In-vitro micro-ecological evaluation method for preclinical living flora drugs

Abstract

The invention relates to the technical field of microbial drugs, in particular to a preclinical in-vitro micro-ecological evaluation method for living flora drugs, which comprises the following steps: S1, preparing a dynamic intestinal tract simulation system comprising an experimental tank, a contrast tank and a blank tank, acquiring fecal microbial bacterial liquid, and adding the fecal microbial bacterial liquid into the experimental tank, the contrast tank and the blank tank; s2, thallus inoculation: obtaining concentrated thalli of a living flora drug, inoculating the concentrated thalli into an experiment tank, purchasing commercial probiotics, and inoculating the commercial probiotics into a contrast tank at the same concentration; s3, time sequence sampling: continuously running for 72 hours, and respectively collecting bacterial liquid in the experimental tank, the contrast tank and the blank tank by using sampling pipes at three time nodes of 24 hours, 48 hours and 72 hours; and S4, multi-dimensional drug effect evaluation: evaluating the drug effect of the living flora drug from two aspects of flora dynamic analysis and metabolic function detection in the bacterial liquid.

Description

Technical Field

[0001] This invention relates to the field of microbial drug technology, and in particular to a method for in vitro microecological evaluation of live microbial flora drugs before clinical trials. 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] When evaluating the mechanism of action of Bifidobacterium drugs, existing research systems face two key challenges: First, there are significant differences between animal models and human gut microbiota (e.g., differences in Bifidobacterium abundance and metabolic pathways of 40%-60%), leading to biases in drug effect predictions. Second, while human clinical trials can reflect the state of the rectal microbiota, it is difficult to obtain information on the colonization dynamics of Bifidobacterium in key intestinal regions and its metabolites (e.g., the yield and distribution of acetic acid / folic acid). These limitations result in a lack of precise data to support the dosage optimization and targeted delivery of Bifidobacterium drugs. Summary of the Invention

[0004] The purpose of this invention is to provide an in vitro microecological evaluation method for live microbial community drugs before clinical trials, so as to solve the problems mentioned in the background art.

[0005] This invention provides a method for preclinical in vitro microecological evaluation of live microbial community drugs, comprising the following steps:

[0006] S1. Prepare a dynamic intestinal simulation system: including experimental tanks, control tanks, and blank tanks. Obtain fecal microbial solution and add it to the experimental tanks, control tanks, and blank tanks.

[0007] S2. Inoculation of bacterial cells: Obtain concentrated bacterial cells of live bacterial flora drugs and inoculate them into experimental tanks. At the same time, purchase commercial probiotics and inoculate them into control tanks at the same concentration.

[0008] S3. Time-series sampling: After running continuously for 72 hours, bacterial solutions were collected from the experimental tank, control tank, and blank tank at three time points: 24h, 48h, and 72h, respectively, using sampling tubes.

[0009] S4. Multidimensional efficacy evaluation: The efficacy of live microbial drugs is evaluated from two aspects: dynamic analysis of the bacterial community in the bacterial solution and detection of metabolic function.

[0010] Preferably, the experimental tank, control tank, and blank tank are respectively equipped with a liquid inlet pipe for inputting acid or alkali solution, a gas inlet pipe for inputting N2, a feed pipe for adding feed liquid, a discharge pipe for discharging fermentation broth, and a stirring device.

[0011] Preferably, the dynamic intestinal simulation system in step S1 further includes:

[0012] The pH control unit is used to maintain the pH value between 6.0 and 7.4 during the fermentation process of the dynamic gut-simulation system;

[0013] Temperature control unit, used to maintain the temperature at 36 to 38°C during the fermentation process of the dynamic gut simulation system;

[0014] The stirring rate control unit is used to control the stirring rate of the stirring device during the fermentation process of the dynamic gut simulation system to be 85 to 115 rpm.

[0015] The anaerobic unit is used to maintain the anaerobic environment during the fermentation process of the dynamic gut microbiota simulation system at O2 < 0.1% and CO8 ≥ 90%.

[0016] Preferably, peristaltic pumps are installed on the infusion pipe, gas infusion pipe, feed pipe, and discharge pipe.

[0017] Preferably, the dynamic intestinal simulation system further includes a central control unit, which controls the infusion tube to input acid or alkali solution to compensate when the pH value shifts by more than 0.3.

[0018] Preferably, the anaerobic unit monitors the O2 concentration every 6 hours. When the O2 concentration exceeds the standard, the central control unit controls the gas supply pipe to input N2 for flushing and inputs palladium catalyst for deoxygenation.

[0019] Preferably, obtaining the fecal microbial inoculum in step S1 includes:

[0020] Weigh fresh feces from healthy individuals, dissolve them in a pH 6.8 buffer solution, filter out solid particles with gauze, and the resulting filtrate is the fecal microbial inoculum.

[0021] Preferably, the concentrated bacterial cells for obtaining the live bacterial flora drug in step S2 include:

[0022] The lyophilized bacterial strains for resuscitating live microbial flora, including Bifidobacterium, were cultured in MRS medium for 16 to 24 hours and then centrifuged to a concentration of 1 × 10⁻⁶. 8 Bifidobacterium cells were collected after CFU / mL concentration.

[0023] Preferably, step S3, which involves collecting bacterial solutions from the experimental, control, and blank containers using sampling tubes, includes:

[0024] Before each use, the sampling tubes are sterilized by steam for 15 to 20 minutes at 0.1 to 0.2 MPa and 115 to 125°C, and then placed in an anaerobic chamber for ultraviolet irradiation for 25 to 30 minutes.

[0025] Preferably, step S4 includes:

[0026] The analysis of bacterial community dynamics in the bacterial culture included: Bifidobacterium abundance, Escherichia coli inhibition rate, Shannon diversity index, and Bacteroides / Firmwallis ratio;

[0027] Metabolic function tests include: acetic acid production, propionic acid production, folic acid production, and β-glucosidase activity.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] The in vitro microecological evaluation method for preclinical live microbiota drugs provided by the embodiments of the present invention involves setting up experimental, control, and blank tanks in a dynamic intestinal simulation system. Concentrated live microbiota drugs are inoculated into the experimental tanks, and commercial probiotics are inoculated into the control tanks. The dynamic intestinal simulation system is maintained stably for 72 hours by pH control, temperature control, stirring rate control, and anaerobic unit. The fermentation broth in the three tanks is sampled sequentially. The efficacy of the live microbiota drugs is evaluated through two aspects: dynamic analysis of the microbiota in the bacterial solution and detection of metabolic function. This method can explore the symbiotic relationship between live microbiota drugs and intestinal microbiota, and thus evaluate the ability of live microbiota drugs to rapidly colonize and continuously inhibit pathogens in the human intestine. Attached Figure Description

[0030] Figure 1 A flowchart of an in vitro microecological evaluation method for preclinical drugs based on live microbial flora provided for embodiments of the present invention. Detailed Implementation

[0031] 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.

[0032] This invention provides a method for preclinical in vitro microecological evaluation of live microbial community drugs, comprising the following steps:

[0033] S1. Prepare a dynamic intestinal simulation system: including experimental tanks, control tanks, and blank tanks. Obtain fecal microbial solution and add it to the experimental tanks, control tanks, and blank tanks.

[0034] S2. Inoculation of bacterial cells: Obtain concentrated bacterial cells of live bacterial flora drugs and inoculate them into experimental tanks. At the same time, purchase commercial probiotics and inoculate them into control tanks at the same concentration.

[0035] S3. Time-series sampling: After running continuously for 72 hours, bacterial solutions were collected from the experimental tank, control tank, and blank tank at three time points: 24h, 48h, and 72h, respectively, using sampling tubes.

[0036] S4. Multidimensional efficacy evaluation: The efficacy of live microbial drugs is evaluated from two aspects: dynamic analysis of the bacterial community in the bacterial solution and detection of metabolic function.

[0037] The in vitro microecological evaluation method for preclinical in vivo microbial community drugs provided by the embodiments of the present invention involves setting up experimental, control, and blank tanks in a dynamic intestinal simulation system. Concentrated in vivo microbial community drugs are inoculated in the experimental tanks, and commercial probiotics (such as Bifidobacterium BB-12) are inoculated in the control tanks. The dynamic intestinal simulation system is maintained stably for 72 hours by pH control, temperature control, stirring rate control, and anaerobic unit. The fermentation broth in the three tanks is sampled sequentially. The efficacy of the in vivo microbial community drugs is evaluated by analyzing the dynamics of the microbial community in the bacterial solution and detecting metabolic functions. This method can explore the symbiotic relationship between the in vivo microbial community drugs and the intestinal microbiota, and thus evaluate the ability of the in vivo microbial community drugs to rapidly colonize the intestine and continuously inhibit pathogens.

[0038] In one embodiment of the present invention, the experimental tank, control tank, and blank tank are respectively equipped with a liquid inlet pipe for inputting acid or alkali solution, a gas inlet pipe for inputting N2, a feed pipe for adding feed liquid, a discharge pipe for discharging fermentation broth, and a stirring device. The above piping configuration maintains the continuous operation of the cell fermentation process.

[0039] Furthermore, in one embodiment of the present invention, the dynamic intestinal simulation system in step S1 further includes:

[0040] The pH control unit is used to maintain the pH value between 6.0 and 7.4 during the fermentation process of the dynamic gut-simulation system;

[0041] Temperature control unit, used to maintain the temperature at 36 to 38°C during the fermentation process of the dynamic gut simulation system;

[0042] The stirring rate control unit is used to control the stirring rate of the stirring device during the fermentation process of the dynamic gut simulation system to be 85 to 115 rpm.

[0043] The anaerobic unit is used to maintain the anaerobic environment during the fermentation process of the dynamic gut microbiota simulation system at O2 < 0.1% and CO2 ≥ 90%.

[0044] By integrating dynamic parameter control and multimodal detection technology through pH control unit, temperature control unit, stirring rate control unit and anaerobic unit, a more realistic human intestinal environment can be simulated and the dynamic intestinal simulation system can be maintained in stable operation.

[0045] In one embodiment of the present invention, peristaltic pumps are installed on the infusion pipe, gas infusion pipe, feed pipe, and discharge pipe. The peristaltic pumps are used to realize the flow of liquids and gases, and the peristaltic frequency of each peristaltic pump is set according to actual needs.

[0046] In one embodiment of the present invention, the dynamic intestinal simulation system further includes a central control unit. When the pH value shifts by more than 0.3, the central control unit controls the infusion tubing to supply acid or alkali for compensation. The central control unit is connected to the peristaltic pumps of each tubing via communication lines, thereby controlling the opening and closing of each peristaltic pump. When a significant pH value shift occurs, the central control unit controls the peristaltic pumps of the acid or alkali infusion tubing to turn on, correspondingly replenishing the acid or alkali to maintain the pH value between 6.0 and 7.4.

[0047] In one embodiment of the present invention, the anaerobic unit monitors the O2 concentration every 6 hours. When the O2 concentration exceeds the standard, the central control unit controls the gas supply pipe to input N2 for flushing and inputs palladium catalyst for deoxygenation.

[0048] When O2 ≥ 0.1%, the central control unit activates the peristaltic pump in the gas supply pipe, injecting N2 (at a flow rate of 5 L / min) for flushing. Simultaneously, a palladium catalyst is introduced for deoxygenation, and the experiment is interrupted to check the anaerobic unit's seal. Placing oxygen indicators (such as methylene blue) in the experimental, control, and blank containers allows for more timely feedback of oxygen content, enabling faster emergency response to oxygen leaks.

[0049] In one embodiment of the present invention, obtaining the fecal microbial inoculum in step S1 includes:

[0050] Fresh feces from healthy individuals were weighed and dissolved in a pH 6.8 buffer solution. The solid particles were then filtered out using gauze. The resulting filtrate is the fecal microbial culture. This fecal microbial culture forms the basis for constructing the gut microbial ecosystem in experiments.

[0051] In one embodiment of the present invention, step S2, which involves obtaining concentrated bacterial cells of a live bacterial flora drug, includes:

[0052] The lyophilized bacterial strains for resuscitating live microbial flora, including Bifidobacterium, were cultured in MRS medium for 16 to 24 hours and then centrifuged to a concentration of 1 × 10⁻⁶. 8 Bifidobacterium cells were collected after CFU / mL concentration. The main objective of this invention is to investigate the ability of Bifidobacterium in live microbial flora drugs to rapidly colonize and inhibit pathogens in the human gut, as well as their effects on improving gut microbiota regulation and metabolic function.

[0053] In one embodiment of the present invention, step S3, which involves collecting bacterial solutions from the experimental tank, control tank, and blank tank using sampling tubes, includes:

[0054] Before each use, the sampling tubes should be steam-sterilized for 15 to 20 minutes at 0.1 to 0.2 MPa and 115 to 125°C, and then irradiated with ultraviolet light in an anaerobic chamber for 25 to 30 minutes. To prevent the introduction of other microorganisms and cross-contamination, the sampling tubes need to undergo strict pre-sterilization and secondary sterilization measures.

[0055] In one embodiment of the present invention, step S4 includes:

[0056] The analysis of bacterial community dynamics in the bacterial culture included: Bifidobacterium abundance, Escherichia coli inhibition rate, Shannon diversity index, and Bacteroides / Firmwallis ratio;

[0057] Metabolic function tests include: acetic acid production, propionic acid production, folic acid production, and β-glucosidase activity.

[0058] Table 1 below shows the data on the dynamic analysis of bacterial communities in the bacterial solutions obtained after time-series sampling in the experimental, control, and blank containers:

[0059] Table 1

[0060]

[0061]

[0062] Table 2 below shows the metabolic function detection data obtained after time-series sampling for the experimental, control, and blank containers:

[0063] Table 2

[0064]

[0065] As shown in Tables 1 and 2, the Bifidobacterium in the live microbial drug of the present invention exhibits significant advantages in terms of microbial regulation and metabolic function, specifically as follows:

[0066] Stronger colonization ability: The abundance of Bifidobacterium reached 42.3±2.1% at 72h, which was significantly higher than that of the control and blank tanks. Its proliferation rate was faster at 48h, indicating that the initial colonization efficiency was higher.

[0067] More efficient pathogen inhibition: The inhibition rate of Escherichia coli reached 84.6±3.3% at 72h, which was 5.4 percentage points higher than that of the control, and the inhibitory effect increased more significantly over time;

[0068] Better ecological stability:

[0069] The Shannon index remained at 3.6 to 3.8, comparable to the control tank, but significantly lower than the blank tank, indicating that Bifidobacterium in the live microbial drug of the present invention can optimize the microbial community structure without disrupting the diversity balance; the Bacteroides / Firmwallis ratio was optimized to 1.3 after 72 hours, which is closer to the ratio of healthy people;

[0070] Better metabolic function:

[0071] The production of short-chain fatty acids (acetic acid and propionic acid) was higher than that of the control tank, indicating a stronger carbon metabolism capacity; the folic acid synthesis was 7.4% higher than that of the control tank at 72h, showing outstanding nutrient synthesis function; the β-glucosidase activity was improved compared with the control tank, indicating that the live microbial drug can enhance β-glucosidase activity and promote dietary fiber degradation.

[0072] In summary, the live microbial flora drug of the present invention has superior probiotic performance compared to commercial probiotics, especially in terms of rapid colonization and sustained inhibition of pathogens, and has clinical translational potential.

[0073] 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 preclinical in vitro microecological evaluation method for live microbial flora drugs, characterized in that, Includes the following steps: S1. Prepare a dynamic intestinal simulation system: including experimental tanks, control tanks, and blank tanks. Obtain fecal microbial solution and add it to the experimental tanks, control tanks, and blank tanks. S2. Inoculation of bacterial cells: Obtain concentrated bacterial cells of live bacterial flora drugs and inoculate them into experimental tanks. At the same time, purchase commercial probiotics and inoculate them into control tanks at the same concentration. S3. Time-series sampling: After running continuously for 72 hours, bacterial solutions were collected from the experimental tank, control tank, and blank tank at three time points: 24h, 48h, and 72h, respectively, using sampling tubes. S4. Multidimensional efficacy evaluation: The efficacy of live microbial drugs is evaluated from two aspects: dynamic analysis of the bacterial community in the bacterial solution and detection of metabolic function.

2. The in vitro microecological evaluation method for preclinical drug development based on live microbiota according to claim 1, characterized in that, The experimental tank, control tank, and blank tank are respectively equipped with a liquid inlet pipe for inputting acid or alkali solution, a gas inlet pipe for inputting N2, a feed pipe for adding feed liquid, a discharge pipe for discharging fermentation broth, and a stirring device.

3. The in vitro microecological evaluation method for preclinical drugs based on live microbiota according to claim 2, characterized in that, The dynamic gut simulation system described in step S1 further includes: The pH control unit is used to maintain the pH value between 6.0 and 7.4 during the fermentation process of the dynamic gut-simulation system; Temperature control unit, used to maintain the temperature at 36 to 38°C during the fermentation process of the dynamic gut simulation system; The stirring rate control unit is used to control the stirring rate of the stirring device during the fermentation process of the dynamic gut simulation system to be 85 to 115 rpm. The anaerobic unit is used to maintain the anaerobic environment during the fermentation process of the dynamic gut microbiota simulation system at O8 < 0.1% and CO8 ≥ 90%.

4. The in vitro microecological evaluation method for preclinical drug development based on live microbiota according to claim 3, characterized in that, Peristaltic pumps are installed on the infusion pipe, gas infusion pipe, feed pipe, and discharge pipe.

5. The in vitro microecological evaluation method for preclinical drug development based on live microbiota according to claim 4, characterized in that, The dynamic intestinal simulation system also includes a central control unit. When the pH value shifts by more than 0.3, the central control unit controls the infusion tube to deliver acid or alkali to compensate.

6. The in vitro microecological evaluation method for preclinical drug development based on live microbiota according to claim 5, characterized in that, The anaerobic unit monitors the O2 concentration every 6 hours. When the O2 concentration exceeds the standard, the central control unit controls the gas supply pipe to input N2 for flushing and inputs palladium catalyst for deoxygenation.

7. The in vitro microecological evaluation method for preclinical drugs based on live microbiota according to claim 6, characterized in that, The step S1 of obtaining fecal microbial inoculum includes: Weigh fresh feces from healthy individuals, dissolve them in a pH 6.8 buffer solution, filter out solid particles with gauze, and the resulting filtrate is the fecal microbial inoculum.

8. The in vitro microecological evaluation method for preclinical drugs based on live microbiota according to claim 7, characterized in that, Step S2, which involves obtaining concentrated bacterial cells of a live bacterial flora drug, includes: The lyophilized bacterial strains for resuscitating live microbial flora, including Bifidobacterium, were cultured in MRS medium for 16 to 24 hours and then centrifuged to a concentration of 1 × 10⁻⁶. 8 Bifidobacterium cells were collected after CFU / mL concentration.

9. The in vitro microecological evaluation method for preclinical drugs based on live microbiota according to claim 8, characterized in that, Step S3, which involves collecting bacterial solutions from the experimental, control, and blank containers using sampling tubes, includes: Before each use, the sampling tubes are sterilized by steam for 15 to 20 minutes at 0.1 to 0.2 MPa and 115 to 125°C, and then placed in an anaerobic chamber for ultraviolet irradiation for 25 to 30 minutes.

10. The in vitro microecological evaluation method for preclinical drug development based on live microbiota according to claim 9, characterized in that, Step S4 includes: The analysis of bacterial community dynamics in the bacterial culture included: Bifidobacterium abundance, Escherichia coli inhibition rate, Shannon diversity index, and Bacteroides / Firmwallis ratio; Metabolic function tests include: acetic acid production, propionic acid production, folic acid production, and β-glucosidase activity.

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