CN119242520A - A high-density culture method of lactic acid bacteria based on pH control system - Google Patents

Abstract

The invention discloses a high-density culture method for lactic acid bacteria based on a pH control system, and relates to the technical field of lactic acid bacteria culture. The invention performs real-time monitoring on the lactic acid bacteria culture stage, adopts a pH sensor and a PID control algorithm, maintains the pH value in the range of 6.7±0.1 during the fermentation process, avoids excessive accumulation of lactic acid, avoids large fluctuations in the pH value, enables lactic acid bacteria to grow efficiently in a stable acid-base environment, greatly improves fermentation efficiency and bacterial yield, provides more effective pH control, accelerates the growth rate of lactic acid bacteria, and maintains high-density bacterial culture, performs automatic adjustment during the fermentation process, provides continuity and stability for the fermentation environment, shortens the fermentation time, significantly increases the biomass of lactic acid bacteria within the same fermentation cycle, improves the yield, and improves the overall production efficiency and economic benefits, the automated pH control system can continuously monitor the environment in the fermentation tank, and automatically adjusts when the pH value changes, reducing the risk of human error.

Description

High-density lactobacillus culture method based on pH control system

Technical Field

The invention relates to the technical field of lactobacillus culture, in particular to a high-density lactobacillus culture method based on a pH control system.

Background

The current high-density culture method of lactic acid bacteria faces a plurality of technical problems, particularly has a critical influence on the growth, metabolism and final yield of the lactic acid bacteria in the control of pH value in the fermentation process.

In the traditional scheme, a large amount of lactic acid is generated by lactic acid bacteria in the fermentation process, so that the pH value of fermentation liquid is rapidly reduced, if the pH value is excessively reduced, the growth and metabolism of the lactic acid bacteria are inhibited, the yield and quality are finally affected, in the traditional fermentation process, the pH value is usually monitored manually by manpower, and is regulated by adding alkaline solution (such as NaOH), the manual adjustment can partially relieve pH fluctuation, but frequent human intervention not only increases operation complexity, but also is easy to cause untimely reaction or inaccurate adjustment, the pH value can be greatly fluctuated in the manual adjustment process, the pH value can not be ensured to be always in the optimal growth range of lactobacillus, the fermentation efficiency is reduced, and finally the stability and the yield consistency of production are affected.

The production efficiency of lactobacillus fermentation is affected by various factors, especially when the pH value, temperature and other parameters in the fermentation process are controlled unstably, the growth rate of lactobacillus can be obviously reduced, the fermentation period is prolonged, the production efficiency is low, and therefore, a high-density lactobacillus culture method based on a pH control system is needed to solve the problems.

Disclosure of Invention

The present invention has been made in view of the above-described problems occurring in the prior art.

The invention provides a high-density lactobacillus culture method based on a pH control system, which solves the problems that the fermentation condition is maintained by mainly monitoring fermentation parameters periodically and manually adjusting temperature and pH value, real-time accurate adjustment cannot be realized, the metabolic state of thalli is often influenced by fluctuation in the fermentation process, and the fermentation time is overlong in the traditional method.

In order to solve the technical problems, the invention provides the following technical scheme:

the invention provides a high-density culture method of lactobacillus based on a pH control system, which comprises the following steps of,

Step S1, a material preparation is performed,

Comprises 3% of soybean peptone, 2% of yeast extract powder, 0.5% of sodium acetate, 0.1% of compound trace element liquid, 0.05% of compound vitamin liquid, 0.2% of trypsin, 0.5% of acid soluble protein (short peptide), 0.2% -0.4% of sodium citrate, 0.02% -0.04% of Mg2+ water-soluble compound, 0.01% -0.02% of Fe2+ water-soluble compound, 93.64% -94.62% of pure water,

Preparing MRS culture medium, adjusting pH to 7.0, and sterilizing the culture medium in an autoclave at 121 ℃ for 15 minutes;

Step S2, setting a fermentation tank,

Transferring the sterilized culture medium to a fermentation tank with a temperature control and pH control system under aseptic conditions;

s3, starting a pH control system and calibrating a pH sensor;

Step S4, real-time monitoring and adjusting,

When fermentation starts, the pH value of the culture solution in the fermentation tank is monitored in real time by the pH sensor,

Step S5, the process is maintained,

The temperature in the fermentation tank is kept constant at 37 ℃, the pH value is kept within a set range by a pH control system,

After the continuous fermentation is carried out for 30 hours, ending the fermentation and collecting the culture solution;

the pH control system is adopted to realize full-automatic monitoring and adjustment of the fermentation environment, the pH value is one of key factors for the growth of lactic acid bacteria in the fermentation process, the fluctuation of the pH value is large in the traditional fermentation method, the growth of thalli is unstable and the fluctuation of the yield and quality is often caused, and the pH value of fermentation liquid can be continuously collected by the pH sensor in real time, so that the inhibition of the growth of thalli caused by the fluctuation of the pH value is avoided.

Further, in step S2, the temperature of the fermenter is adjusted to 37 ℃.

Further, in step S3, the pH controller is set to 6.7±0.1.

Further, in step S4, when the pH value is lower than the preset lower limit value 6.6, the pH controller sends a signal to start the pump connected to the control system.

Further, in step S4, a 20% naoh solution is automatically added from the tank, and the pump is controlled by the flow rate, and an alkaline solution is added to restore the pH to the set range of 6.7±0.1.

Further, the pH control system assembly includes:

the pH sensor is used for detecting the pH value of the fermentation liquid in real time and transmitting data to the pH controller,

The pH controller is used for judging whether the pH value is in a set range according to the input data of the pH sensor, determining whether to start the pump to add acid or alkali, adopting a PID control algorithm to further improve the flexibility and precision of control in pH control, the PID control can dynamically adjust the operation of the pump, and the pH value is kept in a preset range by calculating the error value of the pH value and adding NaOH solution, so that the pH value in the fermentation tank is always in a proper range of lactobacillus, personal errors are reduced, and the pH value can react rapidly when the pH value is slightly changed, thereby effectively improving the growth rate and fermentation efficiency of lactobacillus.

Further, the pH control system assembly further comprises:

the pump and the liquid storage barrel are connected with the pH controller, and acid or alkali solution is added according to the requirement to keep the pH stable.

Further, in the high-density culture process of the lactic acid bacteria of the pH control system, the pH monitoring and adjusting modes comprise:

In the fermentation process, the pH value of the fermentation liquid is continuously collected by the pH sensor, the Kalman filtering is adopted to eliminate noise, the processed data is transmitted to the controller in real time,

And the controller adopts a PID control algorithm to make adjustment judgment according to the pH value information transmitted by the sensor.

Further, in the high-density culture process of the lactic acid bacteria of the pH control system, the pH monitoring and adjusting mode further comprises:

The PID control algorithm calculates the difference between the current pH value and the target pH value, and outputs the difference through the proportional, integral and differential term regulating controller to control the start or stop of the pump, the combination of the pH control system and the fermentation tank improves the automation degree and controllability of the whole fermentation process, provides the best condition for the efficient growth of the lactobacillus based on the combined regulation and control of the temperature and the pH, continuously and automatically regulates, reduces the dependence on manual operation, improves the production efficiency, and also reduces the pollution risk in the operation process.

Further, in the high-density culture process of the lactic acid bacteria of the pH control system, the pH monitoring and adjusting mode further comprises:

The pump calculates the quantity of NaOH solution conveyed by the pump by adopting a flow control algorithm based on the instruction of the controller, and the flow of the pump is regulated in real time according to the deviation of the pH value.

The invention has the beneficial effects that:

According to the invention, the lactobacillus culture stage is monitored in real time, the pH value in the fermentation process is maintained within the range of 6.7+/-0.1 by adopting the pH sensor and the PID control algorithm, the excessive accumulation of lactic acid is reduced, the great fluctuation of the pH value is avoided, the lactobacillus can grow in a stable acid-base environment with high efficiency, and the fermentation efficiency and the thallus yield are greatly improved.

The invention provides more effective pH control, accelerates the growth speed of lactobacillus, maintains high-density thallus culture, automatically adjusts in the fermentation process, provides continuity and stability for the fermentation environment, shortens the fermentation time, obviously increases the biomass of lactobacillus in the same fermentation period, improves the yield, and improves the overall production efficiency and economic benefit.

According to the invention, the automatic pH control system can continuously monitor the environment in the fermentation tank, automatically adjust the pH value when the pH value changes, and inject NaOH solution through the pump to keep the pH value stable, so that the necessity of manual operation is reduced, and the risk of human error is reduced.

According to the invention, the pH control and the temperature control system are integrated, the controllability of the fermentation process is obviously improved, and the lactobacillus can grow in a more suitable environment by the combined regulation and control of the temperature and the pH.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic flow chart of a method for culturing lactobacillus at high density.

Detailed Description

In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.

Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

EXAMPLE 1 high Density culture of lactic acid bacteria

The preparation steps are as follows:

weighing each component according to the capacity of the fermenter, dissolving each component in distilled water, adjusting pH value of the culture medium to 6.7 with a pH meter, sterilizing in an autoclave at 121deg.C for 15min, inoculating lactobacillus into the sterilized culture medium under aseptic condition,

Transferring the culture medium into a fermentation tank, setting the temperature of the fermentation tank to 37 ℃, starting a pH monitoring system, monitoring the pH value of the culture solution in real time, automatically or manually adding 20% NaOH solution when the pH value is lower than 6.6, adjusting and maintaining the pH value at 6.7+/-0.1,

Fermenting under the above conditions for 30 hours, stopping fermenting after 30 hours, and collecting culture solution;

EXAMPLE 2 high Density culture of lactic acid bacteria

Preparing a culture medium, namely 3 percent of soybean peptone, 2 percent of yeast extract powder, 0.5 percent of sodium acetate, 0.1 percent of compound trace element liquid, 0.05 percent of compound vitamin liquid, 0.2 percent of trypsin, 0.5 percent of acid soluble protein (short peptide), 0.2 percent of sodium citrate, 0.02 percent of Mg2+ water-soluble compound, 0.02 percent of Fe2+ water-soluble compound and the balance of pure water;

The preparation steps are as follows:

weighing each component according to the capacity of the fermenter, dissolving each component in distilled water, adjusting pH value of the culture medium to 7.0 with a pH meter, sterilizing in an autoclave at 121deg.C for 15min, inoculating lactobacillus into the sterilized culture medium under aseptic condition,

Transferring the culture medium into a fermentation tank, setting the temperature of the fermentation tank to 37 ℃, starting a pH monitoring system, monitoring the pH value of the culture solution in real time, automatically or manually adding 20% NaOH solution when the pH value is lower than 6.6, adjusting and maintaining the pH value at 7.0+/-0.1,

Fermenting under the above conditions for 30 hours, stopping fermenting after 30 hours, and collecting culture solution;

EXAMPLE 3 high Density culture of lactic acid bacteria

Preparing a culture medium, namely 3 percent of soybean peptone, 2 percent of yeast extract powder, 0.5 percent of sodium acetate, 0.1 percent of compound trace element liquid, 0.05 percent of compound vitamin liquid, 0.2 percent of trypsin, 0.5 percent of acid soluble protein (short peptide), 0.2 percent of sodium citrate, 0.02 percent of Mg2+ water-soluble compound, 0.02 percent of Fe2+ water-soluble compound and the balance of pure water;

The preparation steps are as follows:

Weighing each component according to the capacity of the fermenter, dissolving each component in distilled water, adjusting pH value of the culture medium to 7.5 with a pH meter, sterilizing in an autoclave at 121deg.C for 15min, inoculating lactobacillus into the sterilized culture medium under aseptic condition,

Transferring the culture medium into a fermentation tank, setting the temperature of the fermentation tank to 37 ℃, starting a pH monitoring system, monitoring the pH value of the culture solution in real time, automatically or manually adding 20% NaOH solution when the pH value is lower than 6.6, adjusting and maintaining the pH value at 7.5+/-0.1,

Fermenting under the above conditions for 30 hours, stopping fermenting after 30 hours, and collecting culture solution;

EXAMPLE 4 high Density culture of lactic acid bacteria

Preparing a culture medium, namely 3 percent of soybean peptone, 2 percent of yeast extract powder, 0.5 percent of sodium acetate, 0.1 percent of compound trace element liquid, 0.05 percent of compound vitamin liquid, 0.2 percent of trypsin, 0.5 percent of acid soluble protein (short peptide), 0.2 percent of sodium citrate, 0.02 percent of Mg2+ water-soluble compound, 0.02 percent of Fe2+ water-soluble compound and the balance of pure water;

The preparation steps are as follows:

Weighing each component according to the capacity of the fermenter, dissolving each component in distilled water, adjusting pH value of the culture medium to 8.0 with a pH meter, sterilizing in an autoclave at 121deg.C for 15min, inoculating lactobacillus into the sterilized culture medium under aseptic condition,

Transferring the culture medium into a fermentation tank, setting the temperature of the fermentation tank to 37 ℃, starting a pH monitoring system, monitoring the pH value of the culture solution in real time, automatically or manually adding 20% NaOH solution when the pH value is lower than 6.6, adjusting and maintaining the pH value at 8.0+/-0.1,

Fermenting under the above conditions for 30 hours, stopping fermenting after 30 hours, and collecting culture solution;

EXAMPLE 5 high Density culture of lactic acid bacteria

Preparing a culture medium, namely 3 percent of soybean peptone, 2 percent of yeast extract powder, 0.5 percent of sodium acetate, 0.1 percent of compound trace element liquid, 0.05 percent of compound vitamin liquid, 0.2 percent of trypsin, 0.5 percent of acid soluble protein (short peptide), 0.2 percent of sodium citrate, 0.02 percent of Mg2+ water-soluble compound, 0.02 percent of Fe2+ water-soluble compound and the balance of pure water;

The preparation steps are as follows:

Weighing each component according to the capacity of the fermenter, dissolving each component in distilled water, adjusting pH value of the culture medium to 8.5 with a pH meter, sterilizing in an autoclave at 121deg.C for 15 min, inoculating lactobacillus into the sterilized culture medium under aseptic condition,

Transferring the culture medium into a fermentation tank, setting the temperature of the fermentation tank to 37 ℃, starting a pH monitoring system, monitoring the pH value of the culture solution in real time, automatically or manually adding 20% NaOH solution when the pH value is lower than 6.6, adjusting and maintaining the pH value at 8.5+/-0.1,

Fermenting under the above conditions for 30 hours, stopping fermenting after 30 hours, and collecting culture solution;

experimental example 1 influence of pH conditions on lactic acid bacteria growth experiment

1 Materials and methods

1.1 Medium

Preparing a culture medium, namely 3 percent of soybean peptone, 2 percent of yeast extract powder, 0.5 percent of sodium acetate, 0.1 percent of compound trace element liquid, 0.05 percent of compound vitamin liquid, 0.2 percent of trypsin, 0.5 percent of acid soluble protein (short peptide), 0.2 percent of sodium citrate, 0.02 percent of Mg2+ water-soluble compound, 0.02 percent of Fe2+ water-soluble compound and the balance of pure water;

1.2 reagents and instruments

1.2.1 Reagents

Gram (Gram) staining solution, bromocresol purple indicator, griss reagent (nitrite reagent), diphenylamine-sulfuric acid reagent (nitrate reagent);

1.2.2 instruments

LDZX-50KB type vertical electric heating pressure steam sterilizer (Shanghai Shen An medical equipment factory), FA1004 type electronic balance (Shanghai balance instrument factory), 0.5-10 mu L and 20-200 mu L type micro sample applicator (Simer), ZHJH-2109C type ultra clean bench (Shanghai Zhengcheng analysis instruments Co., ltd.), SPX-250B type intelligent biochemical incubator (Jiangsu altar Jijingji instruments Co., ltd.), E-201-C type PHS-BE (pH meter) (Shanghai precision scientific instruments Co., ltd.), 722 type spectrophotometer (Shanghai third analysis instruments factory), DMBA300LED type electronic microscope (Miao Di Co., ltd.), E221LED type optical microscope (Miao Di Co., ltd.)

1.3 Sample collection and Strain isolation and preservation

1.3.1 Collection of samples

1.3.2 Isolation and purification of lactic acid bacteria

After measuring the temperature and pH value of the sample, separating and purifying lactobacillus, namely, after gradient dilution of the sample, inoculating the sample on an MRS agar culture medium and a BCP culture medium by a streaking method and a coating method respectively, culturing for 24-48 h at 37 ℃, primarily identifying rod-shaped, spherical or chain ball-shaped gram positive bacteria which are produced by catalase (test is negative), non-reduced nitrate, non-liquefied gelatin and sugar fermentation test and do not produce acid as lactobacillus, further purifying, culturing and preserving,

1.4 Experiments and observations at different pH values

The pH value of the solution is monitored in real time by adopting a pH sensor,

The controller is used for receiving the sensor data and reacting, the target pH range can be set,

Automatically adding acid or alkali by a pump to adjust the pH to be respectively 3.5, 4.3, 4.5, 5.7, 6.5, 8.5 and 9.6;

2 results and analysis

2.1 Separation and preliminary identification of lactic acid bacteria in milk

4 Parts of milk collected in the experiment are separated and purified, and the pH value is automatically adjusted;

2.2 pretreatment and results observations of pH Change experiments

The experimental results show that the pH value of the culture solution before and after sterilization is not obviously different under the meta-acid condition, is slightly reduced, is obviously different under the meta-alkali condition, and mainly shows that the pH value is reduced, the pH value before sterilization is appropriately adjusted according to the pretreatment result (shown in table 2) in the experiment, and corresponding sterilization measures are adopted.

TABLE 2pH requirement of culture solution and correction value before sterilization

2.3 Influence of pH on lactic acid bacteria growth

From tables 3 and 4, it can be seen that lactic acid bacteria grow more strains when the pH value is 4.3-8.5, the optimal pH value is 6.5, the pH value is continuously increased along with the extension of the culture time, the number of lactic acid bacteria is continuously increased, the lactic acid bacteria is reduced after reaching the maximum value, the pH value is 3.4 at the minimum value after the reduction, and the cocci SN2 can reduce the pH value to 4.5 when the pH value is 8.5, and meanwhile, the tolerance of the 10 strains to acid and alkali is different from each other according to tables 3 and 4, and the 10 strains have different pH ranges;

TABLE 3pH values before and after lactic acid bacteria growth

Note that the incubation temperature at pH measurement was 20 ℃

TABLE 4 growth pH ranges for the strains

It can be seen that long metabolism is carried out in an acidic environment with low pH, and when the pH of the environment is reduced to a certain extent, the lactic acid bacteria stop growing in the environment with low pH, and meanwhile, the pH also influences the yield of metabolites;

3.1.2 influence of pH on lactic acid bacteria growth

The pH of the culture medium directly influences the growth of lactobacillus, because the concentration of H+ and nutrient substances enter the cell body in a direct relation, the pH influences the absorption of bacteria to the nutrient substances, the pH value is increased, the chemical balance among ions in the original culture medium is moved to generate sediment, the effective components in the culture medium are reduced, the experimental result shows that the optimal pH value of the lactobacillus growth is 6.5, the growth of the thallus is inhibited to a certain extent along with the reduction or the increase of the pH value,

The conclusion is that the growth of the microorganism is subjected to the environmental pH, the influence degree is greatly different under different pH values and temperatures, the optimal pH value of the lactobacillus growth separated by the experiment is about 6.7, and the pH value set by the invention is the optimal pH value.

It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.

Claims (10)

1. A method for culturing lactobacillus at high density based on a pH control system is characterized by comprising the following steps,

Step S1, preparing materials, comprising 3% of soybean peptone, 2% of yeast extract powder, 0.5% of sodium acetate, 0.1% of compound trace element liquid, 0.05% of compound vitamin liquid, 0.2% of trypsin, 0.5% of acid soluble protein, 0.2% -0.4% of sodium citrate, 0.02% -0.04% of Mg2+ water-soluble compound, 0.01% -0.02% of Fe2+ water-soluble compound, 93.64% -94.62% of pure water,

Preparing MRS culture medium, adjusting pH to 7.0, and sterilizing the culture medium in a sterilizer at 121 ℃ for 15 minutes;

S2, arranging a fermentation tank, and transferring the sterilized culture medium to the fermentation tank with a temperature control and pH control system under the aseptic condition;

s3, starting a pH control system and calibrating a pH sensor;

step S4, monitoring and adjusting in real time, when fermentation starts, monitoring the pH value of the culture solution in the fermentation tank in real time by a pH sensor,

And S5, maintaining the fermentation process, maintaining the temperature in the fermentation tank at 37 ℃ constantly, keeping the pH value in a set range by a pH control system, continuously fermenting for 30 hours, ending the fermentation, and collecting the culture solution.

2. The method of high-density culture of lactic acid bacteria by a pH control system according to claim 1, wherein in step S2, the temperature of the fermenter is adjusted to 37 ℃.

3. The method of high-density culture of lactic acid bacteria based on a pH control system according to claim 2, wherein in step S3, the pH controller is set to 6.7±0.1.

4. A method for high-density culture of lactic acid bacteria based on a pH control system according to claim 3, wherein in step S4, when the pH value is lower than a preset lower limit value of 6.6, the pH controller sends a signal to start the pump connected to the control system.

5. The method according to claim 4, wherein in step S4, a 20% NaOH solution is automatically added from a tank, and an alkali solution is added by a pump based on flow control to restore the pH to a set range of 6.7.+ -. 0.1.

6. The method of high-density culture of lactic acid bacteria based on a pH control system according to claim 5, wherein the pH control system assembly comprises:

the pH sensor is used for detecting the pH value of the fermentation liquid in real time and transmitting data to the pH controller,

And the pH controller is used for judging whether the pH value is in a set range according to the input data of the pH sensor and determining whether to start the pump to add acid or alkali.

7. The method of high-density culture of lactic acid bacteria based on a pH control system of claim 6, wherein the pH control system assembly further comprises:

the pump and the liquid storage barrel are connected with the pH controller, and acid or alkali solution is added according to the requirement to keep the pH stable.

8. The method for high-density culture of lactic acid bacteria based on a pH control system according to claim 7, wherein the pH monitoring and adjusting means comprises:

In the fermentation process, the pH value of the fermentation liquid is continuously collected by the pH sensor, the Kalman filtering is adopted to eliminate noise, the processed data is transmitted to the controller in real time,

And the controller adopts a PID control algorithm to make adjustment judgment according to the pH value information transmitted by the sensor.

9. The method for high-density culture of lactic acid bacteria based on a pH control system according to claim 8, wherein in the high-density culture of lactic acid bacteria of the pH control system, the pH monitoring and adjusting means further comprises:

The PID control algorithm calculates the difference between the current pH value and the target pH value, and adjusts the output of the controller through proportional, integral and differential terms to control the start or stop of the pump.

10. The method for high-density culture of lactic acid bacteria based on a pH control system according to claim 9, wherein in the high-density culture of lactic acid bacteria of the pH control system, the pH monitoring and adjusting means further comprises:

The pump calculates the quantity of NaOH solution conveyed by the pump by adopting a flow control algorithm based on the instruction of the controller, and the flow of the pump is regulated in real time according to the deviation of the pH value.