A fermentation method and application of a human intestinal flora

Abstract

The present application relates to a kind of fermentation method and application of human intestinal flora, it relates to the field of microbial fermentation, including the following steps: screening donor: 12-14 years old boy, healthy, no major illness history, no obvious gastrointestinal symptoms, at least 6 months without taking any antibiotic, life is regular;Sample collection and processing: the fresh feces of donor is collected, quickly transferred to anaerobic workstation, weighed, pre-reduced sterile saline is added according to proportion, fully vortex mix evenly;Then filter, collect fecal suspension;Fermentation: after transferring fecal suspension to culture medium, it is moved into multilayer movable coaxial cylindrical ring anaerobic fermentor, sealed fermentation;By constructing dynamic biomimetic fermentation system, the physical structure, chemical environment and physiological activity of human intestinal tract are simulated, the colonization efficiency of intestinal flora in vitro is significantly improved, metabolic activity and the yield and stability of functional product.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial fermentation, in particular to a fermentation method and application of human intestinal flora. BACKGROUND

[0002] In recent years, the role of intestinal microecology in human health and disease has been widely studied and recognized, especially in metabolic syndrome, obesity, inflammatory bowel disease, diabetes and neuropsychiatric diseases, which shows important regulatory potential. Simulating the human intestinal microenvironment and carrying out in vitro artificial intestinal system fermentation has become an important means to explore microbial metabolic mechanisms, evaluate prebiotic functions and develop individualized nutrition strategies. However, the current system for simulating human intestinal flora fermentation in vitro has many problems such as unstable anaerobic control, poor repeatability of flora structure, low efficiency of metabolic product collection, and is difficult to truly reflect the transformation process of in vivo flora ecological structure and functional products. Therefore, developing an anaerobic fermentation system with high stability, high repeatability and precise control of key parameters (such as temperature, pH, anaerobicity) to reconstruct and dynamically maintain a microecological environment similar to the human intestine has important practical significance and industrial value for improving the efficiency and accuracy of intestinal flora-related functional product research. SUMMARY

[0003] The present application provides a fermentation method and application of human intestinal flora to solve the problems of insufficient traditional fermentation microenvironment and unstable flora structure. By constructing a dynamic biomimetic fermentation system, the physical structure, chemical environment and physiological activity of the human intestine are simulated, and the colonization efficiency, metabolic activity and functional product yield and stability of intestinal flora in vitro are improved. It can be widely used in prebiotic screening, microecological function evaluation, nutriomics research and personalized intervention product development.

[0004] The technical solution of the present application to solve the above technical problems is as follows: a fermentation method of human intestinal flora, comprising the following steps:

[0005] a. Screening donors: 12-14 year old boys, healthy, no history of serious illness, no obvious gastrointestinal symptoms, no use of any antibiotics for at least 6 months, regular life;

[0006] b. Sample collection and processing: collect fresh feces of the donor, quickly transfer to an anaerobic workstation, weigh, add pre-reduced sterile normal saline according to the proportion, mix thoroughly by vortex; then filter and collect the fecal suspension;

[0007] c. Fermentation: transfer the fecal suspension to the culture medium and then move it into a multi-layer movable coaxial cylindrical ring anaerobic fermentation tank for sealed fermentation.

[0008] Furthermore, the fermentation medium contains 10.0 g / L peptone, 5.0 g / L glucose, 8.0 g / L disodium hydrogen phosphate, 2.0 g / L potassium dihydrogen phosphate, 20.0 g / L ox bile salt, 0.015 g / L brilliant green, and 1.0-2.0 g / L prebiotics;

[0009] Among them, prebiotics are at least one of inulin, fructooligosaccharides, and β-glucan.

[0010] Furthermore, the fermentation conditions were: temperature 37±0.5℃, pH 5.8-6.8, and gas was introduced to maintain a volume ratio of N2 80%, CO2 15%, and H2 5%.

[0011] Furthermore, the fermenter is made of stainless steel with a diameter of 300 mm. The tank is equipped with multiple layers of coaxial cylindrical rings with a gap of 15 mm. The surface of the cylindrical rings has microgrooves that are 200-300 μm deep and 50 μm wide, with a groove density of 35 grooves / mm².

[0012] Furthermore, the multi-layered movable coaxial cylindrical ring is capable of displacement, including radial displacement, axial displacement, and rotation.

[0013] Among them, radial displacement is generated by the telescopic cylinder moving to the telescopic cylinder A to D of the fixed frame, with an offset amplitude of 10-20μm and a frequency of 0.05 Hz;

[0014] Axial displacement, amplitude of 50-100 μm, frequency of 0.033 Hz;

[0015] The rotation speed is 5-10 rpm, and the rotation direction is bidirectional and alternating, 30 seconds clockwise and 30 seconds counterclockwise.

[0016] Furthermore, during the fermentation process, displacement operations are performed at preset times: 0-20 seconds: radial displacement;

[0017] 20-60 seconds: Axial displacement;

[0018] 60-180 seconds: Alternating rotation in both directions;

[0019] When the butyric acid concentration is detected to be <20mM, repeat the above displacement operation.

[0020] Furthermore, it also includes the renewal period operation: every 72 hours or when the butyric acid synthesis rate increases by <5% for 4 consecutive hours, start a bidirectional alternating rotation at a speed of 25±1 rpm for 90 seconds, while simultaneously performing an axial displacement with an amplitude of 120μm and a frequency of 0.1Hz.

[0021] Furthermore, the displacement is performed in stages after the update period ends:

[0022] Activation period of tactic bacteria 0-30 minutes: radial displacement, amplitude 15μm, frequency 0.05Hz;

[0023] Nutrient delivery period 30-90 minutes: bidirectional alternating rotation with radial displacement, rotation speed 15±0.5 rpm, axial displacement amplitude 80μm, frequency 0.033Hz;

[0024] The synergistic effect of the ditch bacteria is 90-180 minutes: axial coordinated radial displacement, with an axial amplitude of 100μm and a frequency of 0.1Hz, and a radial displacement of 20μm and a frequency of 0.05Hz.

[0025] The application of human gut microbiota obtained by a fermentation method: the fermented human gut microbiota can be used for prebiotic screening, microecological function assessment, nutritional omics research and personalized intervention product development.

[0026] The beneficial effects of this invention are as follows: By combining a multi-layered coaxial cylindrical ring structure with displacement-coordinated control, the physical microenvironment of the human gut is simulated, constructing a dynamic biomimetic fermentation system; by combining a multi-layered coaxial cylindrical ring structure with laser-etched microgrooves to simulate the intestinal environment, the problems of insufficient colonization area and lack of anaerobic microenvironment in traditional fermenters are solved; the displacement-coordinated drive system overcomes the limitations of mass transfer and uneven bacterial distribution in static fermentation; and through prebiotic combinations and strict anaerobic gas control, the bacterial community structure is optimized, metabolic efficiency is improved, and safety and stability are enhanced.

[0027] By initiating mechanical intervention through a dual-trigger renewal period, and employing bidirectional alternating rotation combined with axial vibration, the problem of mass transfer blockage caused by biofilm thickening and decreased metabolic activity of aging bacteria is solved. Through selective peeling, deep cleaning, and metabolic activation, butyric acid production is increased and the structure of anaerobic bacteria is optimized.

[0028] By coordinating the displacement operation in stages according to the functional division of the microbial community, and setting different displacement parameters at different stages, the butyric acid yield was further increased, the proportion of strictly anaerobic bacteria was increased, the survival rate of the microbial community was stabilized, the microbial community structure was optimized, and the fermentation stability was improved. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of a multi-layer coaxial cylindrical ring fermenter according to Embodiment 1 of the present invention;

[0030] Figure 2 This is a top view of the fixing frame in the multi-layer coaxial cylindrical ring fermenter of the present invention. Detailed Implementation

[0031] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0032] In the description of this application, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the stated features. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0033] In the description of this application, the term "for example" is used to mean "used as an example, illustration, or description." Any embodiment described as "for example" in this application is not necessarily to be construed as being more preferred or advantageous than other embodiments. The following description is provided to enable any person skilled in the art to make and use the invention. Details are set forth in the following description for purposes of explanation. It should be understood that those skilled in the art will recognize that the invention can be made without using these specific details. In other instances, well-known structures and processes will not be described in detail to avoid obscuring the description of the invention with unnecessary detail. Therefore, the invention is not intended to be limited to the embodiments shown, but is consistent with the broadest scope of the principles and features disclosed in this application.

[0034] Example 1

[0035] A fermentation method for human gut microbiota, specifically including the following steps:

[0036] a. Donor selection: Boys aged 12-14 years, in good health, with no history of serious illness, no obvious gastrointestinal symptoms, and who have not taken any antibiotics within the past 6 months and have a regular lifestyle;

[0037] b. Sample collection and processing: Fresh feces from the donor were collected, quickly transferred to the anaerobic workstation, weighed, and pre-reduced sterile saline was added in proportion and vortexed thoroughly; then filtered and the fecal suspension was collected.

[0038] The pre-reduced sterile saline solution was prepared by sterilizing a 0.9% sodium chloride solution at 121°C for 20 minutes, followed by degassing and gas replacement treatment in an anaerobic workstation for more than 12 hours; the mixing ratio of fresh feces to the pre-reduced sterile saline solution was 1:10 (w / v).

[0039] c. Fermentation: After transferring the fecal suspension to a culture medium, it is transferred to a multi-layer movable coaxial cylindrical ring anaerobic fermenter and sealed for fermentation;

[0040] The fermentation time of the fecal suspension is 30 to 90 days;

[0041] In the fermentation step, the fecal suspension used is either the fecal suspension from one donor or an equal volume mixture of fecal suspensions from two to five donors.

[0042] Fermentation time is 30 to 90 days;

[0043] The culture medium contains 10.0 g / L peptone, 5.0 g / L glucose, 8.0 g / L disodium hydrogen phosphate, 2.0 g / L potassium dihydrogen phosphate, 20.0 g / L ox bile salts, 0.015 g / L brilliant green, and 1.0-2.0 g / L prebiotics.

[0044] Prebiotics are at least one of inulin, fructooligosaccharides, and beta-glucan;

[0045] The temperature of the fermenter was 37±0.5℃, the pH was 5.8-6.8, and the gas flow rate was controlled to maintain the CO2 / N2 / H2 ratio at the standard level simulating the anaerobic environment of the intestinal lumen, i.e., N2 80%, CO2 15%, H2 5%.

[0046] The fermenter is made of stainless steel and has a diameter of 300 mm. The tank is equipped with multiple layers of coaxial cylindrical rings with a gap of 15 mm. The surface of the cylindrical rings has microgrooves that are 200-300 μm deep and 50 μm wide, with a groove density of 35 grooves / mm².

[0047] Micro-grooves are formed by laser etching;

[0048] like Figure 1 and Figure 2 As shown, the multi-layer coaxial cylindrical ring is capable of displacement, including radial displacement, axial displacement, and rotation.

[0049] Among them, radial displacement is generated by the telescopic cylinder moving to the telescopic cylinder A to D of the fixed frame, with an offset amplitude of 10-20μm and a frequency of 0.05 Hz (1 time / 20s);

[0050] Axial displacement, amplitude of 50-100 μm, frequency of 0.033 Hz (1 time / 30s);

[0051] The rotation speed is 5-10 rpm, and the rotation direction is bidirectional and alternating, 30 seconds clockwise and 30 seconds counterclockwise;

[0052] During the microbial growth cycle, the first 0-20 seconds of fermentation: radial displacement;

[0053] 20-60 seconds: Axial displacement;

[0054] 60-180 seconds: Rotation;

[0055] 180-7200 seconds: No displacement occurs during the recovery period;

[0056] When the butyric acid concentration is <20mM, the above displacement process is performed.

[0057] An experiment was conducted on the technical solution of the above embodiment, wherein the bacterial source was an equal volume of mixed fecal suspension from three healthy 13-year-old boys (diluted with physiological saline at a ratio of 1:10).

[0058] Culture medium: peptone 10.0 g / L, glucose 5.0 g / L, Na2HPO4 8.0 g / L, KH2PO4 2.0 g / L, taurine 20.0 g / L, brilliant green 0.015 g / L, inulin + fructooligosaccharide 1.5 g / L;

[0059] Fermentation environment: 37±0.5℃, pH 6.0±0.1, N2: 80%, CO2: 15%, H2: 5%;

[0060] During fermentation, the radial displacement amplitude was 15 μm and the frequency was 0.05 Hz (3 times / min) for 0-20 seconds; the axial displacement amplitude was 80 μm and the frequency was 0.033 Hz (2 times / min) for 20-60 seconds; and the rotation was 10 rpm for 60-180 seconds, with bidirectional alternating rotation (30 seconds clockwise / 30 seconds counterclockwise). The comparative example used a traditional stainless steel anaerobic fermenter without a cylindrical ring structure.

[0061] The fermentation products were tested and analyzed.

[0062] 1. Butyric acid yield: The butyric acid concentration in the culture broth after fermentation was determined by GC-MS (gas chromatography-mass spectrometry).

[0063] 2. Proportion of strict anaerobic bacteria: The relative abundance of strict anaerobic bacteria (such as Faecalibacterium, Bacteroides, Clostridium IV, etc.) in fermentation products was analyzed by 16S rRNA gene sequencing.

[0064] 3. Viable bacterial count: The proportion of viable bacteria was determined by flow cytometry combined with viable bacterial staining (such as SYBR Green I and PI double staining). The results are shown in Table 1 below.

[0065] Table 1

[0066]

[0067] The technical solutions described in the embodiments of this application have at least the following technical effects or advantages:

[0068] By combining a multi-layered coaxial cylindrical ring structure with displacement-coordinated control, a dynamic biomimetic fermentation system is constructed to simulate the physical microenvironment (peristalsis, nutrient gradient, anaerobic ecology) of the human gut.

[0069] The multi-layered coaxial cylindrical ring structure expands the colonization surface area through geometric progression, significantly increasing the effective contact area compared to the basic tank, simulating the multi-level branching structure of human intestinal villi; the microgrooves etched by laser photolithography simulate the size ratio of colonic crypts, and the 50μm width of the grooves limits the colonization of only 1-3 layers of bacteria in a single groove, thus avoiding the accumulation of dead cells in the deep anaerobic zone. The groove depth (250μm) exceeds the oxygen diffusion limit (about 200μm), naturally forming a strictly anaerobic microzone at the bottom of the tank, meeting the needs of strict anaerobic bacteria such as Faecalibacterium, while the opening of the tank maintains a micro-aerobic environment through gas exchange, meeting the needs of facultative anaerobic bacteria and forming an oxygen gradient;

[0070] Radial displacement (10-20 μm, frequency 0.05 Hz) of multi-layer coaxial cylindrical rings removes dissolved oxygen. Micro-amplitude high-frequency vibrations induce the Bernoulli effect at the liquid-gas interface. As the rings radially displace, the liquid phase undergoes periodic compression and relaxation, causing dissolved oxygen to migrate from the liquid phase to the gas phase due to pressure fluctuations. The 0.05 Hz frequency (20 seconds / cycle) matches the oxygen dissociation kinetic time constant (approximately 18 seconds), achieving a dissolved oxygen removal efficiency of 95%. Simultaneously, the 15 μm amplitude maintains laminar flow, preventing turbulent flow from damaging the bacterial community.

[0071] Axial displacement (50-100 μm, frequency 0.033 Hz) renews the biofilm. The axial displacement simulates the shear force of intestinal peristalsis. The amplitude of 80 μm exceeds the elastic limit of the biofilm (about 50 μm), causing brittle fracture of the senescent bacterial layer. The newly formed bacteria are retained due to the strong adhesion of the extracellular polysaccharides they secrete. The frequency of 0.033 Hz (30 seconds / cycle) allows the extracellular polysaccharide protective layer to be rebuilt within the interval between two vibrations, achieving selective peeling. The senescent bacteria removal rate is 40%, and the loss of viable bacteria is <3%.

[0072] Bidirectional rotation balances nutrition. During alternating rotation, nutrients migrate to the outer ring due to centrifugal force when rotating forward, and metabolic waste accumulates to the inner ring due to centripetal force when rotating counterclockwise. This bidirectional convection increases the mass transfer rate of substrates such as glucose. The rotation speed of 5-10 rpm controls the shear force to be below 1.5 Pa, which is lower than the bacterial damage value, thus avoiding mechanical stress damage.

[0073] The prebiotic combination selectively enriches butyric acid-producing bacteria, promoting increased butyric acid production. The constant temperature of 37°C and the strict anaerobic environment maintain an efficient metabolic pathway and improve energy metabolism.

[0074] Through multidimensional synergistic effects, the overall function of the microbial community is comprehensively enhanced. First, the anaerobic colonization microenvironment created by the microgroove structure leads to a surge in the proportion of strict anaerobic bacteria, laying an ecological foundation for efficient butyrate synthesis. Second, the displacement-driven system composed of radial movement and bidirectional rotation significantly enhances substrate diffusion and product removal efficiency, resulting in a substantial increase in butyrate production. Simultaneously, low-amplitude, high-frequency axial vibration controls mechanical stress below the 1.5 Pa damage threshold while stripping away aging biofilms, improving the survival rate of the microbial community. Finally, the butyrate accumulation driven by the targeted activation of the metabolic network, combined with environmental pH regulation (5.8-6.8) and the bile salt / brilliant green inhibition system, inhibits the production of pathogens.

[0075] The multi-layered coaxial cylindrical rings, combined with their displacement movement and the components of the culture medium, produce a synergistic effect during fermentation. Radial movement removes dissolved oxygen, ensuring the stability of the anaerobic zone at the bottom of the trench and increasing the abundance of strict anaerobic bacteria. Axial movement detaches aging biofilms, releasing polysaccharide fragments that are directionally converted into butyrate precursors by the prebiotic combination. Bidirectional rotation rapidly transports newly generated butyrate throughout the tank, preventing localized low pH that inhibits bacterial activity and forming a metabolic cycle. The increased butyrate concentration further lowers the environmental pH to 5.8-6.0, inhibiting the growth of aerobic pathogens such as Escherichia coli, while simultaneously promoting the secretion of antimicrobial peptides by acid-tolerant bacteria (such as Lactobacillus), achieving niche blockade of pathogens. This dynamic balance significantly improves the survival rate of the bacterial community.

[0076] Example 2

[0077] The above-mentioned Embodiment 1 uses a multi-layer coaxial cylindrical ring structure combined with laser-etched microgrooves to simulate the intestinal environment, solving the problems of insufficient colonization area and lack of anaerobic microenvironment in traditional fermenters; it overcomes the limitations of mass transfer and uneven distribution of microbiota in static fermentation through a displacement-coordinated drive system; and it optimizes the microbiota structure, improves metabolic efficiency and safety and stability through prebiotic combination and strict anaerobic gas control, further improving the overall function of the microbiota based on Embodiment 1.

[0078] Every 72 hours, or when the butyric acid synthesis rate increases by less than 5% for 4 consecutive hours, a renewal period is set, and rotation and axial displacement are initiated. The rotation mode is bidirectional and alternating, with a rotation speed of 25±1 rpm and a time of 90 seconds. Simultaneously, axial displacement is performed, with the amplitude of the axial displacement increased to 120μm and the frequency of 0.1Hz to enhance shear.

[0079] Based on the technical solution of the above embodiment, experiments were conducted. The difference between the technical solution of this embodiment and the technical solution of embodiment one is that an update period is added in this embodiment. Rotation is started every 72 hours or when the butyric acid synthesis rate increases by <5% for 4 consecutive hours. The rotation mode is bidirectional alternation, the rotation speed is 25±1 rpm, and the time is 90 seconds. At the same time, the amplitude of the axial displacement is increased to 120μm and the frequency is 0.1Hz to enhance shear. The detection results of this embodiment are shown in Table 2 below.

[0080] Table 2

[0081]

[0082] The technical solutions described in the embodiments of this application have at least the following technical effects or advantages:

[0083] A periodic renewal mechanism was introduced, initiating high-intensity mechanical intervention through dual triggering conditions (a 72-hour time period or a decrease in metabolic efficiency). Decreased metabolic efficiency refers to a butyrate synthesis rate increase of less than 5% for four consecutive hours, primarily due to excessive biofilm thickening hindering substrate diffusion, interruption of nutrient supply to deeper layers of the microbial community, an increased proportion of senescent bacteria, and decreased metabolic activity leading to a reduction in the expression of the rate-limiting enzyme for butyrate synthesis. When the triggering conditions were met, a bidirectional alternating high-speed rotation of 25±1 rpm was initiated, generating a centrifugal force field >12 N / m² acting on the biofilm. After rotation was initiated, senescent bacteria, due to the degradation of extracellular polysaccharides, experienced reduced cell-substrate binding and were preferentially detached under centrifugal force. Newly formed bacteria secreted novel extracellular polysaccharides rich in β-1,3-glucan, exhibiting enhanced binding force and strong shear resistance, and were thus retained.

[0084] Simultaneously applying a 120μm axial displacement at a frequency of 0.1Hz creates a shear wave propagation effect. When the vibration wave propagates in the liquid phase, it generates micro-fluid vortices in the pores of the biofilm, flushing away aging bacterial fragments and accumulated metabolic waste deep in the gullies, while stimulating new bacteria to secrete adhesion factors to accelerate colonization.

[0085] By setting a renewal period, butyric acid production was increased to 38.2 mM. This was achieved by clearing senescent bacteria to free up colonization space and increasing the proportion of new bacteria. The proportion of strictly anaerobic bacteria was further optimized, and vibration stimulation promoted the secretion of functional proteins by butyric acid-producing bacteria, further increasing their relative abundance. The renewal period also broke the metabolic stagnation period, thereby improving fermentation efficiency and product quality.

[0086] Example 3

[0087] The above-mentioned Example 2 initiates mechanical intervention through a dual-trigger renewal period, using bidirectional alternating rotation combined with axial vibration to solve the problems of mass transfer blockage caused by biofilm thickening and decreased metabolic activity of aging bacteria. Through selective peeling, deep cleaning and metabolic activation, it achieves increased butyric acid production and optimized anaerobic bacterial structure. Based on Example 2, it is further improved to further enhance the overall function of the bacterial community.

[0088] The microbial community in fermentation includes raffinate bacteria and flow-promoting bacteria. Raffinate bacteria include Bacteroides, and flow-promoting bacteria include Pseudomonas.

[0089] After the update period, the 0-30 minute period is the activation period for flow-promoting bacteria, the 30-90 minute period is the nutrient delivery period, and the 90-180 minute period is the synergistic period for crevices bacteria.

[0090] During the activation phase of the tidal bacteria, radial displacement is initiated with an amplitude of 15 μm and a frequency of 0.05 Hz.

[0091] During the nutrient delivery period, the device rotates alternately in both directions while simultaneously displacing radially. The rotation speed is 15±0.5 rpm, the amplitude of the axial displacement is 80μm, and the frequency is 0.033Hz.

[0092] During the synergistic phase of the gully bacteria, axial and radial synergistic displacements are combined, with an axial amplitude of 100 μm and a frequency of 0.1 Hz, and a radial displacement of 20 μm and a frequency of 0.05 Hz.

[0093] Based on Embodiment 2, experiments were conducted on the technical solutions of the above embodiments. The difference between the technical solutions of this embodiment and Embodiment 2 is that, after the update period, the displacement of the crevices bacteria and the flow-tactic bacteria was further adjusted at different times. Specifically, after the update period, 0-30 minutes was the flow-tactic bacteria activation period, 30-90 minutes was the nutrient transport period, and 90-180 minutes was the crevices bacteria enhancement period. During the flow-tactic bacteria activation period, radial displacement was initiated with an amplitude of 15 μm and a frequency of 0.05 Hz. During the nutrient transport period, bidirectional alternating rotation was performed while radial displacement was carried out, with a rotation speed of 15 ± 0.5 rpm and an axial displacement amplitude of 80 μm and a frequency of 0.033 Hz. The detection results of this embodiment are shown in Table 3 below.

[0094] Table 3

[0095]

[0096] The technical solutions described in the embodiments of this application have at least the following technical effects or advantages:

[0097] The displacement operation was coordinated in stages according to the functional division of the bacterial community. During the activation period of tidal bacteria (0-30 minutes), the radial displacement of 15 μm / 0.05 Hz generated a microvortex with a diameter of 50 μm in the ring gap, which reduced the viscous resistance of the liquid. Matching the flagellar movement frequency of Pseudomonas aeruginosa (response frequency 0.04-0.06 Hz), it could cause tidal bacteria to detach from the temporary anchoring point and increase the migration speed to 250 μm / s. At the same time, it removed the metabolic waste attached to its surface and formed a high-density bacterial community band, laying the foundation for nutrient transport.

[0098] During the nutrient transport phase (30-90 minutes), a bidirectional rotation at 15±0.5 rpm generates tangential laminar flow (0.1-0.3 m / s), which is superimposed with unidirectional shear generated by an axial displacement of 80 μm / 0.033 Hz. The rotating flow propels the flow-tactic bacteria to migrate with glucose into the groove area. The axial vibration sawtooth wave removes senescent bacterial debris from the grooves, improving substrate diffusion efficiency and building a material exchange bridge between bacterial communities. The flow-tactic bacteria transport glucose to the groove area, shortening the transport distance.

[0099] During the synergistic phase (90-180 minutes) of Bacteroides, axial displacement generates 0.2-0.5 Pa of shear stress, exceeding the elastic limit of the biofilm of Bacteroides; radial displacement enhances deep trench penetration; axial high-frequency tearing of aging biofilm exposes new colonization sites; radial displacement enhances the substrate's ability to penetrate into microgrooves and promotes butyric acid synthesis.

[0100] Through a three-stage control guided by microbial community function, butyric acid production was further improved. In particular, the substrate diffusion efficiency was improved during the nutrient transport phase, which significantly increased the amount of carbon source entering the metabolic pathway per unit time. In addition, the high-frequency shearing during the synergistic phase precisely activated the metabolic enzyme system of the ditch bacteria, shortening the butyric acid synthesis cycle.

[0101] The microbial community structure was further optimized, and the proportion of strictly anaerobic bacteria was further increased. On the one hand, high-frequency axial vibration selectively eliminated aerobic bacteria; on the other hand, the rapid colonization of new fossicular sites by fossicular bacteria compressed the living space of facultative anaerobes. The uniformity of microbial community distribution was also improved, the annular coverage of fluctuatory bacteria was increased, and the fossicular fossicular ditch anchorage rate was improved.

[0102] Improved stability, stable microbial survival rate, reduced rotation speed, and reduced shear stress, thereby reducing mechanical damage; improved fermentation stability, and avoidance of microbial functional conflicts through three-stage temporal isolation.

[0103] It should be noted that the descriptions of each embodiment in the above embodiments have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0104] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0105] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A fermentation method for human gut microbiota, characterized in that, Includes the following steps: a. Donor selection: Boys aged 12-14 years, in good health, with no history of serious illness, no obvious gastrointestinal symptoms, and who have not taken any antibiotics within the past 6 months and have a regular lifestyle; b. Sample collection and processing: Fresh feces from the donor were collected, quickly transferred to the anaerobic workstation, weighed, and pre-reduced sterile saline was added in proportion and vortexed thoroughly; then filtered and the fecal suspension was collected. c. Fermentation: After transferring the fecal suspension to a culture medium, it is transferred to a multi-layer movable coaxial cylindrical ring anaerobic fermenter and sealed for fermentation; The fermenter is made of stainless steel and has a diameter of 300 mm. The fermenter is equipped with multiple layers of coaxial cylindrical rings with a gap of 15 mm. The surface of the cylindrical rings has microgrooves that are 200-300 μm deep and 50 μm wide, with a groove density of 35 grooves / mm². The multi-layered movable coaxial cylindrical ring is capable of displacement, including radial displacement, axial displacement, and rotation. The radial displacement is generated by the telescopic cylinder moving to the telescopic cylinders A to D of the fixed frame, with an offset amplitude of 10-20 μm and a frequency of 0.05 Hz; the axial displacement has an amplitude of 50-100 μm and a frequency of 0.033 Hz; the rotation speed is 5-10 rpm, and the rotation direction is bidirectional alternating, 30 seconds clockwise / 30 seconds counterclockwise. During fermentation, displacement operations are performed at preset times: 0-20 seconds: radial displacement; 20-60 seconds: axial displacement; 60-180 seconds: bidirectional alternating rotation; when the butyric acid concentration is detected to be <20mM, the above displacement operations are repeated. It also includes the update period operation: every 72 hours or when the butyric acid synthesis rate increases by <5% for 4 consecutive hours, start a bidirectional alternating rotation at a speed of 25±1 rpm for 90 seconds, while simultaneously performing an axial displacement with an amplitude of 120μm and a frequency of 0.1Hz. The displacement will be performed in stages after the update period ends: Activation period of tactic bacteria 0-30 minutes: radial displacement, amplitude 15μm, frequency 0.05Hz; Nutrient delivery period 30-90 minutes: bidirectional alternating rotation with radial displacement, rotation speed 15±0.5 rpm, axial displacement amplitude 80μm, frequency 0.033Hz; The synergistic effect of the ditch bacteria is 90-180 minutes: axial coordinated radial displacement, with an axial amplitude of 100μm and a frequency of 0.1Hz, and a radial displacement of 20μm and a frequency of 0.05Hz.

2. The fermentation method for human intestinal flora according to claim 1, characterized in that, The culture medium used in fermentation contained 10.0 g / L peptone, 5.0 g / L glucose, 8.0 g / L disodium hydrogen phosphate, 2.0 g / L potassium dihydrogen phosphate, 20.0 g / L ox bile salts, 0.015 g / L brilliant green, and 1.0-2.0 g / L prebiotics. Among them, prebiotics are at least one of inulin, fructooligosaccharides, and β-glucan.

3. The fermentation method for human intestinal flora according to claim 1, characterized in that, Fermentation conditions were: temperature 37±0.5℃, pH 5.8-6.8, and gas was introduced to maintain a volume ratio of N2 80%, CO2 15% and H2 5%.

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