise control of the dissolved oxygen content;

[0083] Ventilation system: including air pump and gas flow meter equipment, used to introduce sterile air into the fermentation tank and control the dissolved oxygen content in the fermentation liquid by adjusting the ventilation volume;

[0084] Temperature control system: Although not directly mentioned in the document, it is necessary to maintain a constant temperature during the fermentation process, so a temperature probe and heating / cooling device should be included to maintain the set fermentation temperature;

[0085] The control system includes:

[0086] The data acquisition and processing unit receives signals from the pH electrode and dissolved oxygen electrode, processes and analyzes the data, and sends the processed data to the logic control unit as a basis for formulating the control strategy;

[0087] The logic control unit formulates and executes control strategies based on the data provided by the data acquisition and processing unit; sends control signals to the agitator, ventilation system and acid-base adjustment equipment to achieve precise control of pH value and dissolved oxygen content;

[0088] Human-machine interface: provides a platform for operators to interact with the control system and displays the parameters of the current fermentation process;

[0089] The pH electrode and the pH electrode of the control system measure the pH value in the fermentation liquid in real time and transmit the signal to the data acquisition and processing unit in the control system; the control system determines whether the pH value of the fermentation liquid needs to be adjusted according to the received pH value signal, and adjusts the pH value to the set range by controlling the device for adding acid and alkali solution;

[0090] The dissolved oxygen electrode and the dissolved oxygen electrode of the control system measure the dissolved oxygen content in the fermentation liquid in real time and transmit the signal to the control system; the control system adjusts the speed of the agitator and the ventilation volume of the ventilation system through the logic control unit according to the dissolved oxygen signal to keep the dissolved oxygen content within the set range;

[0091] The agitator of the agitator and ventilation system affects the dissolved oxygen content in the fermentation liquid by adjusting the rotation speed, and is linked to the feedback signal of the dissolved oxygen electrode; the ventilation system further controls the dissolved oxygen content by adjusting the ventilation volume, and works together with the agitator to ensure accurate control of the dissolved oxygen content.

[0092] The oligosaccharide enzyme fermentation process control method based on pH and dissolved oxygen electrode feedback is carried out in the following steps:

[0093] S1, seed culture;

[0094] Slant seed activation: Activate the slant seeds under suitable conditions for 4 hours to prepare for subsequent inoculation;

[0095] Seed culture medium inoculation: inoculate the activated slant seeds into the seed culture medium and culture for 20 hours; during this stage, the shaking speed is controlled at 200 rpm and the temperature is 30°C;

[0096] S2, shake flask fermentation;

[0097] Shake flask preparation: Fill 50 ml of fermentation medium into a 500 ml Erlenmeyer flask;

[0098] Inoculation and cultivation: Inoculate the seeds into the fermentation medium at a 10% inoculation rate, maintain the shaker speed at 200 rpm, set the temperature at 30°C, and cultivate for 30 hours;

[0099] S3, fermentation tank culture;

[0100] S31, fermentation tank preparation: the fermentation tank is filled with 4L of liquid, the inoculation amount is 10% of the fermentation liquid, the ventilation volume is set to 5.0L / min, and the temperature is controlled at 30°C;

[0101] S32, pH value control: Use a pH electrode to measure the pH value of the fermentation liquid online, and adjust the pH value to 5.5 by automatically adding 3 mol / L H2SO4 and NaOH solution;

[0102] S33, dissolved oxygen control:

[0103] Initial control: The initial stirring speed was set at 300 rpm, and the stirring speed was adjusted in real time to maintain a constant dissolved oxygen concentration;

[0104] Early stage: dissolved oxygen levels drop naturally, and no additional control measures are taken;

[0105] Logarithmic growth period: When the dissolved oxygen drops to 15%, the dissolved oxygen level is maintained at 15% by linking the stirring speed with the dissolved oxygen;

[0106] Later stage: Continue to control the dissolved oxygen level at 45% by linking the stirring speed with the dissolved oxygen until the fermentation is completed;

[0107] S4. Monitoring and evaluation;

[0108] S41, determination of bacterial concentration: regularly determine the bacterial concentration in the fermentation broth by dry weight method; the specific operation is to take 50 mL of fermentation broth and filter to obtain bacterial cells, dry to constant weight and weigh, and subtract the weight of the filter paper to obtain the actual bacterial concentration;

[0109] S42, enzyme activity determination: oligosaccharidase activity was determined by spectrophotometry; an appropriate amount of diluted enzyme solution was added to the salicin solution and kept warm, and then the DNS reagent was added to perform a color reaction, and finally the absorbance was measured and the enzyme activity was calculated;

[0110] S5. Optimization and adjustment;

[0111] S51, ventilation ratio optimization: optimize biomass and enzyme activity by changing the ventilation ratio to find the best ventilation conditions;

[0112] S52, linkage optimization of dissolved oxygen and stirring: in the early stage, logarithmic growth stage and late stage of the fermentation process, precise regulation of dissolved oxygen is achieved through linkage control of stirring speed and dissolved oxygen level, thereby increasing biomass and enzyme activity;

[0113] S53. Product synthesis kinetics analysis: Optimize fermentation control by analyzing the relationship between product synthesis rate qP and cell growth rate μ.

[0114] The specific process of step S41 includes:

[0115] S411, sampling: regularly take out 50 mL of fermentation liquid from the fermentation tank as a sample;

[0116] S412, filtration: filtering the sampled fermentation liquid through filter paper to separate the bacteria in the fermentation liquid;

[0117] S413, drying: placing the bacteria on the filter paper in an oven at 100° C. for drying until the bacteria reach a constant weight; maintaining a constant temperature during the drying process to prevent the bacteria from being burned;

[0118] S414. Weighing: weigh the dried filter paper and the bacteria together, and then subtract the weight of the filter paper to obtain the actual dry weight of the bacteria; this dry weight is the concentration of the bacteria in the fermentation liquid.

[0119] The specific process of step S42 includes:

[0120] S421, preparing enzyme solution: taking out an appropriate amount of enzyme solution from the fermentation solution and diluting it appropriately to ensure that the concentration of the enzyme solution is within the measurement range of the spectrophotometer;

[0121] S422, adding substrate: taking a certain amount of 0.5 mL of diluted enzyme solution, adding 1 mL of 1.0% salicin solution; salicin is a common substrate of oligosaccharidase and is used to detect enzyme activity;

[0122] S423, heat preservation reaction: place the mixed solution in a 50°C water bath for a certain period of time of 30 minutes to allow the enzyme and substrate to fully react;

[0123] S424, color reaction: add 3 mL of DNS reagent to the reaction solution, wherein the DNS reagent is 3,5-dinitrosalicylic acid, and then place the solution in a boiling water bath and heat for 10 minutes to color the reaction product; the DNS reagent can react with reducing sugar to generate a colored compound, which is convenient for subsequent absorbance measurement;

[0124] S425, cooling and diluting: cooling the reaction solution after color development to room temperature, and diluting it to 25 mL with water for measurement on a spectrophotometer;

[0125] S426, measuring absorbance: setting the wavelength to 540 nm on the spectrophotometer, and measuring the absorbance of the diluted reaction solution; the absorbance is proportional to the enzyme activity, and the actual enzyme activity is calculated by calibration using a standard curve or a sample with known enzyme activity;

[0126] S427. Calculate enzyme activity: Calculate the activity unit of oligosaccharidase based on the measured absorbance value and the known standard curve or correction factor; the activity unit of oligosaccharidase is the amount of enzyme required to release 1 μmol of reducing sugar from a 1.0% salicin solution per minute under the conditions of 50°C and pH 4.8.

[0127] The specific process of step S51 includes:

[0128] S511, selecting a series of different ventilation ratios as experimental conditions, and performing multiple repeated experiments under each ventilation ratio condition to reduce random errors; and gradually adjusting the ventilation ratio to a predetermined value at different stages during the fermentation process;

[0129] S512, using a dissolved oxygen electrode to monitor the dissolved oxygen content in the fermentation liquid in real time, and simultaneously observing and recording key parameters such as pH value, temperature, and stirring speed during the fermentation process;

[0130] S513. After the fermentation is completed, the bacterial concentration, i.e., biomass, of each experimental group is determined by the dry weight method, and the biomass and enzyme activity data under different ventilation ratios are compared. The influence of ventilation ratio on the fermentation effect is analyzed by statistical methods; it is determined under which ventilation ratio the biomass and enzyme activity are optimal; and repeated experiments are carried out under the selected optimal ventilation ratio to verify.

[0131] The specific process of step S52 includes:

[0132] S521, dissolved oxygen monitoring: when the dissolved oxygen content drops to 15% of the preset value, the linkage control of the stirring speed and the dissolved oxygen level is started;

[0133] S522, adjusting the stirring speed: according to the feedback signal of the dissolved oxygen electrode, automatically adjust the stirring speed to maintain the dissolved oxygen content at 15%; if the dissolved oxygen has an upward trend, appropriately reduce the stirring speed; if the dissolved oxygen has a downward trend, increase the stirring speed;

[0134] S523, maintain stable ventilation: At this stage, the ventilation volume can be kept relatively stable, or fine-tuned as needed to assist the stirring speed in maintaining the dissolved oxygen level;

[0135] S524, dissolved oxygen staged control: according to the fermentation process requirements, the dissolved oxygen level is gradually increased to 45% in the late fermentation period; before each increase in the dissolved oxygen level, the stirring speed is quickly adjusted to make the dissolved oxygen close to the target value, and then gradually stabilized at this level;

[0136] S525, Dynamic Adjustment: In the process of increasing the dissolved oxygen level, continuously monitor the changes in dissolved oxygen, and dynamically adjust the stirring speed and ventilation volume as needed to ensure that the dissolved oxygen is stable within the target range;

[0137] S526. Monitor biomass and enzyme activity: During the process of staged dissolved oxygen increase, take samples regularly to measure biomass and enzyme activity, and evaluate the impact of changes in dissolved oxygen levels on fermentation results.

[0138] The specific process of step S53 includes:

[0139] Samples were taken at different time points of the fermentation, and the product concentration was determined by spectrophotometry; the product synthesis rate qP was calculated based on the change in product concentration; qP was defined as the increase in product concentration per unit time divided by the biomass;

[0140] Samples were taken at different time points of the fermentation to measure the biomass, and the cell growth rate μ was calculated based on the change in biomass; μ is usually defined as the natural logarithmic rate of change of biomass per unit time;

[0141] Construct a mathematical relationship model between product synthesis rate qP and cell growth rate μqP=aμ+β;

[0142] Among them, a represents the size of the product synthesis capacity under the condition of constant cell growth capacity;

[0143] β represents the ability to synthesize products when the cell stops growing;

[0144] The nonlinear regression method is used to estimate the parameters a and β in the model, and the model prediction results are compared with the experimental data to verify the accuracy and reliability of the model;

[0145] The model is used to analyze the changing trends and influencing factors of the product synthesis capacity qp under different fermentation conditions; the fermentation control strategy is adjusted according to the analysis results, and the dissolved oxygen content, pH value, stirring speed, and ventilation volume parameters are adjusted to optimize the product synthesis rate and biomass accumulation; if the model shows that the product synthesis capacity is highest at a certain dissolved oxygen content, then this dissolved oxygen level can be maintained during the fermentation process.

[0146] Example 1. Through this example, the steps of the oligosaccharidase fermentation process control method based on pH and dissolved oxygen electrode feedback are introduced: seed culture, the inclined seeds are activated for 4 hours and then inoculated into the seed culture medium for 20 hours, and the shaking speed is 200r·min -1 , temperature 30℃;

[0147] Shake flask fermentation: 500 ml of fermentation medium was placed in a 500 ml triangular flask, and seeds were added at a 10% inoculation rate. The culture temperature was 30 °C and the shaker speed was 200 r / min. -1 , culture time 30h;

[0148] Fermentation tank culture, the fermentation tank has a liquid volume of 4L, an inoculation volume of 10%, and a ventilation volume of 5.0L·min -1 , temperature 30℃, pH value was measured online by pH electrode, and 3 mol·L -1 H2SO4 and 3mol·L -1 The NaOH solution is automatically adjusted to about 5.5, and the stirring speed is manually controlled in real time to maintain a certain constant dissolved oxygen concentration. The initial speed is 300r·min -1 , adjustment range 10r·min -1 ;

[0149] The bacterial concentration was determined by the dry weight method: 50 mL of fermentation liquid was filtered to obtain bacterial cells, and the bacterial cells were placed in a 100°C oven to dry to constant weight. The mass of the dried filter paper was weighed and subtracted to obtain the bacterial concentration.

[0150] At the same time, the enzyme activity was determined by spectrophotometry. 0.5 mL of the appropriately diluted enzyme solution was added with 1 mL of 1.0% salicin solution, and the mixture was kept in a 50°C water bath for 30 min. 3 mL of DNS reagent was added and the mixture was kept in a boiling water bath for 10 min. After cooling, the mixture was diluted to 25 mL with water and the absorbance was measured at 540 nm.

[0151] The definition of oligosaccharidase activity unit is that the amount of enzyme required to release 1 μmol of reducing sugar from a 1.0% salicin solution in 1 min at 50°C and pH 4.8 is one unit of enzyme activity.

[0152] With the consumption of substrate, the pH of the fermentation liquid dropped rapidly from 5.5 to the lowest point of 1.17 at 18h when glucose utilization was completed, and then basically maintained at this level. The gradually enhanced acidity of the fermentation liquid had a certain inhibitory effect on cell growth until the cell volume reached a maximum value of 12g / L at 18h. In the first 10h of fermentation, the GSH synthesized by the cells was basically distributed inside the cells; after 10h, GSH accumulated outside the cells, and the accumulation amount continued to increase, reaching a maximum value of 86mg / L at 18h. The intracellular GSH content quickly increased from 1 to 1. .27% decreased to 0.46%. After the cells stopped growing, the total amount of extracellular GSH was relatively stable, but the total amount of intracellular GSH was still increasing, indicating that intracellular GSH was still being synthesized. During the entire fermentation process, the total amount of GSH increased. At the end of the fermentation, the total GSH production reached 165 mg / L. The stirring speed was constant and the ventilation ratio was changed to control the dissolved oxygen in the tank. At 38-45h, the biomass was the largest under the ventilation ratio of 1.0vvm, followed by 1.5vvm, and the smallest under 2.0vvm. Due to the difference between 1.5vvm and 2. Under the ventilation ratio of 0vvm, a large amount of foam was produced in the early stage of fermentation, causing some bacteria to adhere to the tank wall with the foam, resulting in biomass loss. The maximum enzyme activity under the ventilation ratio of 2.0vvm was the smallest, and the maximum enzyme activity under the ventilation ratio of 1.0vvm was the largest, but it was almost the same as that under the ventilation ratio of 1.5vvm. According to the characteristics of oligosaccharidase fermentation, the dissolved oxygen in the tank was increased by linking dissolved oxygen with stirring and coordinating with the adjustment of ventilation ratio. The dissolved oxygen was automatically controlled by stirring speed in the early stage of fermentation. The dissolved oxygen in the logarithmic growth period was set to 15%, and the dissolved oxygen in the later stage was controlled by stirring speed. The stirring speed was linked to be controlled at 15%, 30% and 45% respectively until the end of fermentation, and the enzyme activity and biomass were investigated. In the early stage of fermentation, the biological indicators were similar. In the later stage, the stirring was linked with dissolved oxygen and controlled at 15%. The enzyme activity of the bacteria could reach 1435.118U / mL at 108h, and under 30% dissolved oxygen conditions, the enzyme activity of the bacteria reached the maximum at 132h, reaching 2310.686U / mL; under the third control method of 45% dissolved oxygen conditions, the enzyme activity could reach 2381.676U / mL in 120h.

[0153] From the product synthesis kinetics, we know that there is the following relationship between qP and μ: qp=aμ+β, where a represents the size of the product synthesis capacity under a certain cell growth capacity, and β represents the size of the product synthesis capacity when the cell stops growing. According to the experimental results under various DO control modes, the a and β parameters in the above formula are nonlinearly estimated. When DO is constantly controlled at 35%, a and β values ​​are higher. The three more typical groups of kinetic results are further analyzed. The results show that since the actual maximum μ in each fermentation process is not higher than 0.3h, within the μ range, when DO is 35%, the cell can obtain a higher GSH synthesis capacity (qp). By changing the pH value during the fermentation process, it is found that the pH value has an effect on the oligosaccharidase The yield and activity of oligosaccharidase have a significant impact. When the pH value is within a certain range, the yield and activity of oligosaccharidase reach the maximum value. Beyond this range, the yield and activity of oligosaccharidase will decrease. Dissolved oxygen concentration is also an important factor affecting oligosaccharidase fermentation. By adjusting the ventilation volume and stirring speed, it is found that dissolved oxygen concentration also has a significant effect on the yield and activity of oligosaccharidase; within a certain range, increasing the dissolved oxygen concentration can increase the yield and activity of oligosaccharidase, but too high dissolved oxygen concentration may cause oligosaccharidase inactivation. The effect of different fermentation times on oligosaccharidase fermentation. With the extension of fermentation time, the yield of oligosaccharidase gradually increases, but after a certain time, the yield tends to stabilize or even slightly decreases. Too long fermentation time may lead to contamination by foreign bacteria, affecting the quality and activity of oligosaccharidase.

[0154] The specific rate is a relative speed, which is closely related to the biomass. The specific rate is defined by equation group (1) with the unit of h-1, where μ is the specific rate of growth, qs is the specific rate of substrate consumption, and qp is the specific rate of product formation. According to the definition of μ, qs and qp:

[0155]

[0156] When the time interval is very small, μ, qs and qp can be directly calculated using equation (2):

[0157]

[0158] The strains have different requirements for oxygen at different stages. The lag phase of the strains is shorter at low stirring speeds. At higher stirring speeds (400 r / min), the cells have higher μ, qs and qp in the early stage of fermentation, that is, at this stage, high speeds can promote bacterial growth and substrate consumption. After 18 hours, at 400 r / min, the bacterial μ gradually becomes negative. At this time, the bacterial death rate is greater than the growth rate, the bacterial mass increases slowly, and qp gradually decreases to negative values. The main reason is that the bacterial death leads to an increase in dissolved oxygen in the fermentation broth, and 2,3-butanediol begins to reversely synthesize the by-product acetoin. At 350 r / min, higher μ and qp can still be maintained.

[0159] ALA is 5-aminolevulinic acid or its derivatives / analogs, and the substance is an important product or key intermediate in a specific recombinant bacterial fermentation process.

[0160] ALA is an important bioactive substance with a wide range of application prospects, including as a photosensitizer in agriculture to promote plant growth, and as a prodrug in photodynamic therapy for the treatment of skin cancer in medicine. During microbial fermentation, ALA can be synthesized by certain microorganisms through specific metabolic pathways. Therefore, in the fermentation process, the optimization of fermentation conditions such as dissolved oxygen is of great significance for improving the yield and quality of ALA. The fermentation of ALA is greatly affected by dissolved oxygen conditions, so the dissolved oxygen in the fermentation process of recombinant bacteria DALA was optimized. Under the conditions of pH 6.5 in the early stage of fermentation and pH 6.0 in the late stage of stable period, the speed was adjusted to 400r / min and the ventilation volume was 2VVI / 1 in the early stage of fermentation; the speed was 200r / min and the ventilation volume was 1VVITI in the late stage of stable period; the speed was 500r / min and the ventilation volume was 2wm in the early stage of fermentation; the speed was 250r / min and the ventilation volume was 1vm in the late stage of stable period. The dissolved oxygen in the fermentation process was regulated by controlling the speed, and the fermentation results under different dissolved oxygen conditions were investigated. When the pH value was 6.5, ALA could still exist stably in aqueous solution. In the late stage of stable period, the activity of the bacteria began to decline, and a large amount of ALA had accumulated in the fermentation broth. The pH of the fermentation broth was further reduced to 6.0, and the bacteria continued to accumulate ALA. At the same time, when the pH of the fermentation broth was reduced to 6.0, the stability of ALA in the fermentation broth was enhanced, which was conducive to the accumulation of ALA.

Claims

1. Oligosaccharidase fermentation process control system based on pH and dissolved oxygen electrode feedback, characterized in that: It includes a hardware system and a control system, wherein the hardware system includes: pH electrode: used to measure the pH value in the fermentation broth online; Dissolved oxygen electrode: real-time monitoring of dissolved oxygen content in the fermentation liquid, providing feedback for the control system to adjust the stirring speed and ventilation volume; Agitator: Affects the dissolved oxygen level in the fermentation liquid by adjusting the stirring speed, and works in conjunction with the dissolved oxygen electrode to achieve precise control of the dissolved oxygen content; Ventilation system: including air pump and gas flow meter equipment, used to introduce sterile air into the fermentation tank and control the dissolved oxygen content in the fermentation liquid by adjusting the ventilation volume; Temperature control system: Although not directly mentioned in the document, it is necessary to maintain a constant temperature during the fermentation process, so a temperature probe and heating / cooling device should be included to maintain the set fermentation temperature; The control system includes: The data acquisition and processing unit receives signals from the pH electrode and dissolved oxygen electrode, processes and analyzes the data, and sends the processed data to the logic control unit as a basis for formulating the control strategy; The logic control unit formulates and executes control strategies based on the data provided by the data acquisition and processing unit; sends control signals to the agitator, ventilation system and acid-base adjustment equipment to achieve precise control of pH value and dissolved oxygen content; Human-machine interface: provides a platform for operators to interact with the control system and displays the parameters of the current fermentation process; The pH electrode and the pH electrode of the control system measure the pH value in the fermentation liquid in real time and transmit the signal to the data acquisition and processing unit in the control system; the control system determines whether the pH value of the fermentation liquid needs to be adjusted according to the received pH value signal, and adjusts the pH value to the set range by controlling the device for adding acid and alkali solution; The dissolved oxygen electrode and the dissolved oxygen electrode of the control system measure the dissolved oxygen content in the fermentation liquid in real time and transmit the signal to the control system; the control system adjusts the speed of the agitator and the ventilation volume of the ventilation system through the logic control unit according to the dissolved oxygen signal to keep the dissolved oxygen content within the set range; The agitator of the agitator and ventilation system affects the dissolved oxygen content in the fermentation liquid by adjusting the rotation speed, and is linked to the feedback signal of the dissolved oxygen electrode; the ventilation system further controls the dissolved oxygen content by adjusting the ventilation volume, and works together with the agitator to ensure accurate control of the dissolved oxygen content.

2. Oligosaccharidase fermentation process control method based on pH and dissolved oxygen electrode feedback, characterized in that: The oligosaccharidase fermentation process control system based on pH and dissolved oxygen electrode feedback as claimed in claim 1 is carried out according to the following steps: S1, seed culture; Slant seed activation: Activate the slant seeds under suitable conditions for 4 hours to prepare for subsequent inoculation; Seed culture medium inoculation: inoculate the activated slant seeds into the seed culture medium and culture for 20 hours; In this stage, the shaking speed was controlled at 200 rpm and the temperature was 30°C; S2, shake flask fermentation; Shake flask preparation: Fill 50 ml of fermentation medium into a 500 ml Erlenmeyer flask; Inoculation and cultivation: Inoculate the seeds into the fermentation medium at a 10% inoculation rate, maintain the shaker speed at 200 rpm, set the temperature at 30°C, and cultivate for 30 hours; S3, fermentation tank culture; S31, fermentation tank preparation: the fermentation tank is filled with 4L of liquid, the inoculation amount is 10% of the fermentation liquid, the ventilation volume is set to 5.0L / min, and the temperature is controlled at 30°C; S32, pH value control: Use a pH electrode to measure the pH value of the fermentation liquid online, and adjust the pH value to 5.5 by automatically adding 3 mol / L H2SO4 and NaOH solution; S33, dissolved oxygen control: Initial control: The initial stirring speed was set at 300 rpm, and the stirring speed was adjusted in real time to maintain a constant dissolved oxygen concentration; Early stage: dissolved oxygen levels drop naturally, and no additional control measures are taken; Logarithmic growth period: When the dissolved oxygen drops to 15%, the dissolved oxygen level is maintained at 15% by linking the stirring speed with the dissolved oxygen; Later stage: Continue to control the dissolved oxygen level at 45% by linking the stirring speed with the dissolved oxygen until the fermentation is completed; S4. Monitoring and evaluation; S41, determination of bacterial concentration: regularly determine the bacterial concentration in the fermentation broth by dry weight method; the specific operation is to take 50 mL of fermentation broth and filter to obtain bacterial cells, dry to constant weight and weigh, and subtract the weight of the filter paper to obtain the actual bacterial concentration; S42, enzyme activity determination: oligosaccharidase activity was determined by spectrophotometry; an appropriate amount of diluted enzyme solution was added to the salicin solution and kept warm, and then the DNS reagent was added to perform a color reaction, and finally the absorbance was measured and the enzyme activity was calculated; S5. Optimization and adjustment; S51, ventilation ratio optimization: optimize biomass and enzyme activity by changing the ventilation ratio to find the best ventilation conditions; S52, linkage optimization of dissolved oxygen and stirring: in the early stage, logarithmic growth stage and late stage of the fermentation process, precise regulation of dissolved oxygen is achieved through linkage control of stirring speed and dissolved oxygen level, thereby increasing biomass and enzyme activity; S53. Product synthesis kinetics analysis: Optimize fermentation control by analyzing the relationship between product synthesis rate qP and cell growth rate μ.

3. The oligosaccharidase fermentation process control method based on pH and dissolved oxygen electrode feedback according to claim 2, characterized in that: The specific process of step S41 includes: S411, sampling: regularly take out 50 mL of fermentation liquid from the fermentation tank as a sample; S412, filtration: filtering the sampled fermentation liquid through filter paper to separate the bacteria in the fermentation liquid; S413, drying: placing the bacteria on the filter paper in an oven at 100° C. for drying until the bacteria reach a constant weight; maintaining a constant temperature during the drying process to prevent the bacteria from being burned; S414. Weighing: weigh the dried filter paper and the bacteria together, and then subtract the weight of the filter paper to obtain the actual dry weight of the bacteria; this dry weight is the concentration of the bacteria in the fermentation liquid.

4. The oligosaccharidase fermentation process control method based on pH and dissolved oxygen electrode feedback according to claim 1, characterized in that: The specific process of step S42 includes: S421, preparing enzyme solution: taking out an appropriate amount of enzyme solution from the fermentation solution and diluting it appropriately to ensure that the concentration of the enzyme solution is within the measurement range of the spectrophotometer; S422, adding substrate: taking a certain amount of 0.5 mL of diluted enzyme solution, adding 1 mL of 1.0% salicin solution; salicin is a common substrate of oligosaccharidase and is used to detect enzyme activity; S423, heat preservation reaction: place the mixed solution in a 50°C water bath for a certain period of time of 30 minutes to allow the enzyme and substrate to fully react; S424, color reaction: add 3 mL of DNS reagent to the reaction solution, wherein the DNS reagent is 3,5-dinitrosalicylic acid, and then place the solution in a boiling water bath and heat for 10 minutes to color the reaction product; the DNS reagent can react with reducing sugar to generate a colored compound, which is convenient for subsequent absorbance measurement; S425, cooling and diluting: cooling the reaction solution after color development to room temperature, and diluting it to 25 mL with water for measurement on a spectrophotometer; S426, measuring absorbance: setting the wavelength to 540 nm on the spectrophotometer, and measuring the absorbance of the diluted reaction solution; the absorbance is proportional to the enzyme activity, and the actual enzyme activity is calculated by calibration using a standard curve or a sample with known enzyme activity; S427. Calculate enzyme activity: Calculate the activity unit of oligosaccharidase based on the measured absorbance value and the known standard curve or correction factor; the activity unit of oligosaccharidase is the amount of enzyme required to release 1 μmol of reducing sugar from a 1.0% salicin solution per minute under the conditions of 50°C and pH 4.

8.

5. The oligosaccharidase fermentation process control method based on pH and dissolved oxygen electrode feedback according to claim 2, characterized in that: The specific process of step S51 includes: S511, selecting a series of different ventilation ratios as experimental conditions, and performing multiple repeated experiments under each ventilation ratio condition to reduce random errors; and gradually adjusting the ventilation ratio to a predetermined value at different stages during the fermentation process; S512, using a dissolved oxygen electrode to monitor the dissolved oxygen content in the fermentation liquid in real time, and simultaneously observing and recording key parameters such as pH value, temperature, and stirring speed during the fermentation process; S513. After the fermentation is completed, the bacterial concentration, i.e., biomass, of each experimental group is determined by the dry weight method, and the biomass and enzyme activity data under different ventilation ratios are compared. The influence of ventilation ratio on the fermentation effect is analyzed by statistical methods; it is determined under which ventilation ratio the biomass and enzyme activity are optimal; and repeated experiments are carried out under the selected optimal ventilation ratio to verify.

6. The oligosaccharidase fermentation process control method based on pH and dissolved oxygen electrode feedback according to claim 2, characterized in that: The specific process of step S52 includes: S521, dissolved oxygen monitoring: when the dissolved oxygen content drops to 15% of the preset value, the linkage control of the stirring speed and the dissolved oxygen level is started; S522, adjusting the stirring speed: according to the feedback signal of the dissolved oxygen electrode, automatically adjust the stirring speed to maintain the dissolved oxygen content at 15%; if the dissolved oxygen has an upward trend, appropriately reduce the stirring speed; if the dissolved oxygen has a downward trend, increase the stirring speed; S523, maintain stable ventilation: At this stage, the ventilation volume can be kept relatively stable, or fine-tuned as needed to assist the stirring speed in maintaining the dissolved oxygen level; S524, dissolved oxygen staged control: according to the fermentation process requirements, the dissolved oxygen level is gradually increased to 45% in the late fermentation period; before each increase in the dissolved oxygen level, the stirring speed is quickly adjusted to make the dissolved oxygen close to the target value, and then gradually stabilized at this level; S525, Dynamic Adjustment: In the process of increasing the dissolved oxygen level, continuously monitor the changes in dissolved oxygen, and dynamically adjust the stirring speed and ventilation volume as needed to ensure that the dissolved oxygen is stable within the target range; S526. Monitor biomass and enzyme activity: During the process of staged dissolved oxygen increase, take samples regularly to measure biomass and enzyme activity, and evaluate the impact of changes in dissolved oxygen levels on fermentation results.

7. The oligosaccharidase fermentation process control method based on pH and dissolved oxygen electrode feedback according to claim 2, characterized in that: The specific process of step S53 includes: Samples were taken at different time points of the fermentation, and the product concentration was determined by spectrophotometry; the product synthesis rate qP was calculated based on the change in product concentration; qP was defined as the increase in product concentration per unit time divided by the biomass; Samples were taken at different time points of the fermentation to measure the biomass, and the cell growth rate μ was calculated based on the change in biomass; μ is usually defined as the natural logarithmic rate of change of biomass per unit time; Construct a mathematical relationship model between product synthesis rate qP and cell growth rate μqP=aμ+β; Among them, a represents the size of the product synthesis capacity under the condition of constant cell growth capacity; β represents the ability to synthesize products when the cell stops growing; The nonlinear regression method is used to estimate the parameters a and β in the model, and the model prediction results are compared with the experimental data to verify the accuracy and reliability of the model; The model is used to analyze the changing trends and influencing factors of the product synthesis capacity qp under different fermentation conditions; the fermentation control strategy is adjusted according to the analysis results, and the dissolved oxygen content, pH value, stirring speed, and ventilation volume parameters are adjusted to optimize the product synthesis rate and biomass accumulation; if the model shows that the product synthesis capacity is highest at a certain dissolved oxygen content, then this dissolved oxygen level can be maintained during the fermentation process.

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