Production method of engineered bifidobacterium product based on in-vitro continuous fermentation method
By optimizing the feeding strategy and controlling fermentation parameters, and dynamically adjusting the carbon-nitrogen ratio, the problem of limited fermentation scale in existing technologies has been solved, achieving efficient Bifidobacterium fermentation production and supporting its large-scale industrial application.
Patent Information
- Application Number
- CN202510922241.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-04
- Publication Date
- 2025-10-28
AI Technical Summary
The existing technology uses a constant feeding rate during feeding, which cannot effectively supply the rapid growth of microorganisms in the fermenter, resulting in a limited number and scale of microorganisms after fermentation.
Using an in vitro continuous fermentation method, Bifidobacterium strains were selectively modified through gene editing technology. The feeding strategy was optimized, and the ratio of glucose to beef extract in the feed solution was dynamically adjusted. Fermentation parameters were controlled based on the carbon-nitrogen ratio and specific growth rate in the three-stage fermenter to achieve a stable supply of carbon and nitrogen sources.
It improves the efficiency of the fermentation process, significantly increases the number and scale of fermented cells, and supports the large-scale industrial production of Bifidobacterium.
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Figure CN120843392A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial culture technology, and in particular to an engineered method for producing Bifidobacterium products based on in vitro continuous fermentation. Background Technology
[0002] Bifidobacterium, a core symbiotic bacterium of the human gut, possesses multidimensional biological activities. By secreting antimicrobial substances (such as bacteriocins and organic acids), Bifidobacterium can significantly inhibit the proliferation of pathogenic Enterobacteriaceae and fungi. In terms of nutritional metabolism, Bifidobacterium not only synthesizes B vitamins (including essential nutrients such as VB12 and folic acid) but also competitively inhibits the activity of vitamin-degrading bacteria, maintaining host vitamin homeostasis. From a microecological regulation perspective, Bifidobacterium produces short-chain fatty acids through the fermentation of dietary fiber, lowering the colonic pH. This acidic environment not only promotes increased intestinal peristalsis but also increases fecal water content through osmotic pressure effects. Notably, Bifidobacterium metabolites can specifically bind to aryl hydrocarbon receptors (AhR), promoting the biotransformation of indole-like harmful substances, reducing blood ammonia concentration, and exhibiting a significant liver-protective effect. In the field of immune regulation, Bifidobacterium cell wall components (such as lipoteichoic acid) can activate the Toll-like receptor signaling pathway in dendritic cells, increasing the secretion of intestinal sIgA. Epidemiological studies have shown that long-term supplementation with specific strains can reduce the risk of colorectal cancer, and the mechanism involves the biodegradation of carcinogens (such as nitrosamines) and the downregulation of pro-inflammatory factors.
[0003] Traditional microbial fermentation processes employ continuous fermentation, which extends the logarithmic growth phase of microorganisms in batch fermentation, maintaining stable growth and specific growth rates, and keeping cell concentration, total cell mass, and culture medium volume constant in the fermenter. However, existing technologies typically use a constant feeding rate, which cannot effectively support the rapid growth of microorganisms in the fermenter, thus limiting the number and scale of cells after fermentation. Summary of the Invention
[0004] The purpose of this invention is to provide an engineered method for producing Bifidobacterium products based on in vitro continuous fermentation, so as to solve the problems mentioned in the background art.
[0005] This invention provides a method for producing engineered Bifidobacterium products based on in vitro continuous fermentation, comprising the following steps:
[0006] S1. Strain selection and preparation: Bifidobacterium original strains were isolated from the intestines of healthy human patients. Gene editing technology was used to directionally modify the strains to enhance their immunotherapeutic ability. After passage culture, genetic stability was observed, and candidate strains were screened.
[0007] S2. Culture medium design and sterilization: Prepare modified MRS culture medium, adjust the pH to 5.6 to 6.0, and then sterilize by autoclaving.
[0008] S3. Fermentation process control: In the primary seed tank, the freeze-dried candidate strain is inoculated into modified MRS medium and cultured in an anaerobic environment at 33 to 38°C for 18 to 24 hours; it is then transferred to the secondary propagation tank for scale-up culture for 12 to 16 hours, with nitrogen gas introduced to maintain the anaerobic environment; it is then transferred to the tertiary fermenter for fermentation culture, with parameters controlled in the tertiary fermenter and continuous fermentation method used for stable operation, and feeding strategy optimized until the fermentation endpoint is reached;
[0009] S4. Product separation and purification: The fermentation broth was separated into three phases using a disc centrifuge. After the viable bacteria concentration reached 1x10^11 CFU / mL, it was collected and then washed three times with phosphate buffer pre-cooled to 4°C to remove culture medium residues and metabolic byproducts. After freeze-drying, Bifidobacterium freeze-dried powder was obtained.
[0010] Preferably, the raw materials for the modified MRS culture medium in step S2 include:
[0011] Yeast extract 1% to 1.5%, glucose 2.4% to 3.2%, beef extract 1% to 1.5%, L-cysteine hydrochloride 0.15% to 0.2%, dipotassium hydrogen phosphate 0.2% to 0.5%, sodium chloride 0.5% to 1%, magnesium sulfate 0.05% to 0.1%, vitamin solution 1% to 1.5%, balance being water.
[0012] Preferably, the high-pressure steam sterilization in step S2 includes:
[0013] Sterilize with steam for 15 to 20 minutes at 0.1 to 0.2 MPa and 115 to 125°C, then allow to cool naturally.
[0014] Preferably, step S3, controlling the parameters inside the tertiary fermenter, includes:
[0015] The pH value inside the three-stage fermenter is monitored, and when the pH value drops below 5.6, calcium carbonate solution is automatically added to raise the pH value to 5.6 to 6.0.
[0016] Preferably, step S3, controlling the parameters inside the tertiary fermenter, further includes:
[0017] A gradient temperature control strategy was adopted: initially maintaining 35℃ to promote cell proliferation, raising the temperature to 38℃ in the middle stage to stimulate the secretion of metabolites, and lowering the temperature to 34℃ in the later stage to prolong the stationary period.
[0018] Preferably, during the fermentation process, the stirring speed in the tertiary fermenter is set to 85 to 115 rpm.
[0019] Preferably, the optimized feeding strategy in step S3 includes:
[0020] Detecting OD in the three-stage fermenter 600 Value, when OD 600 When the ratio is >2.0, add the same feed solution as the modified MRS medium to the tertiary fermenter, dynamically adjust the ratio of glucose to beef extract in the feed solution, and maintain a stable carbon-nitrogen ratio R in the tertiary fermenter.
[0021] Preferably, the addition of the same feed solution as the modified MRS culture medium to the tertiary fermenter includes:
[0022] OD during the detection period 600 The value exhibits an exponential growth trend. The specific growth rate of the microorganisms in the tertiary fermenter is calculated using the following formula:
[0023]
[0024] In equation (1), μ represents the specific growth rate, and OD 600 OD0 represents the optical density of the fermentation broth obtained from the current detection, and OD0 represents the initial optical density of the fermentation broth.
[0025] Calculate the feeding rate using the following formula:
[0026]
[0027] In equation (2), F represents the feeding rate, V represents the volume of the three-stage fermenter, X represents the current cell concentration, and Y represents the culture medium conversion coefficient.
[0028] Preferably, the dynamic adjustment of the ratio of glucose to beef extract in the feed solution includes:
[0029] Obtain the current carbon concentration C in the tertiary fermenter. t and nitrogen concentration N t And the carbon concentration C in the feed solution 补 and nitrogen concentration N 补 Set the following constraints:
[0030]
[0031] During the feeding process, the carbon concentration C in the feed solution is used as the reference. 补 and nitrogen concentration N 补 One of them is a fixed ratio, and the ratio of the other is adjusted according to formula (3).
[0032] Preferably, the freeze-dried Bifidobacterium powder obtained in step S4 includes:
[0033] After pre-freezing for 2 to 4 hours at -65 to -45°C and a vacuum of 3 to 10 Pa, the product is then re-frozen for 10 to 30 hours at -65°C and a vacuum of 1 to 7 Pa.
[0034] Compared with the prior art, the beneficial effects of the present invention are:
[0035] The engineered Bifidobacterium product production method based on in vitro continuous fermentation provided by the embodiments of the present invention optimizes the feeding strategy. During the fermentation culture, the feeding rate of the feed solution added to the tertiary fermenter is controlled according to the specific growth rate of the current cells in the tertiary fermenter and fully considering the exponential changes in the logarithmic growth phase of the cells. This ensures a more effective and sufficient supply of carbon and nitrogen sources, guaranteeing the rapid growth of cells in the fermenter. At the same time, the ratio of glucose to beef extract in the feed solution is dynamically adjusted, thereby keeping the carbon-nitrogen ratio R in the tertiary fermenter stable. This enables the continuous fermentation process to operate efficiently for a long time, increasing the number and scale of cells after fermentation. Attached Figure Description
[0036] Figure 1 A flowchart of an engineered Bifidobacterium product production method based on in vitro continuous fermentation, provided for embodiments of the present invention. Detailed Implementation
[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0038] This invention provides a method for producing engineered Bifidobacterium products based on in vitro continuous fermentation, such as... Figure 1 As shown, it includes the following steps:
[0039] S1. Strain selection and preparation: Bifidobacterium original strains were isolated from the intestines of healthy human patients. Gene editing technology was used to directionally modify the strains to enhance their immunotherapeutic ability. After passage culture, genetic stability was observed, and candidate strains were screened.
[0040] S2. Culture medium design and sterilization: Prepare modified MRS culture medium, adjust the pH to 5.6 to 6.0, and then sterilize by autoclaving.
[0041] S3. Fermentation process control: In the primary seed tank, the freeze-dried candidate strain is inoculated into modified MRS medium and cultured in an anaerobic environment at 33 to 38°C for 18 to 24 hours; it is then transferred to the secondary propagation tank for scale-up culture for 12 to 16 hours, with nitrogen gas introduced to maintain the anaerobic environment; it is then transferred to the tertiary fermenter for fermentation culture, with parameters controlled in the tertiary fermenter and continuous fermentation method used for stable operation, and feeding strategy optimized until the fermentation endpoint is reached;
[0042] S4. Product separation and purification: The fermentation broth was separated into three phases using a disc centrifuge. After the viable bacteria concentration reached 1x10^11 CFU / mL, it was collected and then washed three times with phosphate buffer pre-cooled to 4°C to remove culture medium residues and metabolic byproducts. After freeze-drying, Bifidobacterium freeze-dried powder was obtained.
[0043] In one embodiment of the present invention, the raw materials for the modified MRS culture medium in step S2 include:
[0044] Yeast extract 1% to 1.5%, glucose 2.4% to 3.2%, beef extract 1% to 1.5%, L-cysteine hydrochloride 0.15% to 0.2%, dipotassium hydrogen phosphate 0.2% to 0.5%, sodium chloride 0.5% to 1%, magnesium sulfate 0.05% to 0.1%, vitamin solution 1% to 1.5%, balance being water.
[0045] Among them, glucose serves as the main carbon source, beef extract as the main nitrogen source, L-cysteine hydrochloride as an antioxidant, dipotassium hydrogen phosphate as the phosphorus source, sodium chloride as the sodium source, magnesium sulfate as the magnesium source, and vitamin solution provides the vitamins required by Bifidobacteria, which helps their growth and reproduction.
[0046] Furthermore, in one embodiment of the present invention, the high-pressure steam sterilization in step S2 includes:
[0047] Sterilize with steam for 15 to 20 minutes at 0.1 to 0.2 MPa and 115 to 125°C, then allow to cool naturally.
[0048] In one embodiment of the present invention, step S3, controlling the parameters inside the three-stage fermenter, includes:
[0049] The pH value inside the tertiary fermenter is monitored. When the pH value drops below 5.6, calcium carbonate solution is automatically added to raise the pH value to 5.6 to 6.0. Maintaining a relatively constant pH value within the tertiary fermenter during fermentation is crucial to promote cell proliferation.
[0050] In one embodiment of the present invention, step S3, which involves controlling the parameters within the tertiary fermenter, further includes:
[0051] A gradient temperature control strategy was adopted: initially maintaining 35℃ to promote cell proliferation, raising the temperature to 38℃ in the middle stage to stimulate metabolite secretion, and then lowering the temperature to 34℃ in the later stage to prolong the stationary period. This gradient temperature control method can improve the fermentation process's adaptability and tolerance to high temperatures.
[0052] In one embodiment of the invention, the stirring speed in the tertiary fermenter is set to 85 to 115 rpm during fermentation. Maintaining a low stirring speed can prevent shear force from damaging the cell cells.
[0053] In one embodiment of the present invention, the optimized feeding strategy in step S3 includes:
[0054] Detecting OD in the three-stage fermenter 600 Value, when OD 600 When the ratio is >2.0, add the same feed solution as the modified MRS medium to the tertiary fermenter, dynamically adjust the ratio of glucose to beef extract in the feed solution, and maintain a stable carbon-nitrogen ratio R in the tertiary fermenter.
[0055] Furthermore, in one embodiment of the present invention, the addition of the same feed solution as the modified MRS culture medium raw material to the tertiary fermenter includes:
[0056] OD during the detection period 600 The value exhibits an exponential growth trend. The specific growth rate of the microorganisms in the tertiary fermenter is calculated using the following formula:
[0057]
[0058] In equation (1), μ represents the specific growth rate, and OD 600 OD0 represents the optical density of the fermentation broth obtained from the current detection, and OD0 represents the initial optical density of the fermentation broth.
[0059] Calculate the feeding rate using the following formula:
[0060]
[0061] In equation (2), F represents the feeding rate, V represents the volume of the three-stage fermenter, X represents the current cell concentration, and Y represents the culture medium conversion coefficient.
[0062] Through the above technical solution, the feed rate F of the feed liquid added to the tertiary fermenter is adjusted based on the specific growth rate μ of the cells in the tertiary fermenter and taking into full account the exponential changes in the logarithmic growth phase of the cells. This ensures a more effective and sufficient supply of carbon and nitrogen sources, thereby guaranteeing the rapid growth of the cells in the fermenter.
[0063] It should be noted that when using continuous fermentation to cultivate microorganisms, the fermentation liquid is discharged from the fermenter at the same flow rate while feeding is being done. In the embodiments of the present invention, the discharge rate of the three-stage fermenter changes synchronously with the feeding rate F and remains consistent.
[0064] Furthermore, in one embodiment of the present invention, the dynamic adjustment of the ratio of glucose to beef extract in the feed solution includes:
[0065] Obtain the current carbon concentration C in the tertiary fermenter. t and nitrogen concentration N t And the carbon concentration C in the feed solution 补 and nitrogen concentration N 补 Set the following constraints:
[0066]
[0067] During the feeding process, the carbon concentration C in the feed solution is used as the reference. 补 and nitrogen concentration N 补 One of them is a fixed ratio, and the ratio of the other is adjusted according to formula (3).
[0068] In an embodiment of the present invention, R in formula (3) is 4:1 to 6:1;
[0069] By dynamically adjusting the ratio of glucose to beef extract in the feed solution, the carbon-nitrogen ratio R in the tertiary fermenter is kept stable, enabling the continuous fermentation process to operate efficiently for a long time and increasing the number and scale of cells after fermentation.
[0070] In one embodiment of the present invention, the freeze-dried Bifidobacterium powder obtained in step S4 includes:
[0071] After pre-freezing for 2 to 4 hours at -65 to -45°C and a vacuum of 3 to 10 Pa, the product is then re-frozen for 10 to 30 hours at -65°C and a vacuum of 1 to 7 Pa.
[0072] The technical solution of the present invention is further illustrated below through specific embodiments:
[0073] Example 1:
[0074] S1. Strain selection and preparation: Bifidobacterium original strains were isolated from the intestines of healthy human patients. Gene editing technology was used to directionally modify the strains to enhance their immunotherapeutic ability. After passage culture, genetic stability was observed, and candidate strains were screened.
[0075] S2. Culture Medium Design and Sterilization: Prepare modified MRS culture medium. The raw materials of the modified MRS culture medium include: 1% yeast extract, 2.4% glucose, 1% beef extract, 0.15% L-cysteine hydrochloride, 0.2% dipotassium hydrogen phosphate, 0.5% sodium chloride, 0.05% magnesium sulfate, and 1% vitamin solution; the balance is water. After adjusting the pH to 5.8, sterilize by steaming at 0.15 MPa and 120℃ for 15 to 20 minutes, and then allow it to cool naturally.
[0076] S3. Fermentation process control: In the primary seed tank, the freeze-dried candidate strain was inoculated onto modified MRS medium and cultured in an anaerobic environment at 33-38℃ for 18-24 hours; it was then transferred to the secondary propagation tank for scale-up culture for 12-16 hours, with nitrogen gas introduced to maintain the anaerobic environment; it was then transferred to the tertiary fermenter for fermentation culture, and the pH value in the tertiary fermenter was monitored. When the pH value dropped below 5.6, calcium carbonate solution was automatically added to raise the pH value to 5.8; a gradient temperature control strategy was adopted: initially maintaining 35℃ to promote cell proliferation, raising the temperature to 38℃ in the middle stage to stimulate the secretion of metabolites, and lowering the temperature to 34℃ in the later stage to prolong the stationary period;
[0077] Detecting OD in the three-stage fermenter 600 Value, when OD 600 When the value is greater than 2.0, the corresponding feeding rate F is calculated. The same feed solution as the modified MRS medium is added to the tertiary fermenter. During the feeding process, the carbon concentration C in the feed solution is used as the reference. 补 and nitrogen concentration N 补 One component is kept in a fixed ratio, while the ratio of the other component is adjusted accordingly to maintain a stable carbon-nitrogen ratio R in the tertiary fermenter until the end of fermentation.
[0078] S4. Product Separation and Purification: The fermentation broth was separated into three phases using a disc centrifuge. After the viable cell concentration reached 1x10^11 CFU / mL, the broth was collected and then washed three times countercurrently with phosphate buffer pre-cooled to 4°C to remove culture medium residues and metabolic byproducts. After pre-freezing at -65 to -45°C and a vacuum of 3-10 Pa for 2 to 4 hours, it was then second-frozen at -65°C and a vacuum of 1-7 Pa for 10 to 30 hours to obtain Bifidobacterium lyophilized powder.
[0079] Comparative Example 1:
[0080] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 uses a constant feeding rate to add feed liquid to the three-stage fermenter, and the ratio of glucose to beef extract in the feed liquid is fixed.
[0081] Example 2:
[0082] The difference between Example 2 and Example 1 is that the raw materials for the modified MRS culture medium in Example 2 include:
[0083] Yeast extract 1.3%, glucose 2.8%, beef extract 1.3%, L-cysteine hydrochloride 0.18%, dipotassium hydrogen phosphate 0.4%, sodium chloride 0.8%, magnesium sulfate 0.08%, vitamin solution 1.3%, balance water.
[0084] Comparative Example 2:
[0085] The difference between Comparative Example 2 and Example 2 is that Comparative Example 2 uses a constant feeding rate to add feed liquid to the tertiary fermenter, and the ratio of glucose to beef extract in the feed liquid is fixed.
[0086] Example 3:
[0087] The difference between Example 3 and Example 1 is that the raw materials for the improved MRS culture medium in Example 3 include:
[0088] Yeast extract 1.5%, glucose 3.2%, beef extract 1.5%, L-cysteine hydrochloride 0.2%, dipotassium hydrogen phosphate 0.5%, sodium chloride 1%, magnesium sulfate 0.1%, vitamin solution 1.5%, balance water.
[0089] Comparative Example 3:
[0090] The difference between Comparative Example 3 and Example 3 is that Comparative Example 3 uses a constant feeding rate to add feed liquid to the three-stage fermenter, and the ratio of glucose to beef extract in the feed liquid is fixed.
[0091] Table 1 below shows the relevant data collected after fermentation for each example and comparative example, based on the same fermentation time:
[0092] Table 1
[0093]
[0094]
[0095] As can be seen from Table 1, the optimized feeding strategy adopted in this invention, compared with the use of constant feeding rate and feeding ratio in the prior art, can effectively improve the cell growth rate, carbon source conversion rate and nitrogen source conversion rate, thereby significantly increasing the number and scale of cells after fermentation. This is beneficial to the large-scale industrial production of Bifidobacterium and is of great significance for promoting production capacity improvement.
[0096] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the equivalents of the appended claims.
Claims
1. A method for producing engineered Bifidobacterium products based on in vitro continuous fermentation, characterized in that, The following steps are involved: S1. Strain selection and preparation: Bifidobacterium original strains were isolated from the intestines of healthy human patients. Gene editing technology was used to directionally modify the strains to enhance their immunotherapeutic ability. After passage culture, genetic stability was observed, and candidate strains were screened. S2. Culture medium design and sterilization: Prepare modified MRS culture medium, adjust the pH to 5.6 to 6.0, and then sterilize by autoclaving. S3. Fermentation process control: In the primary seed tank, the freeze-dried candidate strain is inoculated into modified MRS medium and cultured in an anaerobic environment at 33 to 38°C for 18 to 24 hours; it is then transferred to the secondary propagation tank for scale-up culture for 12 to 16 hours, with nitrogen gas introduced to maintain the anaerobic environment; it is then transferred to the tertiary fermenter for fermentation culture, with parameters controlled in the tertiary fermenter and continuous fermentation method used for stable operation, and feeding strategy optimized until the fermentation endpoint is reached; S4. Product separation and purification: The fermentation broth was separated into three phases using a disc centrifuge. After the viable bacteria concentration reached 1x10^11 CFU / mL, it was collected and then washed three times with phosphate buffer pre-cooled to 4°C to remove culture medium residues and metabolic byproducts. After freeze-drying, Bifidobacterium freeze-dried powder was obtained.
2. The method for producing engineered Bifidobacterium products based on in vitro continuous fermentation according to claim 1, characterized in that, The raw materials for the modified MRS culture medium mentioned in step S2 include: Yeast extract 1% to 1.5%, glucose 2.4% to 3.2%, beef extract 1% to 1.5%, L-cysteine hydrochloride 0.15% to 0.2%, dipotassium hydrogen phosphate 0.2% to 0.5%, sodium chloride 0.5% to 1%, magnesium sulfate 0.05% to 0.1%, vitamin solution 1% to 1.5%, balance being water.
3. The method for producing engineered Bifidobacterium products based on in vitro continuous fermentation according to claim 2, characterized in that, The high-pressure steam sterilization described in step S2 includes: Sterilize with steam for 15 to 20 minutes at 0.1 to 0.2 MPa and 115 to 125°C, then allow to cool naturally.
4. The method for producing engineered Bifidobacterium products based on in vitro continuous fermentation according to claim 3, characterized in that, Step S3, controlling the parameters inside the tertiary fermenter, includes: The pH value inside the three-stage fermenter is monitored, and when the pH value drops below 5.6, calcium carbonate solution is automatically added to raise the pH value to 5.6 to 6.
0.
5. The method for producing engineered Bifidobacterium products based on in vitro continuous fermentation according to claim 4, characterized in that, Step S3, controlling the parameters inside the tertiary fermenter, also includes: A gradient temperature control strategy was adopted: initially maintaining 35℃ to promote cell proliferation, raising the temperature to 38℃ in the middle stage to stimulate the secretion of metabolites, and lowering the temperature to 34℃ in the later stage to prolong the stationary period.
6. The method for producing engineered Bifidobacterium products based on in vitro continuous fermentation according to claim 5, characterized in that, During the fermentation process, the stirring speed in the three-stage fermenter is set to 85 to 115 rpm.
7. The method for producing engineered Bifidobacterium products based on in vitro continuous fermentation according to claim 6, characterized in that, The optimized feeding strategy described in step S3 includes: Detecting OD in the three-stage fermenter 600 Value, when OD 600 When the ratio is >2.0, add the same feed solution as the modified MRS medium to the tertiary fermenter, dynamically adjust the ratio of glucose to beef extract in the feed solution, and maintain the carbon-nitrogen ratio R in the tertiary fermenter.
8. The method for producing engineered Bifidobacterium products based on in vitro continuous fermentation according to claim 7, characterized in that, The addition of the same feed solution as the modified MRS medium to the tertiary fermenter includes: OD during the detection period 600 The value exhibits an exponential growth trend. The specific growth rate of the microorganisms in the tertiary fermenter is calculated using the following formula: In equation (1), μ represents the specific growth rate, and OD 600 OD0 represents the optical density of the fermentation broth obtained from the current detection, and OD0 represents the initial optical density of the fermentation broth. Calculate the feeding rate using the following formula: In equation (2), F represents the feeding rate, V represents the volume of the three-stage fermenter, X represents the current cell concentration, and Y represents the culture medium conversion coefficient.
9. The method for producing engineered Bifidobacterium products based on in vitro continuous fermentation according to claim 8, characterized in that, The dynamic adjustment of the ratio of glucose to beef extract in the feed solution includes: Obtain the current carbon concentration C in the tertiary fermenter. t and nitrogen concentration N t And the carbon concentration C in the feed solution 补 and nitrogen concentration N 补 Set the following constraints: During the feeding process, the carbon concentration C in the feed solution is used as the reference. 补 and nitrogen concentration N 补 One of them is a fixed ratio, and the ratio of the other is adjusted according to formula (3).
10. The method for producing engineered Bifidobacterium products based on in vitro continuous fermentation according to claim 9, characterized in that, The freeze-dried Bifidobacterium powder obtained in step S4 includes: After pre-freezing for 2 to 4 hours at -65 to -45°C and a vacuum of 3 to 10 Pa, the product is then re-frozen for 10 to 30 hours at -65°C and a vacuum of 1 to 7 Pa.