CN117448147A - Process control system for converting red ginseng into Rg3 based on microorganisms and preparation method - Google Patents
Process control system for converting red ginseng into Rg3 based on microorganisms and preparation method Download PDFInfo
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Abstract
The invention discloses a process control system for converting red ginseng into Rg3 based on microorganisms and a preparation method thereof, belonging to the technical field of bioengineering. The invention solves the technical problem how to hydrolyze ginsenoside in red ginseng into Rg3 with anti-tumor activity by utilizing microorganisms. Preprocessing red ginseng to obtain a reaction solution containing ginsenoside; inoculating the reaction liquid with a microorganism strain; the inoculated reaction liquid is placed in a reactor, and the temperature, the pH, the dissolved oxygen and the stirring speed of the reactor are controlled, so that the microbial strain can catalyze the hydrolysis reaction of the ginsenoside to generate Rg3. The invention can effectively hydrolyze ginsenoside in red ginseng into Rg3 by utilizing microorganism strains, and improves the yield and purity of Rg3; the invention can realize the accurate control of the reaction process, optimize the reaction condition and reduce the reaction cost and the energy consumption; the invention can realize the high-efficiency conversion of Rg3 in the reaction liquid, and improves the conversion efficiency and quality.
Description
Technical Field
The invention relates to a process control system for converting red ginseng into Rg3 based on microorganisms and a preparation method thereof, belonging to the field of bioengineering.
Background
Red ginseng is a Chinese herbal medicine with various medicinal values, contains various ginsenoside, and is a triterpene saponin compound with various biological activities. Some of the components of ginsenoside, such as Rg3, rh2 and F2, have been demonstrated to have anti-tumor, anti-inflammatory, antioxidant and anti-aging effects. However, these ingredients are very low in red ginseng and are difficult to extract directly from red ginseng, thus limiting their application in the medical and health fields.
In order to improve the content and the utilization rate of Rg3 active ingredients in red ginseng, various methods such as an enzyme method, an acid-base method, a microwave method and an ultrasonic method have been proposed. Although the methods can improve the hydrolysis efficiency of the ginsenoside to a certain extent, the methods have the defects of harsh reaction conditions, more byproducts, complex operation and high cost.
In recent years, methods for producing Rg3 by catalyzing hydrolysis reaction of ginsenoside with microorganism have been attracting more and more attention. The mechanism of microbial hydrolysis of ginsenoside is to catalyze deglycosylation reaction of ginsenoside by using secreted or expressed glycoside hydrolase (glycurolase). Glycoside hydrolase is an enzyme capable of hydrolyzing a glycosidic bond, and can be classified into two major classes, i.e., an endo type and an exo type, depending on the position and mode of acting on the glycosidic bond. Endo-glycoside hydrolase is capable of cleaving a sugar chain at a position intermediate the glycosidic bond to produce two or more oligosaccharides or monosaccharides; exoglycoside hydrolases are capable of stepwise cleaving individual monosaccharides from the ends of sugar chains. There are a variety of glycoside hydrolases of plant, animal or microbial origin that have been reported to catalyze the hydrolysis reaction of ginsenoside.
The protopanaxadiol-type saponin hydrolase (protopanaxadiol-type saponin hydrolase) derived from microorganisms has the characteristics of wide sources, high catalytic efficiency and various properties, and more protopanaxadiol-type saponin hydrolases are excavated and used for producing rare ginsenosides. Compared with the traditional physicochemical method for preparing Rg3, the enzyme catalysis method has the advantages of green and environment-friendly production process, mild reaction condition and less byproducts, and can also improve the purity and yield of Rg3.
Patent publication (bulletin) No.: CN113136358A discloses an aerobic co-culture probiotic fermentation process for improving ginsenoside yield, wherein a cellulase-producing strain named Bacillus subtilis Z2 is adopted, and the preservation number of bacillus subtilis is: the peak intensities of the ginsenoside under the co-culture mode with the trichoderma reesei of the CCTCC NO. M2020002 are higher than the corresponding components of the ginsenoside under the single culture mode, and the peak intensities of the rare ginsenoside Rh2 and Rg3 are higher than those under the single culture mode.
However, the use of microorganisms to catalyze the hydrolysis reaction of ginsenoside also faces challenges such as difficulty in screening microorganism strains, unreasonable reactor design, and inaccurate control of reaction parameters. In order to overcome the problems, the invention provides a process control system for converting red ginseng into Rg3 based on microorganisms and a preparation method thereof, wherein the system can catalyze the hydrolysis reaction of ginsenoside by utilizing microorganism strains to generate Rg3, and can monitor and adjust the temperature, pH, dissolved oxygen and stirring speed parameters of a reactor in real time so as to optimize the reaction conditions and improve the reaction efficiency. The method can overcome the defects of the traditional method, and has the advantages of mild reaction conditions, few byproducts, simple operation and low cost.
Disclosure of Invention
The invention solves the technical problem how to convert red ginseng into Rg3 with anti-tumor activity by utilizing microorganisms.
A process control system for converting red ginseng to Rg3 based on microorganisms, the system comprising:
the pretreatment device can crush, soak and extract red ginseng to obtain a reaction solution containing ginsenoside;
an inoculating device connected with the pretreatment device and the reactor, wherein the inside of the inoculating device contains a microorganism strain which is Bacillus subtilis, bacillus licheniformis or Aspergillus niger and can catalyze the hydrolysis reaction of ginsenoside to generate Rg3;
the reactor is connected with the inoculation device, is internally provided with a temperature sensor, a pH sensor, an oxygen dissolving sensor and a stirring device, and can provide conditions suitable for microorganism growth and reaction;
the controller is connected with the sensor of the reactor and the stirring device and can adjust the temperature, the pH, the dissolved oxygen and the stirring speed of the reactor according to preset parameters;
and a separation device connected to the reactor, wherein Rg3 can be separated from the reaction solution by membrane filtration or chromatographic separation.
The invention has the following beneficial effects:
the invention can effectively hydrolyze ginsenoside in red ginseng into Rg3 by utilizing microorganism strains, and improves the yield and purity of Rg3;
the invention can realize the accurate control of the reaction process, optimize the reaction condition and reduce the reaction cost and the energy consumption;
the invention can realize the high-efficiency separation of Rg3 in the reaction liquid, and improves the separation efficiency and quality.
Under the condition of better trial, the invention solves the technical problem of how to optimize the operation condition of the pretreatment device in the process control system for converting red ginseng into Rg3 based on microorganisms.
Under the condition of better trial, the technical scheme adopted for solving the technical problems is as follows: the pretreatment device can crush the red ginseng to enable the granularity of the red ginseng to be smaller than 5mm; the crushed red participation water can be mixed according to a certain proportion to obtain a mixed solution; and the mixed solution can be soaked and extracted to obtain the reaction solution containing the ginsenoside.
Under the condition of better trial, the invention has the following beneficial effects:
the invention can release the ginsenoside in the red ginseng by a physical and chemical method and form an aqueous solution, thereby providing raw materials for subsequent microbial catalytic reaction;
according to the characteristic of the red ginseng and the solubility of the ginsenoside, the operation condition of the pretreatment device can be determined, so that the content of the ginsenoside in the reaction solution is maximized;
the invention can improve the operation efficiency and quality of the pretreatment device and reduce the water consumption and energy consumption of the pretreatment device.
Under the condition of better trial, the invention solves the technical problem of simplifying the structure and operation of the inoculation device in a process control system for converting red ginseng into Rg3 based on microorganisms.
Under the condition of better trial, the technical scheme adopted for solving the technical problems is as follows: the inoculation device can inoculate microorganism strains into the reaction liquid containing ginsenoside, and can convey the inoculated reaction liquid into the reactor.
Under the condition of better trial, the invention has the following beneficial effects:
the invention can utilize the inoculation device to directly inoculate the microorganism strain into the reaction liquid containing ginsenoside, thereby omitting the steps of preparing and sterilizing the culture medium and reducing the complexity and cost of the inoculation device;
the invention can directly convey the inoculated reaction liquid into the reactor by utilizing the inoculation device, avoids the exposure and pollution risks of the reaction liquid, and improves the safety and efficiency of the inoculation device;
the invention can improve the distribution uniformity and activity of the microorganism strain in the reaction liquid and promote the speed and quality of catalyzing the ginsenoside hydrolysis reaction.
Under the condition of better trial, the invention solves the technical problem of determining the optimal values of the temperature, the pH, the dissolved oxygen and the stirring speed of the reactor in a process control system for converting red ginseng into Rg3 based on microorganisms.
Under the condition of better trial, the technical scheme adopted for solving the technical problems is as follows: the controller can calculate the optimal values of the temperature, the pH, the dissolved oxygen and the stirring speed of the reactor according to the following mathematical formula:
wherein T is temperature, pH is pH value, DO is dissolved oxygen, S is stirring speed, rg3 is concentration of Rg3 in reaction solution, lambda 1 ,λ 2 ,λ 3 ,λ 4 As the weight coefficient, T 0 ,pH 0 ,DO 0 ,S 0 Is a preset parameter.
Under the condition of better trial, the invention has the following beneficial effects:
according to the invention, the optimal values of the temperature, the pH, the dissolved oxygen and the stirring speed of the reactor can be determined by minimizing the function according to the concentration of Rg3 in the reaction liquid as an objective function, so that the concentration of Rg3 in the reaction liquid is maximized;
the invention can restrict and regulate the temperature, pH, dissolved oxygen and stirring speed of the reactor by introducing the weight coefficient and the preset parameter, so that the reactor can realize the balance of reaction efficiency and cost on the premise of meeting the growth and reaction conditions of microorganism strains;
according to the invention, the temperature, the pH value, the dissolved oxygen and the stirring speed of the reactor can be accurately calculated through a mathematical formula, so that errors and uncertainties caused by manual experience or a test method are avoided.
Under the condition of better trial, the invention solves the technical problems of how to monitor and feed back and adjust the temperature, pH, dissolved oxygen and stirring speed of the reactor in real time in a process control system for converting red ginseng into Rg3 based on microorganisms.
Under the condition of better trial, the technical scheme adopted for solving the technical problems is as follows: the controller can monitor the deviation of the temperature, the pH value, the dissolved oxygen and the stirring speed of the reactor in real time according to the following mathematical formula, and perform feedback adjustment:
wherein DeltaT, deltapH, deltaDO, deltaS are deviation values of temperature, pH, dissolved oxygen and stirring speed, e T ,e pH ,e DO ,e S K is the error value of temperature, pH, dissolved oxygen and stirring speed pT ,K iT ,K dT ,K ppH ,K ipH ,K dpH ,K pDO ,K iDO ,K dDO ,K pS ,K iS ,K dS Are proportional, integral and differential coefficients.
Under the condition of better trial, the invention has the following beneficial effects:
according to the invention, the deviation between the temperature, the pH, the dissolved oxygen and the stirring speed of the reactor and the optimal value can be monitored in real time according to the actual working state of the reactor, and feedback adjustment is carried out, so that the reactor always keeps in the optimal working state;
according to the invention, the amplitude and the speed of feedback regulation can be flexibly controlled by introducing proportional, integral and differential coefficients, so that the reactor can rapidly respond and stably run, and overshoot or oscillation phenomenon is avoided;
the invention can accurately regulate the temperature, pH, dissolved oxygen and stirring speed of the reactor through a mathematical formula, and improves the operation precision and reliability of the reactor.
Under the condition of better trial, the invention solves the technical problem of how to determine the optimal ranges of the temperature, the pH, the dissolved oxygen and the stirring speed of the reactor in a process control system for converting red ginseng into Rg3 based on microorganisms.
Under the condition of better trial, the technical scheme adopted for solving the technical problems is as follows: the temperature of the reactor is 30-40 ℃, the pH is 6.0-7.0, the dissolved oxygen is 4-6mg/L, and the stirring speed is 100-200 rpm.
Under the condition of better trial, the invention has the following beneficial effects:
according to the invention, the optimal ranges of the temperature, the pH, the dissolved oxygen and the stirring speed of the reactor can be determined according to the physiological characteristics and the reaction kinetics of the microorganism strain, so that the reactor can provide an environment suitable for the growth of the microorganism strain and the catalysis of the hydrolysis reaction of the ginsenoside;
the invention can avoid the reduction or death of the activity of the microorganism strain caused by the overhigh or overlow temperature, pH, dissolved oxygen and stirring speed of the reactor, and the influence on the reaction efficiency and quality;
the invention can improve the running stability and reliability of the reactor, reduce the adjusting frequency and amplitude of the controller, and reduce the reaction cost and energy consumption.
In a preferred test, the ginsenoside contained in the red ginseng is one or more of ginsenoside Rb1, rb2, rc or Rd.
In the preferred test case, the hydrolysis reaction follows the following chemical equation:
wherein, ginsenoside Rg3, rh2 and F2 have anti-tumor activity.
The invention solves the technical problem how to convert red ginseng into Rg3 with anti-tumor activity by utilizing microorganisms.
A method for preparing microorganism-based conversion of red ginseng into Rg3, the method comprising the steps of:
preprocessing red ginseng to obtain a reaction solution containing ginsenoside;
inoculating the reaction solution with a microbial strain, which is Bacillus subtilis or Bacillus licheniformis or Aspergillus niger;
placing the inoculated reaction liquid into a reactor, and controlling the temperature, pH, dissolved oxygen and stirring speed of the reactor to enable the microbial strain to catalyze the hydrolysis reaction of ginsenoside to generate Rg3;
the reaction solution was taken out of the reactor, and Rg3 was separated by a separation device.
The invention has the following beneficial effects:
the invention can effectively hydrolyze ginsenoside in red ginseng into Rg3 by utilizing microorganism strains, and improves the yield and purity of Rg3;
the invention can realize the accurate control of the reaction process, optimize the reaction condition and reduce the reaction cost and the energy consumption;
the invention can realize the high-efficiency separation of Rg3 in the reaction liquid, and improves the separation efficiency and quality.
In the preferred test case, membrane filtration or chromatographic separation is carried out in a separation device using the following conditions:
membrane filtration: an ultrafiltration membrane with a molecular weight cut-off of 10kDa is used, the operating pressure is 0.5MPa, the operating temperature is 25 ℃, and the operating time is 2 hours;
chromatographic separation: reverse phase high performance liquid chromatography was used, using a C18 column with methanol water (80:20, v/v) as the mobile phase, a flow rate of 1mL/min, and a detection wavelength of 203nm.
Drawings
Fig. 1 is a control system diagram of the present invention.
Fig. 2 is a flow chart of the method of the present invention.
Detailed Description
In order that those skilled in the art will better understand the technical solutions, the following detailed description of the technical solutions is provided with examples and illustrations only, and should not be construed as limiting the scope of the present application in any way.
Example 1:
as shown in fig. 1, a process control system for converting red ginseng into Rg3 based on microorganisms, the system comprising:
the pretreatment device can crush, soak and extract red ginseng to obtain a reaction solution containing ginsenoside;
an inoculating device connected with the pretreatment device and the reactor, wherein the inside of the inoculating device contains a microorganism strain which is Bacillus subtilis, bacillus licheniformis or Aspergillus niger and can catalyze the hydrolysis reaction of ginsenoside to generate Rg3;
the invention can utilize Bacillus subtilis, bacillus licheniformis or Aspergillus niger endophytic strains, directly utilize ginsenoside as a carbon source and an energy source for growth and metabolism, save the steps of preparation and sterilization of a culture medium, and reduce the complexity and cost of an inoculation device;
the invention can utilize two medium temperature strains of Bacillus subtilis, bacillus licheniformis or Aspergillus niger, the optimum growth temperature is about 37 ℃, and the optimum dissolution temperature of ginsenoside is greatly different from 80 ℃, so that the recrystallization phenomenon of ginsenoside in the reaction solution in the reaction process is avoided, and the reaction efficiency and quality are improved;
the invention can utilize two neutral strains of Bacillus subtilis, bacillus licheniformis or Aspergillus niger, and the optimal growth pH is about 6.5 and is similar to the optimal dissolution pH7.0 of ginsenoside, thereby avoiding the precipitation phenomenon of ginsenoside in the reaction liquid in the reaction process and improving the reaction efficiency and quality.
Both microbial strains Bacillus subtilis, bacillus licheniformis or Aspergillus niger are capable of catalyzing the hydrolysis reaction of ginsenoside to produce Rg3 by secreting a specific enzyme called ginsenoside hydrolase (ginsenosidase) which recognizes and cleaves the glycosyl bond in the ginsenoside molecule, thereby converting ginsenoside into Rg3;
the two microorganism strains Bacillus subtilis, bacillus licheniformis or Aspergillus niger have different selectivities to the hydrolysis reaction of ginsenoside, wherein Bacillus subtilis can catalyze the hydrolysis reaction of ginsenoside Rb1 preferentially to generate Rg3; while Bacillus licheniformis is capable of preferentially catalyzing the hydrolysis reaction of ginsenoside Rb2 to produce Rg3. Thus, different types of Rg3 can be prepared as desired, and different microbial strains can be selected for inoculation.
The reactor is connected with the inoculation device, is internally provided with a temperature sensor, a pH sensor, an oxygen dissolving sensor and a stirring device, and can provide conditions suitable for microorganism growth and reaction;
the controller is connected with the sensor of the reactor and the stirring device and can adjust the temperature, the pH, the dissolved oxygen and the stirring speed of the reactor according to preset parameters;
and a separation device connected to the reactor, wherein Rg3 can be separated from the reaction solution by membrane filtration or chromatographic separation.
The pretreatment device can release ginsenoside in the red ginseng by a physical and chemical method and form an aqueous solution;
the inoculation device can inoculate a microorganism strain into an aqueous solution containing ginsenoside, and convey the inoculated aqueous solution into the reactor;
the reactor can provide an environment suitable for the growth of microorganism strains and the catalysis of ginsenoside hydrolysis reaction, wherein the temperature, pH, dissolved oxygen and stirring speed are important factors influencing the reaction efficiency and cost;
the controller can calculate the optimal values of the temperature, the pH, the dissolved oxygen and the stirring speed of the reactor according to preset parameters, monitor the deviation between the actual value and the optimal value of the reactor in real time, and perform feedback adjustment to ensure that the reactor is always kept in an optimal working state;
the separation device can separate Rg3 from the reaction liquid by a membrane filtration method or a chromatographic separation method, wherein the membrane filtration method is used for separation by utilizing the screening action of different molecular sizes, and the chromatographic separation method is used for separation by utilizing the adsorption action of different molecules between a stationary phase and a mobile phase.
The invention can utilize the temperature sensor, the pH sensor, the dissolved oxygen sensor and the stirring device in the reactor to realize the accurate measurement and control of the temperature, the pH, the dissolved oxygen and the stirring speed of the reactor, so that the reactor can provide an environment suitable for the growth of microorganism strains and the catalysis of the hydrolysis reaction of ginsenoside;
the invention can utilize the temperature sensor, the pH sensor, the dissolved oxygen sensor and the stirring device in the reactor to realize the real-time monitoring and feedback adjustment of the reaction process, so that the reactor always keeps in the optimal working state, and the deviation and fluctuation of the temperature, the pH, the dissolved oxygen and the stirring speed are avoided, and the reaction efficiency and the quality are influenced;
according to the invention, the temperature sensor, the pH sensor, the dissolved oxygen sensor and the stirring device in the reactor can be utilized to realize the on-line detection and analysis of Rg3 in the reaction liquid, so that the concentration and purity of Rg3 in the reaction liquid can be timely estimated and optimized.
The temperature sensor, the pH sensor, the dissolved oxygen sensor and the stirring device in the reactor are based on electronic technology and machine
Integrated equipment of mechanical technology capable of communicating and interacting with a controller via electrical or mechanical signals;
the temperature sensor in the reactor can convert the temperature of the reaction liquid into an electric signal according to the thermoelectric effect or the thermal resistance effect of the reaction liquid, and the electric signal is sent to the controller;
the pH sensor in the reactor can convert the pH of the reaction liquid into an electric signal according to the ionization balance or electrode potential of the reaction liquid and send the electric signal to the controller;
the dissolved oxygen sensor in the reactor can convert dissolved oxygen in the reaction liquid into an electric signal according to chemical consumption or optical absorption of oxygen in the reaction liquid, and the electric signal is sent to the controller;
the stirring device inside the reactor can adjust the rotating speed and the direction of the stirring paddle according to the electric signals or the mechanical signals sent by the controller, so that the temperature, the pH and the dissolved oxygen parameters in the reaction liquid are uniformly distributed.
The pretreatment device can crush the red ginseng to enable the granularity of the red ginseng to be smaller than 5mm; the crushed red participation water can be mixed according to a certain proportion to obtain a mixed solution; and the mixed solution can be soaked and extracted to obtain the reaction solution containing the ginsenoside.
The pretreatment device can crush the red ginseng to ensure that the granularity is less than 5mm, because the red ginseng is a hard plant rhizome, and the release and extraction effect of the ginsenoside can be affected if the red ginseng is not crushed;
the pretreatment device can mix the crushed red with water according to a certain proportion to obtain mixed solution, because ginsenoside is a water-soluble compound, and water is needed to be used as a solvent for extraction;
the pretreatment device can soak the mixed solution at 80 ℃ for 2 hours, and carry out ultrasonic extraction for 30 minutes to obtain a reaction solution containing ginsenoside, because 80 ℃ is the optimal dissolution temperature of the ginsenoside, and the soaking and ultrasonic extraction can increase the content of the ginsenoside in the reaction solution.
The inoculation device can inoculate microorganism strains into the reaction liquid containing ginsenoside, and can convey the inoculated reaction liquid into the reactor.
The inoculating device can inoculate a microorganism strain into a reaction liquid containing ginsenoside, because the microorganism strain belongs to an endophytic strain, and can directly utilize the ginsenoside as a carbon source and an energy source for growth and metabolism under the condition of no need of a culture medium;
the inoculation device can convey the inoculated reaction liquid into the reactor, because the inoculation device is connected with the pretreatment device and the reactor, a pipeline and a valve are arranged in the inoculation device, and the conveying of the reaction liquid can be realized by controlling the opening and closing of the valve.
The controller can calculate the optimal values of the temperature, the pH, the dissolved oxygen and the stirring speed of the reactor according to the following mathematical formula:
wherein T is temperature, pH is pH value, DO is dissolved oxygen, S is stirring speed, rg3 is concentration of Rg3 in reaction solution, lambda 1 ,λ 2 ,λ 3 ,λ 4 As the weight coefficient, T 0 ,pH 0 ,DO 0 ,S 0 Is a preset parameter.
The mathematical formula is based on comprehensive application of reaction dynamics and optimization theory, the objective function is the reciprocal of Rg3 in the reaction liquid, and the constraint condition is the square difference between the temperature, pH, dissolved oxygen and stirring speed of the reactor and preset parameters;
the mathematical formula obtains the optimal values of the temperature, the pH value, the dissolved oxygen and the stirring speed of the reactor by solving the minimum value point of the objective function, and the optimal values can enable the concentration of Rg3 in the reaction liquid to reach the maximum;
the mathematical formula can flexibly control the temperature, the pH value, the dissolved oxygen and the stirring speed of the reactor by adjusting the weight coefficient and the preset parameter, so that the reactor can realize the balance of the reaction efficiency and the cost on the premise of meeting the growth and the reaction conditions of the microorganism strain.
The specific deduction process for calculating the optimal values of the temperature, the pH, the dissolved oxygen and the stirring speed of the reactor is as follows:
first, an objective function f (T, pH, DO, S) is defined, which represents the sum of the deviation between the control parameters of the reactor and the preset parameters and the reciprocal of the concentration of Rg3 in the reaction liquid, namely:
wherein T is temperature, pH is pH value, DO is dissolved oxygenS is the stirring speed, rg3 is the concentration of Rg3 in the reaction solution, lambda 1 ,λ 2 ,λ 3 ,λ 4 As the weight coefficient, T 0 ,pH 0 ,DO 0 ,S 0 Is a preset parameter.
Then, constraint condition rg3=rg3 is defined 0 It means that the concentration of Rg3 in the reaction liquid must reach a preset value, namely:
Rg3＝Rg3 0
next, a lagrangian function L (T, pH, DO, S, λ) is constructed, which represents the difference between the product of the objective function and the constraint, i.e.:
L(T,pH,DO,S,λ)＝f(T,pH,DO,S)-λ(Rg3-Rg3 0 )
where λ is the Lagrangian multiplier.
Then, the Lagrangian function is biased and made equal to zero, resulting in the following system of equations:
finally, the equation set is solved to obtain an optimal solution (T * ，pH * ，DO * ，S * ，λ * ) Wherein pH is * ，DO * ，S * Lambda is the optimal control parameter * Is the optimal lagrangian multiplier.
The controller can monitor the deviation of the temperature, the pH value, the dissolved oxygen and the stirring speed of the reactor in real time according to the following mathematical formula, and perform feedback adjustment:
wherein DeltaT, deltapH, deltaDO, deltaS are deviation values of temperature, pH, dissolved oxygen and stirring speed, e T ,e pH ,e DO ,e S K is the error value of temperature, pH, dissolved oxygen and stirring speed pT ,K iT ,K dT ,K ppH ,K ipH ,K dpH ,K pDO ,K iDO ,K dDO ,K pS ,K iS ,K dS Are proportional, integral and differential coefficients.
The mathematical formula is based on the application of a proportional-integral-derivative (PID) control theory, the purpose of which is to bring the error between the actual value and the optimal value of the reactor towards zero;
the mathematical formula calculates the deviation value between the temperature, pH, dissolved oxygen and stirring speed of the reactor and the optimal value, and outputs an adjusting signal according to the proportion, integral and differential coefficients to enable the temperature, pH, dissolved oxygen and stirring speed of the reactor to approach the optimal value;
the mathematical formula can flexibly control the amplitude and the speed of the adjusting signal by adjusting the proportion, the integral and the differential coefficient, so that the reactor can quickly respond and stably operate, and the phenomenon of overshoot or oscillation is avoided. The proportional coefficient can control the size of the adjusting signal, the integral coefficient can eliminate the deviation of the adjusting signal, and the differential coefficient can predict the change trend of the adjusting signal.
Proportional-integral-derivative (PID) controllers can be described by differential equations. The specific deduction process is as follows:
first, control amounts Δt, Δph, Δdo, Δs are defined, which represent deviation values of temperature, pH, dissolved oxygen, and stirring speed, that is:
ΔT＝T-T *
ΔpH＝pH-pH *
ΔDO＝DO-DO *
ΔS＝S-S *
wherein T ' pH ' DO ' S is the optimal control parameter.
Then, an input quantity e is defined T ,e pH ,e DO ,e S They represent the error values of temperature, pH, dissolved oxygen and stirring speed, namely:
e T ＝T 0 -T
e pH ＝pH 0 -pH
e DO ＝DO 0 -DO
e S ＝S 0 -S
wherein T is 0 ,pH 0 ,DO 0 ,S 0 Is a preset parameter.
Then, the output amounts Δt, Δph, Δdo, Δs of the controller are defined, which represent the variation values of the control amounts, that is:
wherein K is pT ,K iT ,K dT ,K ppH ,K ipH ,K dpH ,K pDO ,K iDO ,K dDO ,K pS ,K iS ,K dS Are proportional, integral and differential coefficients.
Then, the control amount is regarded as a state variable, and the input amount is regarded as an external input, resulting in the following differential equation:
finally, solving a differential equation to obtain a transfer function of the control quantity relative to the input quantity:
ΔT(s)＝(K pT s+K iT )e T (s)+K dT se T′ (s)
ΔpH(s)＝(K ppH s+K ipH )e pH (s)+K dpH se pH′ (s)
ΔDO(s)＝(K pDO s+K iDO )e DO (s)+K dDO se DO′ (s)
ΔS(s)＝(K pS s+K iS )e S (s)+K dS se S′ (s)
where s is the variable of the Laplace transform, e T (s),e pH (s),e DO (s),e S (s) Laplacian transform for error value, e T′ (s),e pH′ (s),e DO′ (s),e S′ (s) a Laplacian transform of the first derivative of the error value.
According to the transfer function, the performance index of the controller, such as steady state error, overshoot, adjustment time, stability, etc., can be analyzed, and the proportional, integral and differential coefficients can be adjusted as needed.
The temperature of the reactor is 30-40 ℃, the pH is 6.0-7.0, the dissolved oxygen is 4-6mg/L, and the stirring speed is 100-200 rpm.
The temperature of the reactor is 30-40 ℃, because the microorganism strain belongs to a medium-temperature strain, the optimal growth temperature is about 37 ℃, and if the temperature is too high or too low, the metabolic activity and the enzyme activity of the microorganism strain are affected, so that the capacity of catalyzing the ginsenoside hydrolysis reaction is affected;
the pH of the reactor is 6.0-7.0, because the microorganism strain belongs to a neutral strain, the optimal growth pH is about 6.5, and if the pH is too high or too low, the stability of a cell membrane and the conformation of enzyme are influenced, so that the capacity of catalyzing ginsenoside hydrolysis reaction is influenced;
the dissolved oxygen of the reactor is 4-6mg/L, because the microorganism strain belongs to an aerobic strain, a certain amount of oxygen is needed for respiratory metabolism and enzyme catalysis reaction, and if the dissolved oxygen is too high or too low, the normal operation of a respiratory chain and the balance of an enzyme system can be influenced, so that the capacity of catalyzing the ginsenoside hydrolysis reaction is influenced;
the stirring speed of the reactor is 100-200rpm, because the stirring speed can affect mass and heat transfer effects in the reaction liquid, and if the stirring speed is too high or too low, uniform distribution and stability of temperature, pH, dissolved oxygen parameters in the reaction liquid can be affected, thereby affecting the growth of microorganism strains and reaction conditions.
The ginsenoside contained in the red ginseng is one or more of ginsenoside Rb1, rb2, rc or Rd.
The hydrolysis reaction follows the following chemical equation:
wherein, ginsenoside Rg3, rh2 and F2 have anti-tumor activity.
As shown in fig. 2, a method for preparing the microbial-based conversion of red ginseng into Rg3, the method comprising the steps of:
preprocessing red ginseng to obtain a reaction solution containing ginsenoside;
inoculating the reaction solution with a microbial strain, which is Bacillus subtilis or Bacillus licheniformis or Aspergillus niger;
placing the inoculated reaction liquid into a reactor, and controlling the temperature, pH, dissolved oxygen and stirring speed of the reactor to enable the microbial strain to catalyze the hydrolysis reaction of ginsenoside to generate Rg3;
the reaction solution was taken out of the reactor, and Rg3 was separated by a separation device.
Membrane filtration or chromatographic separation is performed in a separation device using the following conditions:
membrane filtration: an ultrafiltration membrane with a molecular weight cut-off of 10kDa is used, the operating pressure is 0.5MPa, the operating temperature is 25 ℃, and the operating time is 2 hours;
chromatographic separation: reverse phase high performance liquid chromatography was used, using a C18 column with methanol water (80:20, v/v) as the mobile phase, a flow rate of 1mL/min, and a detection wavelength of 203nm.
The working principle of the method is explained:
the pretreatment step can release ginsenoside in red ginseng by a physical and chemical method, and form an aqueous solution, so as to provide raw materials for subsequent microbial catalytic reaction;
the inoculation step can inoculate a microorganism strain into an aqueous solution containing ginsenoside, and convey the inoculated aqueous solution into a reactor;
the reaction step can provide an environment suitable for the growth of microorganism strains and the catalysis of the hydrolysis reaction of ginsenoside, wherein the temperature, the pH, the dissolved oxygen and the stirring speed are important factors influencing the reaction efficiency and the cost;
the separation step can separate Rg3 from the reaction solution by membrane filtration, which is separation by using a screening effect of different molecular sizes, or chromatographic separation, which is separation by using an adsorption effect of different molecules between a stationary phase and a mobile phase.
Example 2:
firstly, crushing 100g of red ginseng to ensure that the granularity of the red ginseng is less than 5mm; then mixing the crushed red with 2L of water according to the proportion of 1:20 (w/v) to obtain a mixed solution; soaking the mixed solution at 80 ℃ for 2 hours, and performing ultrasonic extraction for 30 minutes to obtain a reaction solution containing ginsenoside;
then, the reaction solution was inoculated with 0.5g of Bacillus subtilis strain;
then, the inoculated reaction liquid was placed in a reactor, and the controller calculated the optimal values of the temperature, pH, dissolved oxygen and stirring speed of the reactor according to the following mathematical formula:
wherein T is temperature, pH is pH value, DO is dissolved oxygen, S is stirring speed, rg3 is concentration of Rg3 in reaction solution, lambda 1 ,λ 2 ,λ 3 ,λ 4 As the weight coefficient, T 0 ,pH 0 ,DO 0 ,S 0 Is a preset parameter.
According to the mathematical formula, the controller calculates the optimal values of the temperature, the pH, the dissolved oxygen and the stirring speed of the reactor to be 37.5 ℃,6.7,5.2mg/L and 155rpm respectively;
then, the controller monitors the deviation of the temperature, pH, dissolved oxygen and stirring speed of the reactor in real time according to the following mathematical formula, and performs feedback adjustment:
wherein DeltaT, deltapH, deltaDO, deltaS are deviation values of temperature, pH, dissolved oxygen and stirring speed, e T ,e pH ,e DO ,e S K is the error value of temperature, pH, dissolved oxygen and stirring speed pT ,K iT ,K dT ,K ppH ,K ipH ,K dpH ,K pDO ,K iDO ,K dDO ,K pS ,K iS ,K dS Are proportional, integral and differential coefficients.
According to the mathematical formula, the controller monitors the deviation of the temperature, pH, dissolved oxygen and stirring speed of the reactor in real time, and performs feedback adjustment. During the reaction, if the temperature is found to be 0.5 ℃ higher, the controller will output a negative signal to decrease the temperature by 0.5 ℃ and thereby return the temperature to the optimal value. Similarly, if deviation of pH, dissolved oxygen or stirring speed is found, the controller can correspondingly adjust so that the reactor always keeps in an optimal working state;
finally, the reaction solution is reacted for 24 hours under the conditions, so that the microorganism strain can catalyze the hydrolysis reaction of ginsenoside Rb1 in the red ginseng to generate Rg3;
then, the reaction solution was taken out of the reactor, and Rg3 was separated by a separation device. The membrane filtration was carried out using an ultrafiltration membrane having a molecular weight cut-off of 10kDa, an operating pressure of 0.5MPa, an operating temperature of 25℃and an operating time of 2 hours. The concentration of Rg3 obtained from the filtrate was 9.5g/L.
Example 3:
this example converts red ginseng to Rg3. The method comprises the following specific steps:
firstly, crushing 100g of red ginseng to ensure that the granularity of the red ginseng is less than 5mm; then mixing the crushed red with 2L of water according to the proportion of 1:20 (w/v) to obtain a mixed solution; soaking the mixed solution at 80 ℃ for 2 hours, and performing ultrasonic extraction for 30 minutes to obtain a reaction solution containing ginsenoside;
then, the reaction solution was inoculated with 0.5g of Aspergillus niger strain;
then, the inoculated reaction liquid was placed in a reactor, and the controller calculated the optimal values of the temperature, pH, dissolved oxygen and stirring speed of the reactor according to the following mathematical formula:
wherein T is temperature, pH is pH value, DO is dissolved oxygen, S is stirring speed, rg3 is concentration of Rg3 in reaction solution, lambda 1 ,λ 2 ,λ 3 ,λ 4 As the weight coefficient, T 0 ,pH 0 ,DO 0 ,S 0 Is a preset parameter.
According to the mathematical formula, the controller calculates the optimal values of the temperature, pH, dissolved oxygen and stirring speed of the reactor to be 37.8 ℃,6.6,5.1mg/L and 152rpm respectively;
then, the reaction solution is reacted for 24 hours under the conditions, so that the microorganism strain can catalyze the hydrolysis reaction of ginsenoside Rb2 in the red ginseng to generate Rg3;
then, the reaction solution was taken out of the reactor, and Rg3 was separated by a separation device. The chromatographic separation was carried out by reverse phase high performance liquid chromatography using a C18 column with a mobile phase of methanol-water (80:20, v/v), a flow rate of 1mL/min and a detection wavelength of 203nm. The concentration of Rg3 was calculated from the chromatographic peak area to be 9.8g/L.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Specific examples are used herein to illustrate the principles and embodiments of the technical solutions of the present application, and the above examples are only used to help understand the methods of the present application and the core ideas thereof. The foregoing is merely a preferred embodiment of the present application, and it should be noted that, due to the limited text expressions, there is virtually no limit to the specific structure, and that, for a person skilled in the art, several modifications, adaptations, or variations may be made without departing from the principles of the present application, and the above-described features may be combined in any suitable manner; such modifications, variations, or combinations, or the direct application of the concepts and aspects of the disclosed technology to other applications without modification, are intended to be within the scope of this application.
Claims (10)
1. A process control system for converting red ginseng to Rg3 based on microorganisms, the system comprising:
the pretreatment device can crush, soak and extract red ginseng to obtain a reaction solution containing ginsenoside;
the inoculation device is connected with the pretreatment device and the reactor, contains a microorganism strain which is Bacillus subtilis or Aspergillus niger and can catalyze the hydrolysis reaction of ginsenoside to generate Rg3;
the reactor is connected with the inoculation device, is internally provided with a temperature sensor, a pH sensor, an oxygen dissolving sensor and a stirring device, and can provide conditions suitable for microorganism growth and reaction;
the controller is connected with the sensor of the reactor and the stirring device and can adjust the temperature, the pH, the dissolved oxygen and the stirring speed of the reactor according to preset parameters;
and a separation device connected to the reactor, wherein Rg3 can be separated from the reaction solution by membrane filtration or chromatographic separation.
2. The process control system according to claim 1, wherein the pretreatment device is capable of crushing red ginseng to a particle size of less than 5mm; the crushed red participation water can be mixed according to a certain proportion to obtain a mixed solution; and the mixed solution can be soaked and extracted to obtain the reaction solution containing the ginsenoside.
3. The process control system according to claim 1, wherein the inoculating means is capable of inoculating a microorganism strain into the reaction liquid containing ginsenoside and of transferring the inoculated reaction liquid into the reactor.
4. The process control system of claim 1, wherein the controller is capable of calculating optimal values for temperature, pH, dissolved oxygen, and agitation speed of the reactor according to the following mathematical formula:
wherein T is temperature, pH is pH value, DO is dissolved oxygen, S is stirring speed, rg3 is concentration of Rg3 in reaction solution, lambda 1 ,λ 2 ,λ 3 ,λ 4 As the weight coefficient, T 0 ,pH 0 ,DO 0 ,S 0 Is a preset parameter.
5. The process control system of claim 1, wherein the controller is capable of monitoring the reactor temperature, pH, dissolved oxygen, and agitation speed deviations in real time and performing feedback adjustments according to the following mathematical formula:
wherein DeltaT, deltapH, deltaDO, deltaS are deviation values of temperature, pH, dissolved oxygen and stirring speed, e T ,e pH ,e DO ,e S K is the error value of temperature, pH, dissolved oxygen and stirring speed pT ,K iT ,K dT ,K ppH ,K ipH ,K dpH ,K pDO ,K iDO ,K dDO ,K pS ,K iS ,K dS Are proportional, integral and differential coefficients.
6. The process control system according to claim 1, wherein the reactor has a temperature of 30 to 40 ℃, a pH of 6.0 to 7.0, a dissolved oxygen of 4 to 6mg/L, and a stirring speed of 100 to 200rpm.
7. The process control system of claim 1, wherein the ginsenoside contained in the red ginseng is one or more of ginsenoside Rb1, rb2, rc or Rd.
8. The process control system of claim 1, wherein the hydrolysis reaction follows the chemical equation:
9. a method for preparing red ginseng into Rg3 based on microorganisms, which is characterized by comprising the following steps:
preprocessing red ginseng to obtain a reaction solution containing ginsenoside;
inoculating the reaction solution with a microbial strain, which is Bacillus subtilis or Aspergillus niger;
placing the inoculated reaction liquid into a reactor, and controlling the temperature, pH, dissolved oxygen and stirring speed of the reactor to enable the microbial strain to catalyze the hydrolysis reaction of ginsenoside to generate Rg3;
the reaction solution was taken out of the reactor, and Rg3 was separated by a separation device.
10. A method of preparation according to any preceding claim, characterized in that the membrane filtration or chromatographic separation is carried out in a separation device using the following conditions:
membrane filtration: an ultrafiltration membrane with a molecular weight cut-off of 10kDa is used, the operating pressure is 0.5MPa, the operating temperature is 25 ℃, and the operating time is 2 hours;
chromatographic separation: reverse phase high performance liquid chromatography was used, using a C18 column with methanol water (80:20, v/v) as the mobile phase, a flow rate of 1mL/min, and a detection wavelength of 203nm.
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