A probiotic composition for modulating gut microbiota and uses thereof

Abstract

The present application relates to the technical fields of fermentation engineering and microbial application, and discloses a probiotic composition for regulating intestinal microorganisms and application, which comprises the following steps: putting animal Bifidobacterium freeze-dried powder and porous amorphous auxiliary materials into a mixing kettle, controlling first-order shearing rotating speed to complete premixing, so that the animal Bifidobacterium powder is adsorbed in the pores of the porous amorphous auxiliary materials; adding Lactobacillus freeze-dried powder and adjusting second-order rotating speed to complete blending, so that the Lactobacillus powder is filled in the gap between the porous amorphous auxiliary material particles to construct a spatial isolation structure; and determining the overall water activity of the finished product material through an online water activity detection sensor and linking and adjusting the shunt, wherein the present application changes the water adsorption potential energy distribution state in the multiphase heterogeneous system, blocks the path of water interfacial mass transfer during the storage period, and improves the normal temperature stability of the finished product.

Description

Technical Field

[0001] This invention belongs to the fields of fermentation engineering and microbial application technology, and particularly relates to a probiotic composition for regulating intestinal microorganisms and its application. Background Technology

[0002] Currently, in the preparation process of microbial agents, controlling the humidity of the mixing environment and reducing the initial water activity of solid powder are means to extend the shelf life of live bacteria. Generally, closed-loop water control processes such as overall material drying combined with nitrogen-filled and sealed packaging are used to inhibit the metabolic activity inside the bacterial cells and maintain the stability of the solid material system during the shelf life at room temperature. For compound probiotic preparations, the retention rate of compound strain activity directly determines the engineering value of the finished product.

[0003] However, the surface composition of freeze-dried powder particles from different bacterial genera varies, exhibiting nonlinear isothermal hygroscopic characteristics. This leads to a difference in water adsorption potential energy within the solid heterogeneous system. During long-term storage, this potential energy difference triggers spontaneous phase migration and cross-interface mass transfer redistribution of free water molecules. Water molecules continuously accumulate on the surface of sensitive strains with slow hygroscopic rates but high binding energy levels, resulting in an abnormally high local water activity in these strains. This induces an unexpected phase transition from a crystalline to a fluid state in the cell membrane lipid bilayer, incorrectly activating dormant intracellular enzyme systems and causing spontaneous inactivation of the sensitive strains within the shelf life. This disrupts the synergistic balance between the compound bacterial genera. To address the aforementioned water migration defects, adding excessive amounts of initial live bacteria significantly increases production costs, while extreme drying processes can easily cause physical dehydration damage to bacterial cells and induce particle hygroscopicity. The rebound is due to the fact that static compounding methods that rely on overall material drying are insufficient to curb moisture migration within the surface heterogeneous system. Furthermore, the dynamic moisture control methods in the mixing process are inadequate. For example, Chinese invention patent application CN112106916A discloses a compound probiotic solid beverage and its preparation method, which reduces the overall water activity of the mixed materials by increasing the drying temperature of the excipients. This approach relies excessively on conventional hot air drying and fails to understand the deep mass transfer mechanism caused by differences in hygroscopic kinetics between different bacterial genera. Due to the lack of surface-scale spatial isolation barriers, it cannot prevent the spontaneous redistribution of moisture to sensitive strains during storage. The mixing process is an open-loop operation, lacking adaptive and linked sensing of the moisture content of the materials under mixing conditions. In continuous industrial production, fluctuations in environmental humidity can easily cause the finished product's water activity to deviate from the design window.

[0004] Therefore, the technical problem to be solved by this invention is how to construct a spatial physical barrier to restrict the cross-interface mass transfer of water based on the differences in hygroscopic kinetics among the compound bacteria, block the local enrichment and migration of free water during storage, and establish an adaptive closed-loop control system for mixing control and material moisture content perception. Summary of the Invention

[0005] This invention aims to solve the problems of local microenvironmental water enrichment and inactivation of sensitive strains due to cross-interface water mass transfer in composite strain powder systems, as well as the lack of closed-loop adaptive regulation in the mixing and water control process.

[0006] In this technical solution, a probiotic composition for regulating intestinal microbiota includes: Lyophilized Bifidobacterium animalis powder with a mass percentage of 15% to 25% and an initial water activity of 0.12 to 0.15; Porous amorphous excipients with a mass percentage of 45% to 55% and an initial water activity of 0.01 to 0.09, the median particle size of the porous amorphous excipients being 50 μm to 150 μm and the average pore size of the micropores being 2 nm to 10 nm; and lyophilized Lactobacillus acidophilus powder with a mass percentage of 5% to 15% and an initial water activity of 0.15 to 0.18. Lyophilized powder of *Lactobacillus plantarum* with a mass percentage of 5% to 15% and an initial water activity of 0.15 to 0.18, and lyophilized powder of *Lactobacillus rhamnosus* with a mass percentage of 5% to 15% and an initial water activity of 0.15 to 0.18; The lyophilized Bifidobacterium animalis powder is distributed within the micropores of the porous amorphous excipient; the lyophilized Lactobacillus acidophilus powder, the lyophilized Lactobacillus plantarum powder, and the lyophilized Lactobacillus rhamnosus powder are distributed within the interparticle spaces of the porous amorphous excipient. The porous amorphous excipient has hydrophilic hydroxyl groups on its microporous surface; the single-dose live bacteria addition of the probiotic composition product is 30 billion CFU to 60 billion CFU, and the water activity of the probiotic composition product is 0.10 to 0.13.

[0007] Preferably, based on 100% of the total weight of the probiotic composition, it includes 5% to 10% by weight of Lactobacillus reuteri lyophilized powder with an initial water activity of 0.15 to 0.18; the Lactobacillus reuteri lyophilized powder is distributed in the interparticle spaces of the porous amorphous excipient.

[0008] Preferably, the ratio of the initial water activity of the porous amorphous excipient to the water activity of the finished probiotic composition is 1:1.1 to 1:1.5.

[0009] Preferably, the total pore volume of the micropores in the porous amorphous excipient is between 0.2 cm³ / g and 0.5 cm³ / g.

[0010] Preferably, in the probiotic composition, the ratio of the live bacteria count of Bifidobacterium animalis freeze-dried powder to the total live bacteria count of Lactobacillus acidophilus freeze-dried powder, Lactobacillus plantarum freeze-dried powder, and Lactobacillus rhamnosus freeze-dried powder is 1:1 to 1:2.

[0011] Preferably, the probiotic composition is sealed in a nitrogen-filled aluminum foil bag, and the nitrogen volume percentage of the nitrogen-filled aluminum foil bag is 98% to 99%.

[0012] Preferably, the solid phase transition temperature of the lyophilized Bifidobacterium animalis powder is 26°C to 35°C.

[0013] Preferably, the probiotic composition is in the form of a solid powder.

[0014] Preferably, the porous amorphous excipient is microcrystalline cellulose.

[0015] Application of a probiotic composition for regulating gut microbiota: Using a probiotic composition for regulating gut microbiota in the preparation of products that improve irritable bowel syndrome and gastrointestinal health.

[0016] Compared with existing technologies, the probiotic composition for regulating intestinal microbiota of the present invention has the following advantages: 1. In a probiotic composition for regulating intestinal microorganisms, a sequential step blending process based on the differences in hygroscopic kinetics of bacterial strains is used. Initially, Bifidobacterium animalis powder with low water activity is preferentially pre-mixed with porous amorphous excipients. This allows the Bifidobacterium animalis powder to be physically anchored within the adsorption pores on the surface of the excipient particles. Subsequently, Lactobacillus powder introduced through blending freely fills the external gaps between the excipient particles. This utilizes the spatial occupancy of the excipient particles to construct a three-dimensional physical barrier between the Bifidobacterium animalis and Lactobacillus strains, altering the distribution of water adsorption potential within the multiphase heterogeneous system. This blocks the kinetic pathway of cross-interface mass transfer of water to the Bifidobacterium animalis encapsulation space during room temperature storage, reducing its tendency for local water accumulation and maintaining the stability of water distribution within the multi-strain coexisting solid material system.

[0017] 2. By relying on the coordinating hydrophilic groups on the surface pores of porous amorphous excipient particles to preferentially capture free water molecules, a water binding energy level ladder is established at the physical interface. Combined with the inhibitory effect of three-dimensional physical isolation barriers on cross-interface water mass transfer, the aggregation dynamics of free water on the surface of sensitive strains are weakened. This avoids the unexpected phase transition of the cell membrane lipid bilayer from crystalline to fluid state caused by the increase in local environmental water activity in Bifidobacterium animalis. It maintains the dormant state of the intracellular metabolic enzyme system of sensitive strains during room temperature storage, spontaneously delaying its irreversible inactivation process. It overcomes the synergistic attenuation defect caused by the heterogeneity of the hygroscopic characteristics of the powder material. It can ensure the stability of high viable count of fermented products during long shelf life without excessively increasing the drying limit of the material.

[0018] 3. An online water activity detection feedback loop is set at the discharge end of the mixing vessel to dynamically track the overall water activity status of the finished powder material output from the mixing vessel. An automatic discharge and flow direction switching control response based on a preset water activity threshold range is established. When the water activity is within the preset qualified range, single-bag nitrogen-filled and sealed packaging is opened for dispensing. When the water activity exceeds the preset upper limit, the flow path is automatically switched to the circulating airflow drying path. This achieves closed-loop fine control of the water content of multiphase microbial powder materials, avoids the response delay and risk of whole batch material inactivation caused by the traditional batch sampling mode, reduces the probability of powder embrittlement and hygroscopic rebound caused by over-drying, improves the adaptive adjustment capability of process parameters in the production process, and ensures the uniformity of live bacteria addition and product quality among the finished products in each bag. Attached Figure Description

[0019] Figure 1 This is a flow chart of the sequential blending process of the probiotic composition of the present invention; Figure 2 This is the control logic diagram of the automated fermentation control system of the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0021] A probiotic composition for regulating gut microbiota, comprising: Lyophilized Bifidobacterium animalis powder with a mass percentage of 15% to 25% and an initial water activity of 0.12 to 0.15; Porous amorphous excipients with a mass percentage of 45% to 55% and an initial water activity of 0.01 to 0.09, the median particle size of the porous amorphous excipients being 50 μm to 150 μm and the average pore size of the micropores being 2 nm to 10 nm; and lyophilized Lactobacillus acidophilus powder with a mass percentage of 5% to 15% and an initial water activity of 0.15 to 0.18. Lyophilized powder of *Lactobacillus plantarum* with a mass percentage of 5% to 15% and an initial water activity of 0.15 to 0.18, and lyophilized powder of *Lactobacillus rhamnosus* with a mass percentage of 5% to 15% and an initial water activity of 0.15 to 0.18; The lyophilized Bifidobacterium animalis powder is distributed within the micropores of the porous amorphous excipient; the lyophilized Lactobacillus acidophilus powder, the lyophilized Lactobacillus plantarum powder, and the lyophilized Lactobacillus rhamnosus powder are distributed within the interparticle spaces of the porous amorphous excipient. The porous amorphous excipient has hydrophilic hydroxyl groups on its microporous surface; the single-dose live bacteria addition of the probiotic composition product is 30 billion CFU to 60 billion CFU, and the water activity of the probiotic composition product is 0.10 to 0.13.

[0022] Preferably, based on 100% of the total weight of the probiotic composition, it includes 5% to 10% by weight of Lactobacillus reuteri lyophilized powder with an initial water activity of 0.15 to 0.18; the Lactobacillus reuteri lyophilized powder is distributed in the interparticle spaces of the porous amorphous excipient.

[0023] Preferably, the ratio of the initial water activity of the porous amorphous excipient to the water activity of the finished probiotic composition is 1:1.1 to 1:1.5.

[0024] Preferably, the total pore volume of the micropores in the porous amorphous excipient is between 0.2 cm³ / g and 0.5 cm³ / g.

[0025] Preferably, in the probiotic composition, the ratio of the live bacteria count of Bifidobacterium animalis freeze-dried powder to the total live bacteria count of Lactobacillus acidophilus freeze-dried powder, Lactobacillus plantarum freeze-dried powder, and Lactobacillus rhamnosus freeze-dried powder is 1:1 to 1:2.

[0026] Preferably, the probiotic composition is sealed in a nitrogen-filled aluminum foil bag, and the nitrogen volume percentage of the nitrogen-filled aluminum foil bag is 98% to 99%.

[0027] Preferably, the solid phase transition temperature of the lyophilized Bifidobacterium animalis powder is 26°C to 35°C.

[0028] Preferably, the probiotic composition is in the form of a solid powder.

[0029] Preferably, the porous amorphous excipient is microcrystalline cellulose.

[0030] Application of a probiotic composition for regulating gut microbiota: Using a probiotic composition for regulating gut microbiota in the preparation of products that improve irritable bowel syndrome and gastrointestinal health.

[0031] Example 1: A specific application of the composition of the present invention is as follows: 20% (by mass) of freeze-dried Bifidobacterium animalis powder with an initial water activity of 0.12 is added to a mixing vessel. 50% (by mass) of microcrystalline cellulose is added to the vessel. The median particle size of the microcrystalline cellulose is 100 μm, the average pore size is 6 nm, the initial water activity is 0.05, and the micropore surface has hydrophilic hydroxyl groups. Before adding the materials, the solid-state phase transition temperature of the cell membrane lipid bilayer of the freeze-dried Bifidobacterium animalis powder, where the lipid bilayer transitions from an ordered crystalline state to a disordered fluid state, is determined by differential scanning calorimetry (DSC). The test conditions are set to scan from 0°C to 60°C at a temperature change rate of 5°C / min under a pure nitrogen protective atmosphere. The initial transition temperature point of the endothermic peak is extracted, and the solid-state phase transition temperature is measured to be between 26°C and 35°C. The variable temperature range has clear engineering significance for water control and stability: if the phase transition temperature is below 26℃, when the powder is stored at room temperature, slight external temperature fluctuations can easily trigger an unexpected phase transition of the cell membrane from a crystalline to a fluid state, activating the intracellular dormant enzyme system and causing it to spontaneously inactivate within the shelf life; if the phase transition temperature is above 35℃, it indicates that excessive physical dehydration occurred inside the bacterial cells during the early fermentation freeze-drying process, resulting in excessive embrittlement of the cell membrane skeleton. Under the first-order shear premixing condition, it is extremely susceptible to mechanical shear tearing damage, which reduces the survival rate of live bacteria. Under the conditions of 15% relative humidity and 20℃ ambient temperature, the first-order time-sequential premixing was carried out at a shear speed of 40r / min for 18min, allowing the freeze-dried Bifidobacterium animalis powder to enter and embed in the micropores on the surface of the microcrystalline cellulose particles.

[0032] 10% by mass of lyophilized Lactobacillus acidophilus powder, 10% by mass of lyophilized Lactobacillus plantarum powder, and 10% by mass of lyophilized Lactobacillus rhamnosus powder were added to the reactor. The initial water activity of the three Lactobacillus powders was 0.16. The shear speed of the mixing reactor was adjusted to 25 r / min for two-stage sequential mixing for 12 min, so that the Lactobacillus powders were evenly distributed in the external gaps between the microcrystalline cellulose particles. The water activity of the finished product was measured to be 0.11 in real time by an online water activity detection sensor at the discharge port. The system controlled the discharge valve to open and transported the material to a vacuum nitrogen-filled sealed packaging machine. Quantitative dispensing was performed under the condition of 98% nitrogen volume percentage. The amount of live bacteria added per bag was 45 billion CFU. The resulting solid powder was used to prepare products for improving irritable bowel syndrome.

[0033] Example 2: The probiotic composition for regulating intestinal microbiota of the present invention was used in a gradient control experiment to verify the effect of a specific water activity range and the adsorption characteristics of porous amorphous excipients on the synergistic survival rate of the complex microbial community. The experimental design included the sample group of the present invention, a partially missing control group, and an out-of-range control group. The sample group of the present invention contained 20% by mass of lyophilized Bifidobacterium animalis powder with an initial water activity of 0.13, and 50% by mass of porous microcrystalline cellulose excipient with an average pore size of 6 nm and a median particle size of 10 nm. In a drying workshop with a relative humidity of 18% and an ambient temperature of 22℃, the two groups of materials were added to a mixing vessel. The first-order shear speed was set to 50 r / min, and premixing was performed for 18 min. This allowed the freeze-dried Bifidobacterium animalis powder to enter and anchor within the micropores of porous microcrystalline cellulose, completing the physical shielding construction of the bacterial strain. During this physical shielding construction process, the surface geometry of the intact Bifidobacterium animalis cell is typically in the micrometer range, while the average pore size of the micropores in porous microcrystalline cellulose is in the nanometer range. The first-order shear premixing... Specifically, the mechanism involves high-frequency shear friction causing the surface layer of nanoscale sugar and protein protective matrix debris adhering to the periphery of the Bifidobacterium animalis freeze-dried powder to peel off. These nanoscale debris and the accompanying subcellular particles preferentially fill and mechanically embed themselves within the 2nm to 10nm micropores on the surface of porous microcrystalline cellulose. Meanwhile, the intact bacterial cells at the micrometer scale are firmly adsorbed and anchored to the periphery of the micropores through hydrogen bonds formed by hydrogen bonds of hydrophilic hydroxyl groups embedded within these micropores, using these embedded nanoscale debris as a physical link. At the mouth, thus achieving a stable distribution of Bifidobacterium animalis in the microporous arrangement layer of excipients in terms of overall and surface transformation, eliminating the mass transfer discontinuity caused by differences in spatial geometric scale. 10% by mass of Lactobacillus acidophilus, 10% of Lactobacillus plantarum and 10% of Lactobacillus rhamnosus freeze-dried powder were added to the reactor. The initial water activity of each Lactobacillus powder was 0.16. The second-order shear speed was set to 25 r / min and the mixture was mixed for 12 min to make the Lactobacillus powder uniformly distributed in the external gaps between the porous microcrystalline cellulose particles.

[0034] Real-time monitoring using an online water activity sensor at the discharge end determined the overall water activity of the finished powder to be 0.12. The partially deficient control group eliminated the sequential premixing step and employed a one-step high-speed mixing method. The out-of-range control group used excipients with an average micropore size of 15 nm and an initial water activity of 0.25. After 90 days of storage at room temperature, the viable retention rate of *Bifidobacterium animalis* in the sample group of this invention was 87.5%. The partially deficient control group had a viable retention rate of 61.2% due to the lack of a water trans-interface mass transfer barrier. The out-of-range control group had a viable retention rate of 61.2% due to the absence of adsorption sites on the excipients. Saturation leads to localized moisture enrichment in the microenvironment of the bacterial community, with a retention rate of 55.8%. Data confirms that this invention, through a time-step process and a micropore anchoring mechanism, constructs a barrier to moisture migration, maintaining the stability of moisture distribution within the multi-strain coexisting solid material system. Testing the storage stability of the finished product under different initial water activity to finished product water activity ratios revealed that, according to the principle of multiphase non-equilibrium thermodynamic water mass transfer equilibrium, the rate of moisture migration between components within the system depends on the difference in hygroscopic energy levels. When the ratio is less than 1:1.1, the porous amorphous excipient has a high initial water activity and micropores... The hydrophilic hydroxyl groups on the surface of the gap tend to be water-saturated, failing to form a binding energy level ladder to capture free water molecules. This leads to water accumulation in the microenvironment surrounding the freeze-dried Bifidobacterium animalis powder, resulting in a viable bacterial retention rate of 54.2% after 90 days of storage at room temperature. When the ratio is higher than 1:1.5, the excipients are in a dry state, and the high water migration and adsorption potential energy causes excessive dehydration damage to the Bifidobacterium animalis powder, triggering an unexpected phase transition in the cell membrane and inactivation, resulting in a viable bacterial retention rate of 58.0%. When the ratio is controlled within the working window of 1:1.1 to 1:1.5, the water mass transfer path... With the pathway blocked, the viable bacterial retention rate remained above 86%. The results of the synergistic survival test of different strain combinations showed that the two-phase combination containing only Bifidobacterium animalis and porous amorphous excipients had a viable bacterial retention rate of 61.2% after 90 days of storage at room temperature; while the complete combination consisting of Bifidobacterium animalis, porous amorphous excipients, and freeze-dried Lactobacillus powder had a viable bacterial retention rate of 87.5%. The space filling of Lactobacillus powder in the interparticle gaps and the micropore anchoring of porous amorphous excipients created a three-dimensional physical isolation barrier, which delayed the inactivation of sensitive strains.

[0035] Example 3: This invention provides a probiotic composition and application for regulating intestinal microbiota. It solves the problem of microbial activity loss caused by fluctuations in environmental humidity during production and filling by introducing multi-dimensional environmental parameters for adaptive closed-loop control. The system is equipped with an online water activity sensing module. This module uses dual-point high-sensitivity water activity sensors distributed at the bottom of the mixing tank and the discharge pipeline to collect real-time microenvironment water activity data of the material at different processing stages. The sampling period is 500ms. The water activity regulation logic set by this invention is as follows: When the sensor detects that the overall water activity of the finished material is within the preset safe range of 0.08 to 0.12, the control system maintains continuous operation of the nitrogen filling and packaging process; when the water activity value exceeds the upper limit of the threshold of 0.15, the controller automatically triggers a secondary airflow circulation drying program, introducing drying gas into the mixing tank at a flow rate of 30m³ / h until the sensor provides feedback. When the water activity drops below 0.12, in the aforementioned closed-loop refined control, the safety range of the sensors at the bottom of the reactor and the discharge pipeline is set to 0.08 to 0.12. This is to proactively pre-compensate for the physical moisture regain in the subsequent packaging flow path. Specifically, during the process of qualified materials being output from the discharge end of the mixing reactor and transported to the nitrogen-filled sealing packaging machine via pipeline, under continuous production conditions with a relative humidity of 25%, the microenvironment water activity of the material will increase in stages from 0.01 to 0.02 due to overall exposure and equilibrium moisture absorption. Therefore, when the water activity of the material output from the mixing reactor is locked in the unit frequency band of 0.08 to 0.12, the water activity inside each bag of finished product after quantitative packaging can precisely converge and stabilize within the design protection window of 0.10 to 0.13, thereby achieving absolute self-consistency and coordination between the mixing and water control state of the post-fermentation treatment section and the final index boundary of the finished product.

[0036] When the water activity value falls below the threshold of 0.05, the controller instructs to slow down the material stirring speed and activate the micro-humidification spray device to spray micron-level water mist into the material, preventing cell brittleness caused by excessively low water activity. This control mechanism ensures that even under extreme production conditions with a relative humidity of 25%, the internal water activity of the finished product is still limited to the design window of 0.08 to 0.15 through real-time response to environmental disturbances. This embodiment is verified by multi-dimensional comparative data: under fluctuating environmental humidity conditions, the probiotic composition using this closed-loop adaptive control has a viable bacterial retention rate of 86.4% after 90 days of storage at room temperature, while the control group without adaptive control has a viable bacterial retention rate of only 52.7% due to moisture fluctuations, confirming the stability of this invention in complex industrial environments.

[0037] Example 4: In the industrial production scenario of probiotic fermentation engineering, it is necessary to solve the technical problem of local microenvironment moisture enrichment and activity inactivation caused by inconsistent moisture absorption rates of multiple strains during the shelf life at room temperature through standardized engineering calibration procedures. Porous silica with a median particle size of 60μm and an average micropore diameter of 3nm is used as the adsorption carrier. Before being added to the mixing vessel, this carrier needs to be dehumidified by a vacuum drying device at 105℃ to calibrate the initial water activity of the porous silica to below 0.02. In this industrial calibration scenario, the core purpose of selecting porous silica as the adsorption carrier is to utilize the physical properties of inorganic silica—non-swelling upon heating and high structural rigidity—to provide a standardized inorganic control carrier for the automated fermentation control system, thereby accurately calibrating the reference zero point and control response of the online water activity detection sensor at the discharge end. Because the average pore size (3nm) and hydrophilic hydroxyl binding energy level of the porous silica surface after drying at 105℃ are in the same multiphase non-equilibrium thermodynamic water transfer orbit as the microcrystalline cellulose defined in the core of this invention, the process control parameters of the linkage vessel temperature regulation unit calibrated on this inorganic carrier can be seamlessly mapped and equivalently applied to the actual industrial continuous production conditions using microcrystalline cellulose as the main and auxiliary materials. The mixing vessel is started, and 15% by mass of freeze-dried Bifidobacterium animalis powder with an initial water activity of 0.12 and 55% by mass of pretreated porous silica are added into the vessel. The stirring speed is set to 40r / min, and the premixing is continued for 15min. The strong affinity of the microporous structure of the porous silica surface for free water molecules is used to achieve the physical encapsulation of Bifidobacterium animalis powder.

[0038] 10% by mass of *Lactobacillus acidophilus*, 10% of *Lactobacillus plantarum*, and 10% of *Lactobacillus rhamnosus* freeze-dried powder were added to the reactor. The initial water activity of each *Lactobacillus* powder was 0.15. The stirring speed was adjusted to 20 r / min for secondary mixing, and the mixture was stirred for 10 min to distribute the *Lactobacillus* powder in the external pores of the excipient particles. The process was controlled by an online water activity detection sensor at the discharge end. If the detected value deviated from the baseline set point of 0.10, the control system would link with the reactor temperature adjustment unit to make micro-adjustments to lock the water activity of the finished material near the baseline of 0.10. After 90 days of storage at room temperature (25°C), the viable retention rate of *Bifidobacterium animalis* in the sample group of this invention was 89.2%. The viable retention rate of the deletion control group dropped sharply to 58.6% due to the lack of physical barriers. The viable retention rate of the control group with excessive addition was 64.3% due to the excessive total pore volume of the excipients and the heat generated during the mixing process causing thermal denaturation.

[0039] Example 5: On a production line employing an automated fermentation control system, to avoid the risk of product pH runaway due to differences in the acid production metabolic characteristics of different strains, an offline compensation parameter calibration procedure based on real-time monitoring of metabolites was constructed. In an offline simulated reactor, the initial inoculum was set to 10^8 CFU per milliliter. By changing the initial concentration gradient of the substrate glucose (10 g / L, 20 g / L, and 30 g / L), the acid production rate curve under this specific fermentation environment was measured. Data mining techniques were used to perform first-order difference calculations on the curves to pinpoint the metabolic acid production inflection point during fermentation, i.e., the instantaneous time point at which the acid production rate transitions from linear growth to a plateau. Based on the basic physicochemical environment corresponding to this inflection point, a quaternary correlation matrix was established, including the relationship between stirring speed, aeration rate, feed rate, and acid production rate. This matrix was used as the basic parameter before system operation. The specific steps of the first-order differential calculation are as follows: The system continuously collects pH data in the reactor at a sampling period of 500ms. The pH difference between two adjacent sampling periods is divided by the time interval to extract the current acid production rate in real time. When the rate of change obtained from three consecutive differential calculations enters the preset zero-growth fluctuation zone, it is determined to be the metabolic acid production inflection point. The quaternary correlation matrix is ​​a piecewise linear interpolation data table constructed inside the controller with stirring speed, aeration rate, and feeding rate as three-dimensional independent variables and acid production rate as a one-dimensional dependent variable response. In the formal fermentation stage, the system uses the real-time collected pH decay rate as the target input, uses the interpolation data table for table lookup and weight matching, and directly outputs the corresponding feeding and aeration compensation commands under the current physical conditions, thereby realizing the transparency and explicitness of the algorithm control path.

[0040] When the production line enters the formal fermentation stage, the system collects the pH decay rate in the reactor in real time as an input parameter, and calculates the optimal alkali replenishment flow rate required to maintain the acid-base balance of the fermentation broth based on the aforementioned preset parameter matrix. In the high-load production test, the initial substrate glucose concentration was increased to 50 g / L. Due to the excess substrate, the acid production rate exceeded the upper limit of the alkali replenishment kinetics. The system triggered the overload protection logic at the 6th hour of fermentation and actively reduced the feeding rate to prevent the inhibition of microbial metabolism. Finally, the alkali replenishment action and the change trend of the acid production metabolic rate showed a high positive correlation, proving that the control logic can effectively deal with the metabolic imbalance caused by substrate concentration fluctuations during the fermentation process.

[0041] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit of this application and the scope of protection of this invention, and all of these forms are within the protection scope of this application.

Claims

1. A probiotic composition for regulating intestinal microbiota, characterized in that, include: Lyophilized Bifidobacterium animalis powder with a mass percentage of 15% to 25% and an initial water activity of 0.12 to 0.15; Porous amorphous excipients with a mass percentage of 45% to 55% and an initial water activity of 0.01 to 0.09, the median particle size of the porous amorphous excipients being 50 μm to 150 μm and the average pore size of the micropores being 2 nm to 10 nm; and lyophilized Lactobacillus acidophilus powder with a mass percentage of 5% to 15% and an initial water activity of 0.15 to 0.

18. Lyophilized powder of *Lactobacillus plantarum* with a mass percentage of 5% to 15% and an initial water activity of 0.15 to 0.18, and lyophilized powder of *Lactobacillus rhamnosus* with a mass percentage of 5% to 15% and an initial water activity of 0.15 to 0.18; The lyophilized Bifidobacterium animalis powder is distributed within the micropores of the porous amorphous excipient; the lyophilized Lactobacillus acidophilus powder, the lyophilized Lactobacillus plantarum powder, and the lyophilized Lactobacillus rhamnosus powder are distributed within the interparticle spaces of the porous amorphous excipient. The porous amorphous excipient has hydrophilic hydroxyl groups on its microporous surface; the single-dose live bacteria addition of the probiotic composition product is 30 billion CFU to 60 billion CFU, and the water activity of the probiotic composition product is 0.10 to 0.

13.

2. The probiotic composition for regulating intestinal microbiota according to claim 1, characterized in that, The probiotic composition comprises, by weight 100%, 5% to 10% by mass of Lactobacillus reuteri lyophilized powder with an initial water activity of 0.15 to 0.18; the Lactobacillus reuteri lyophilized powder is distributed in the interparticle spaces of the porous amorphous excipient.

3. The probiotic composition for regulating intestinal microbiota according to claim 1, characterized in that, The ratio of the initial water activity of the porous amorphous excipient to the water activity of the finished probiotic composition is 1:1.1 to 1:1.

5.

4. The probiotic composition for regulating intestinal microbiota according to claim 1, characterized in that, The total pore volume of the micropores in the porous amorphous excipients is 0.2 cm³ / g to 0.5 cm³ / g.

5. The probiotic composition for regulating intestinal microbiota according to claim 1, characterized in that, In the probiotic composition, the ratio of the live bacteria count of Bifidobacterium animalis freeze-dried powder to the total live bacteria count of Lactobacillus acidophilus freeze-dried powder, Lactobacillus plantarum freeze-dried powder, and Lactobacillus rhamnosus freeze-dried powder is 1:1 to 1:

2.

6. The probiotic composition for regulating intestinal microbiota according to claim 1, characterized in that, The probiotic composition is sealed in a nitrogen-filled aluminum foil bag, and the nitrogen volume percentage of the nitrogen-filled aluminum foil bag is 98% to 99%.

7. The probiotic composition for regulating intestinal microbiota according to claim 1, characterized in that, The solid phase transition temperature of the freeze-dried Bifidobacterium animalis powder is 26℃ to 35℃.

8. The probiotic composition for regulating intestinal microbiota according to claim 1, characterized in that, The probiotic composition is in the form of a solid powder.

9. The probiotic composition for regulating intestinal microbiota according to claim 1, characterized in that, The porous amorphous excipient is microcrystalline cellulose.

10. The application of a probiotic composition for regulating intestinal microbiota, characterized in that, The probiotic composition for regulating gut microbiota as described in claim 1 is used to prepare products that improve irritable bowel syndrome and gastrointestinal health.

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