A bacillus coagulans body management composition for modulating the metabolic environment

Abstract

The application discloses a Bacillus coagulans body weight management composition for adjusting a metabolic environment, relates to the technical field of probiotics and functional food, and comprises Bacillus coagulans, animal bifidobacterium, lactobacillus plantarum and cell-free supernatant metabolic filtrate produced by fermentation of the Bacillus coagulans, wherein the animal bifidobacterium and the lactobacillus plantarum are dispersed in the metabolic filtrate to form a mixture. The application is designed by pre-incubation of strain metabolic filtrate and multi-strain time sequence cooperation, so that the three strains form a progressive functional cooperation system in the intestinal tract, effectively improve the intestinal colonization efficiency and storage stability of the strains, can safely and efficiently adjust the intestinal metabolic environment of a host, realize the core function of body weight management, and can be widely applied to preparation and production of functional food, dietary supplements and body weight management related products.

Description

Technical Field

[0001] This invention relates to the field of probiotics and functional food technology, specifically to a Bacillus coagulans weight management composition for regulating the metabolic environment. Background Technology

[0002] The application of probiotics in weight management has become a research hotspot in functional foods and pharmaceutical technology in recent years. The gut microbiota structure is closely related to host energy metabolism, fat accumulation, and inflammatory states. Regulating the gut microecological environment through exogenous supplementation with specific probiotics has become one of the important technical pathways for improving metabolic syndrome and assisting in weight management. Existing research shows that different strains have their own functional characteristics in regulating metabolism, and the combined application of multiple strains is gradually becoming a major research direction in this field.

[0003] Currently, some existing technologies use compositions containing Bacillus coagulans, Bifidobacterium animalis, and Lactobacillus plantarum, which regulate intestinal flora balance and promote lipid metabolism through the synergistic effect of these three strains. This approach involves direct application of a simple physical mixture of the three probiotics, relying on the natural colonization and function of each strain in the intestine. However, this approach does not consider the differences in germination and colonization time among different strains in the intestine. Strictly anaerobic Bifidobacterium animalis has a low survival rate in the aerobic or microaerobic proximal intestinal environment, affecting its enrichment effect in the distal colon. Other existing technologies use pH-sensitive coating materials to encapsulate probiotics and deliver them to specific sites in the intestine. This approach achieves spatially targeted delivery of probiotics through physical encapsulation, improving the survival rate of strains in the target intestinal segment. However, this approach relies on exogenous carrier materials, and each strain continues to function independently after reaching the target site. It does not address the issue of synergistic effects between strains through metabolites, nor does it utilize the metabolites of one strain to create favorable conditions for the colonization of other strains.

[0004] While existing technologies have proposed the technical approach of multi-strain combined application and targeted delivery, the following core issues remain: how to enable three probiotic strains with different functions to form a functionally synergistic and sequentially progressive organic whole in the gut, so that the strains that colonize first can create a suitable microenvironment for the strains that colonize later through their metabolites, and enable the strains that colonize later to efficiently perform their function of regulating energy absorption in a low-inflammatory environment, thereby achieving a progressive synergy from environment creation and inflammation clearance to energy regulation, has not yet been effectively solved. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Bacillus coagulans weight management composition for regulating the metabolic environment. The composition comprises Bacillus coagulans, Bifidobacterium animalis, Lactobacillus plantarum, and their metabolic filtrates as a probiotic composition. By dispersing Bifidobacterium animalis and Lactobacillus plantarum in the Bacillus coagulans metabolic filtrate to form a mixture, the cross-protection and functional timing coordination are achieved by utilizing the metabolic products of the strains themselves, so that the three strains can colonize the intestine sequentially and work synergistically.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a Bacillus coagulans weight management composition for regulating the metabolic environment, the composition comprising: Bacillus coagulans; Animal Bifidobacterium; Lactobacillus plantarum; And the cell-free supernatant metabolic filtrate produced by fermentation of the aforementioned Bacillus coagulans; The animal bifidobacteria and the plant lactobacillus are dispersed in the cell-free supernatant metabolic filtrate produced by the fermentation of Bacillus coagulans to form a mixture.

[0007] By dispersing Bifidobacterium animalis and Lactobacillus plantarum into a mixture in the cell-free supernatant metabolic filtrate produced by Bacillus coagulans fermentation, Bifidobacterium animalis and Lactobacillus plantarum are protected by the metabolic filtrate during processing and storage. At the same time, the two strains are pre-exposed to metabolic signaling molecules produced by the pioneer bacteria before entering the intestine, thus achieving cross-protection and functional integration between the strains.

[0008] Furthermore, the mixture is obtained by dispersing the bacterial sludge of Bifidobacterium animalis and the bacterial sludge of Lactobacillus plantarum together in the cell-free supernatant metabolic filtrate produced by the fermentation of Bacillus coagulans, followed by incubation and drying.

[0009] By dispersing Bifidobacterium animalis and Lactobacillus plantarum sludge together in metabolic filtrate, incubating them, and then drying them, the active ingredients in the metabolic filtrate can fully act on the two strains. The incubation process induces the strains to produce an environmental adaptation response, thereby improving the tolerance of the two strains to the subsequent intestinal environment.

[0010] Furthermore, the composition is formed by physically mixing bacterial powder containing Bifidobacterium animalis and Lactobacillus plantarum obtained after incubation and drying with spore powder of Bacillus coagulans.

[0011] By physically mixing bacterial powders containing Bifidobacterium animalis and Lactobacillus plantarum with spore powders of Bacillus coagulans, and utilizing the acid-resistant properties of Bacillus coagulans spores and their oxygen-consuming germination at the anterior end of the intestine, an anaerobic environment is created for the colonization of strictly anaerobic Bifidobacterium animalis in the distal colon, thus achieving the temporal colonization of the three strains in different segments of the intestine.

[0012] Furthermore, the cell-free supernatant metabolic filtrate is obtained by centrifuging or filtering Bacillus coagulans after fermentation culture to remove bacterial cells.

[0013] By fermenting and culturing Bacillus coagulans, and then removing the bacterial cells by centrifugation or filtration, a cell-free supernatant metabolic filtrate is obtained. This process retains active ingredients such as short-chain fatty acids, catalase, and amino acids in the metabolic filtrate, while avoiding the influence of live bacteria on subsequent processing. This ensures the stability and reproducibility of the metabolic filtrate as a functional preservative.

[0014] Furthermore, in the composition, Bacillus coagulans germinates at the anterior end of the intestine, consuming free oxygen and providing an anaerobic environment for the colonization of Bifidobacterium animalis and Lactobacillus plantarum. Lactobacillus plantarum colonizes in the middle of the small intestine, while Bifidobacterium animalis colonizes in the distal colon.

[0015] By germinating in the proximal intestinal tract, Bacillus coagulans consumes free oxygen, providing an anaerobic environment for the colonization of Bifidobacterium animalis and Lactobacillus plantarum. Lactobacillus plantarum colonizes in the middle of the small intestine, while Bifidobacterium animalis colonizes in the distal colon. This allows the three strains to occupy dominant ecological niches in different segments of the intestine, transforming the competitive relationship between strains into a spatial subordination relationship.

[0016] Furthermore, the cell-free supernatant metabolic filtrate produced by the fermentation of Bacillus coagulans contains short-chain fatty acids, catalase, and amino acids.

[0017] The short-chain fatty acids, catalase, and amino acids contained in the metabolic filtrate provide antioxidant protection and nutritional support to Bifidobacterium animalis and Lactobacillus plantarum dispersed in the filtrate. At the same time, the short-chain fatty acids act as signaling molecules to induce intestinal cells to prepare in advance, creating conditions for subsequent colonization of the strains.

[0018] Furthermore, the ratio of viable Bacillus coagulans, Bifidobacterium animalis, and Lactobacillus plantarum in the composition is 1:1:1.

[0019] By setting the ratio of viable Bacillus coagulans, Bifidobacterium animalis, and Lactobacillus plantarum to 1:1:1, the three strains are kept in a balanced quantitative relationship in the composition, ensuring that the pioneer bacteria germinate in sufficient quantity to create an anaerobic environment, the backbone bacteria colonize in sufficient quantity to exert anti-inflammatory effects, and the core bacteria accumulate in sufficient quantity to regulate energy absorption.

[0020] Furthermore, the *Lactobacillus plantarum* strain is a strain that secretes anti-inflammatory factors.

[0021] By using Lactobacillus plantarum as a strain that secretes anti-inflammatory factors, it can efficiently colonize the small intestinal mucosa in the micro-anaerobic environment created by Bacillus coagulans, secrete anti-inflammatory factors to repair the intestinal barrier, reduce the level of metabolic endotoxins, and clear inflammatory barriers for Bifidobacterium animalis to perform its energy regulation function.

[0022] Furthermore, the animal Bifidobacterium is a strain that regulates energy absorption.

[0023] Animal Bifidobacterium, as a strain that regulates energy absorption, accumulates in large quantities in the colonic environment after inflammation levels have decreased. It utilizes food residue fermentation to produce conjugated linoleic acid and short-chain fatty acids, which promote thermogenesis in adipose tissue through the intestinal fat axis, thus efficiently regulating energy absorption and consumption.

[0024] Furthermore, the Bacillus coagulans is a strain with a spore structure and is resistant to gastric acid.

[0025] By using Bacillus coagulans, a strain with a spore structure and tolerance to gastric acid, it can resist gastric acid and germinate rapidly after entering the small intestine in spore form. It first consumes residual oxygen in the intestine to reduce the redox potential. At the same time, the germination process releases active substances similar to metabolic filtrate, forming a microenvironmental acclimatization field in situ in the intestine.

[0026] Compared with existing technologies, this Bacillus coagulans weight management composition for regulating the metabolic environment has the following beneficial effects: I. This invention involves incubating Bifidobacterium animalis and Lactobacillus plantarum in the cell-free supernatant metabolic filtrate produced by Bacillus coagulans fermentation, and then physically mixing them with Bacillus coagulans spore powder to construct a composition. This enables three probiotic strains with different functions to form a synergistic and progressively developing organic whole in the host's intestine. The first colonized strain creates a suitable growth microenvironment for the later colonized strain through its metabolites, achieving a progressive synergistic effect from creating an anaerobic environment in the intestine, clearing inflammation, to regulating energy metabolism. This effectively solves the technical problem that existing multi-strain compositions cannot achieve organic synergy between strains, and significantly improves the composition's regulatory effect on the intestinal metabolic environment and its weight management efficacy.

[0027] II. This invention utilizes the cell-free supernatant metabolic filtrate produced by Bacillus coagulans fermentation as a protective matrix to pre-incubate Bifidobacterium animalis and Lactobacillus plantarum. This allows the two anaerobic strains to receive sufficient antioxidant protection and nutritional support during processing and storage, inducing environmental adaptation responses in the strains and significantly improving their storage stability and intestinal tolerance. It achieves efficient intestinal delivery of the strains without relying on exogenous encapsulation carrier materials, simplifying the composition preparation process, reducing production costs, and ensuring the stability and reproducibility of the composition's effects.

[0028] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0030] Figure 1 This is a flowchart illustrating the preparation process of the composition of the present invention. Detailed Implementation

[0031] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0032] The Bacillus coagulans weight management composition for regulating the metabolic environment provided in this specific embodiment involves publicly available standard strains that do not require biopreservation. Specifically, the Bacillus coagulans, Bifidobacterium animalis, and Lactobacillus plantarum were purchased from the American Center for Type Culture Collection (ACC), respectively, and the composition was purchased from the German Microbiological Culture Collection (DMC).

[0033] The culture medium formulation used in this specific embodiment is as follows: The activation medium for Bacillus coagulans was MRS broth medium with the following specific formula: 10.0 g peptone, 8.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1.0 g Tween 80, and distilled water to a final volume of 1000 mL. The pH was 6.2 ± 0.2, and the medium was autoclaved at 121°C for 15 min. The solid medium was prepared by adding 15.0 g agar to the above formula.

[0034] The activated culture medium for Bifidobacterium animalis was a modified MRS broth medium, with 0.5 g of cysteine ​​hydrochloride added to the above MRS broth medium. The pH value was 6.0±0.2. The medium was autoclaved at 121℃ for 15 min. The culture environment was anaerobic, with anaerobic conditions of 85% nitrogen, 10% carbon dioxide and 5% hydrogen.

[0035] The activation medium for Lactobacillus plantarum was prepared using MRS broth medium, which has the same formulation as the activation medium for Bacillus coagulans, with a pH of 6.0±0.2 and autoclaved at 121℃ for 15 min.

[0036] The instruments used in this specific embodiment are all conventional commercial instruments in the field, including anaerobic incubators, high-speed refrigerated centrifuges, vacuum freeze dryers, constant temperature shakers, high-pressure steam sterilizers, pH meters, viable cell counting plates, high-performance liquid chromatographs, enzyme-linked immunosorbent assay (ELISA) readers, portable microelectrode detectors, three-dimensional motion mixers, and 50L mechanically stirred fermenters.

[0037] Example 1 This embodiment provides a complete preparation method for a Bacillus coagulans weight management composition that regulates the metabolic environment, clarifying the preparation parameters and process steps of each component of the composition, and verifying the basic performance of the composition. In this embodiment, the total viable count of the composition is not less than 1.0 × 10^11 CFU / g, and the ratio of viable Bacillus coagulans, Bifidobacterium animalis, and Lactobacillus plantarum is 1:1:1.

[0038] like Figure 1 As shown, the first step is the fermentation culture of Bacillus coagulans and the preparation of cell-free supernatant metabolic filtrate.

[0039] Specifically, glycerol preservation tubes of *Bacillus coagulans* were streaked onto *Bacillus coagulans* solid culture plates in a clean bench and incubated at 37°C for 18 hours to obtain single colonies. Plump, well-defined single colonies were selected and inoculated into 50 mL of *Bacillus coagulans* activation medium and incubated at 37°C and 180 rpm for 12 hours to obtain primary seed culture. The primary seed culture was inoculated at 2% of the total culture volume into 2 L of *Bacillus coagulans* activation medium and incubated at 37°C and 180 rpm for 24 hours to obtain secondary seed culture. The secondary seed culture was then inoculated at 5% of the total culture volume into a 50 L fermenter containing *Bacillus coagulans* fermentation medium. The fermentation medium had the same formulation as the activation medium. Fermentation conditions were 37°C, tank pressure 0.05 MPa, aeration rate 1.0 vvm, stirring speed 200 rpm, and a fermentation period of 48 hours. After fermentation, the spore count was measured, and fermentation was terminated when the spore count reached 95% or higher.

[0040] The fermented Bacillus coagulans broth was transferred to a high-speed refrigerated centrifuge and centrifuged at 8000 rpm for 20 min at 4°C to remove the bacterial precipitate. The supernatant was collected. The supernatant was then filtered through a 0.22 μm microporous membrane for two stages to completely remove residual bacterial cells and spores, yielding a cell-free metabolic filtrate. The prepared cell-free metabolic filtrate was sealed and stored at 4°C for no more than 72 h. Testing showed that the cell-free metabolic filtrate prepared in this embodiment contained a total short-chain fatty acid content of not less than 1.2 g / L, a catalase activity of not less than 80 U / mL, and a total free amino acid content of not less than 0.5 g / L.

[0041] The second step is the preparation of a bacterial sludge mixture of Bifidobacterium animalis and Lactobacillus plantarum.

[0042] Specifically, glycerol preservation tubes of *Bifidobacterium animalis* were streaked onto *Bifidobacterium animalis* solid culture medium plates in a clean bench and incubated at 37°C for 48 hours to obtain single colonies. A single colony was picked and inoculated into 50 mL of *Bifidobacterium animalis* activation medium and incubated at 37°C for 24 hours to obtain primary seed culture. The primary seed culture was inoculated at 3% of the total culture into 2 L of *Bifidobacterium animalis* activation medium and incubated at 37°C for 36 hours to obtain secondary seed culture. The secondary seed culture was then inoculated at 5% of the total culture into a 50 L fermenter containing *Bifidobacterium animalis* fermentation medium. Fermentation conditions were: 37°C, 0.05 MPa, anaerobic environment, stirring speed 50 rpm, and fermentation period 48 hours. After fermentation, the fermentation broth was centrifuged at 4°C and 6000 rpm for 15 minutes, the supernatant was discarded, the bacterial precipitate was collected, and the bacterial precipitate was washed twice with sterile physiological saline to obtain Bifidobacterium animalis sludge.

[0043] Glycerol preservation tubes of *Lactobacillus plantarum* were streaked onto *Lactobacillus plantarum* solid culture plates in a clean bench and incubated at 37°C for 24 hours to obtain single colonies. A single colony was picked and inoculated into 50 mL of *Lactobacillus plantarum* activation medium and incubated at 37°C and 180 rpm for 12 hours to obtain primary seed culture. The primary seed culture was inoculated at 2% of the culture into 2 L of *Lactobacillus plantarum* activation medium and incubated at 37°C and 180 rpm for 18 hours to obtain secondary seed culture. The secondary seed culture was inoculated at 5% of the culture into *Lactobacillus plantarum* fermentation medium in a 50 L fermenter. Fermentation conditions were 37°C, tank pressure 0.05 MPa, aeration rate 0.5 vvm, stirring speed 150 rpm, and fermentation period 36 hours. After fermentation, the fermentation broth was centrifuged at 4°C and 6000 rpm for 15 minutes. The supernatant was discarded, and the bacterial precipitate was collected. The precipitate was washed twice with sterile physiological saline to obtain *Lactobacillus plantarum* sludge.

[0044] The third step is the incubation of bacterial strains and the preparation of compound bacterial powder.

[0045] Specifically, the cell-free supernatant metabolic filtrate prepared in the first step was placed in a sterile stirred tank, with the stirring speed controlled at 50 rpm and the temperature controlled at 25℃. Bifidobacterium animalis and Lactobacillus plantarum sludge were added to the metabolic filtrate at a mass-to-volume ratio of 1:10, i.e., 100g of Bifidobacterium animalis sludge and 100g of Lactobacillus plantarum sludge were added to every 1000mL of metabolic filtrate. After all the sludge was added, the stirred tank was sealed, and nitrogen gas was introduced to replace the air inside the tank to maintain an anaerobic environment. The stirring speed was increased to 100 rpm, the temperature was adjusted to 37℃, and the mixture was incubated at this constant temperature for 4 hours.

[0046] After incubation, the bacterial suspension in the tank was pre-frozen at -40℃ for 12 hours. Following pre-freezing, the suspension was transferred to a vacuum freeze dryer at -55℃ and a vacuum of 10 Pa for 36 hours to obtain a dried compound bacterial powder. Viable cell count testing showed that the ratio of viable Bifidobacterium animalis to Lactobacillus plantarum in the compound bacterial powder was 1:1, and the total viable cell count was not less than 2.0 × 10^11 CFU / g.

[0047] The fourth step is to prepare Bacillus coagulans spore powder and mix it with the final composition.

[0048] Specifically, the fermentation broth of *Bacillus coagulans* after the first fermentation step was taken and heat-treated in a water bath at 80℃ for 10 minutes to kill the vegetative cells and retain the spores. The heat-treated fermentation broth was centrifuged at 4℃ and 8000 rpm for 20 minutes, the supernatant was discarded, and the spore precipitate was collected. The spore precipitate was washed twice with sterile physiological saline to obtain spore mud. The spore mud was then subjected to vacuum freeze-drying: pre-freezing temperature -40℃, pre-freezing time 12 hours, cold trap temperature -55℃, vacuum degree 10 Pa, drying time 24 hours, to obtain *Bacillus coagulans* spore powder. Viable cell count testing showed that the viable number of *Bacillus coagulans* in the spore powder was not less than 2.0 × 10^11 CFU / g, and the spore rate was not less than 98%.

[0049] The compound bacterial powder prepared in step three was physically mixed with Bacillus coagulans spore powder at a mass ratio of 1:1 in a three-dimensional motion mixer for 30 minutes at a speed of 15 rpm. The mixing environment was room temperature, dry, and sterile to obtain the final Bacillus coagulans weight management composition for regulating the metabolic environment. Viable cell count testing showed that the ratio of viable Bacillus coagulans, Bifidobacterium animalis, and Lactobacillus plantarum in the final composition was 1:1:1, and the total viable cell count was not less than 1.0 × 10^11 CFU / g.

[0050] The composition prepared in this embodiment was subjected to a storage stability test. The composition was sealed in an aluminum foil bag and stored at 25°C and 60% relative humidity for 6 months, with the viable count measured monthly. After 6 months of storage, the total viable count retention rate of the three strains in the composition was not less than 85%, and the viable count retention rate of Bifidobacterium animalis was not less than 82%.

[0051] This embodiment clarifies the specific process parameters and quality control standards for each step. By incubating with metabolic filtrate, the storage stability of anaerobic strains is significantly improved. By controlling the ratio of the three strains and coordinating the process, the synergistic function of the strains is achieved, providing a complete and replicable technical path for the large-scale production and application of the composition.

[0052] Example 2 This embodiment, based on Example 1, verifies the colonization timing and spatial distribution characteristics of the three bacterial strains in the host gut, clarifies the regulatory effect of the composition on the intestinal metabolic environment, and verifies the technical solution regarding the colonization site and environment creation function of the strains. This embodiment uses SPF-grade C57BL / 6J mice as experimental animals to fully record the colonization dynamics and metabolic environment changes of the strains in different segments of the gut.

[0053] The composition was prepared entirely using the preparation process and parameters of Example 1. The ratio of viable Bacillus coagulans, Bifidobacterium animalis, and Lactobacillus plantarum in the prepared composition was 1:1:1, and the total viable count was 1.2 × 10^11 CFU / g.

[0054] The experimental animals were grouped and treated as follows: Specifically, 60 SPF-grade male C57BL / 6J mice, 6 weeks old and weighing 18-22g, were selected and housed in an SPF-grade animal room at a temperature of 22±2℃ and a relative humidity of 50±5%, with a 12-hour light-dark cycle. They had free access to food and water and were acclimatized for 1 week before the experiment began.

[0055] Mice were randomly divided into 6 groups of 10 mice each: a blank control group, and groups 2h, 6h, 12h, 24h, and 48h after gavage administration of the composition. The blank control group was administered sterile saline by gavage, while the other groups were administered the suspension of the composition prepared in this embodiment by gavage at a dose of 0.2 mL per mouse per day. The concentration of the composition suspension was 1.0 × 10^9 CFU / mL. Gavage was performed for 3 consecutive days. After the last gavage, the mice were sacrificed at the corresponding time points, and the contents and mucosal tissue of different intestinal segments were collected, including the duodenum, jejunum, ileum, proximal colon, and distal colon.

[0056] For the detection of bacterial colonization and intestinal environmental parameters, specifically, the collected intestinal contents and mucosal tissues were serially diluted with sterile physiological saline, and viable bacteria were counted using selective media corresponding to the corresponding strains. Bacillus coagulans was counted using MRS solid medium supplemented with 50 μg / mL nalidixic acid, after heat treatment in an 80℃ water bath for 10 min, and then plated and incubated at 37℃ for 24 h. Lactobacillus plantarum was counted using MRS solid medium supplemented with 50 μg / mL vancomycin and anaerobic incubated at 37℃ for 48 h. Bifidobacterium animalis was counted using modified MRS solid medium supplemented with 100 μg / mL mupirocin and anaerobic incubated at 37℃ for 72 h.

[0057] Simultaneously, the redox potential, free oxygen content, short-chain fatty acid content, and inflammatory factor levels were measured in different segments of the intestine. Redox potential and free oxygen content were detected in situ using a portable microelectrode detector, short-chain fatty acid content was detected using high-performance liquid chromatography (HPLC), and the levels of inflammatory factors IL-6 and TNF-α were detected using enzyme-linked immunosorbent assay (ELISA).

[0058] Specifically, no colonization of the three target bacterial strains was detected in the intestines of mice in the blank control group. Two hours after gavage, *Bacillus coagulans* was detected only in the duodenum and jejunum; no effective colonization of the other strains was detected. Six hours after gavage, the abundance of *Bacillus coagulans* in the entire small intestine reached 1.2 × 10^7 CFU / g of contents, and the free oxygen content in the corresponding region decreased from 5.2 mg / L before gavage to 0.8 mg / L, while the redox potential decreased from +120 mV to -180 mV, successfully creating an anaerobic microenvironment. Twelve hours after gavage, the abundance of *Lactobacillus plantarum* in the jejunum and ileum reached 8.5 × 10^6 CFU / g of contents, and the levels of IL-6 and TNF-α in the corresponding region decreased by 42% and 38%, respectively, compared to the blank control group, achieving an improvement in the intestinal inflammatory environment. 24 hours after gavage, the abundance of Bifidobacterium animalis in the distal colon reached 9.2 × 10^6 CFU / g contents, and the abundance remained at 7.8 × 10^6 CFU / g contents at 48 hours. The total amount of short-chain fatty acids in the corresponding segment was 65% higher than that in the blank control group.

[0059] It is understood that the experimental results of this embodiment fully verify the technical solution regarding the colonization site and functional sequence of the strains. Bacillus coagulans, as a pioneer strain, germinates first in the anterior end of the intestine, consuming free oxygen and providing an anaerobic environment for the colonization of subsequent strains. Lactobacillus plantarum, as a backbone strain, colonizes in the middle of the small intestine and secretes anti-inflammatory factors to improve the intestinal inflammatory state. Bifidobacterium animalis, as a core strain, colonizes in the distal colon and regulates energy absorption and metabolism. The three strains form a synergistic system with temporal progression and spatial succession.

[0060] This embodiment, through animal experiments, fully verified the colonization timing and spatial distribution characteristics of the three strains in the composition of the present invention in the intestine, and clarified the stepwise regulatory effect of the composition on the intestinal metabolic environment. The experimental results of this embodiment demonstrate that the composition of the present invention can form a functionally synergistic and temporally progressive organic whole in the intestine, solving the technical problems of low strain colonization efficiency and weak synergistic effect in the prior art.

[0061] Example 3 This embodiment, based on Example 1, verifies the efficacy of the composition in weight management and metabolic environment regulation in high-fat diet-induced obese mice, clarifying the actual effects of the composition in weight control, body fat reduction, and improvement of metabolic indicators, and providing complete experimental data support for the application of the composition. This embodiment uses a high-fat diet-induced obese mouse model, strictly controlling experimental variables to ensure the objectivity and reproducibility of the experimental results.

[0062] The composition was prepared entirely using the preparation process and parameters of Example 1. The ratio of viable Bacillus coagulans, Bifidobacterium animalis, and Lactobacillus plantarum in the prepared composition was 1:1:1, and the total viable count was 1.2 × 10^11 CFU / g.

[0063] The experimental animals were grouped and treated as follows: Specifically, 80 SPF-grade male C57BL / 6J mice, 4 weeks old and weighing 12-14g, were selected and housed in an SPF-grade animal room at a temperature of 22±2℃ and a relative humidity of 50±5%, with a 12-hour light-dark cycle and free access to food and water. After one week of acclimatization, the mice were divided into a basal diet group (n=10) and a high-fat diet group (n=70). The basal diet group was given a normal maintenance diet, while the high-fat diet group was given a 60% high-fat diet. This feeding was continued for 8 weeks to establish an obese mouse model.

[0064] Eight weeks later, the high-fat diet group showed an average weight increase of over 35% compared to the basal diet group, indicating successful model establishment. The successfully established obese mice were randomly divided into five groups of ten mice each: a model control group, a low-dose group, a medium-dose group, a high-dose group, and a positive control group. The basal diet group and the model control group were administered sterile saline via gavage. The low-dose, medium-dose, and high-dose groups were administered the suspension of the composition prepared in this embodiment via gavage at doses of 1.0 × 10^8 CFU / mouse / day, 1.0 × 10^9 CFU / mouse / day, and 1.0 × 10^10 CFU / mouse / day, respectively. The positive control group was administered orlistat via gavage at a dose of 60 mg / kg body weight / day. During the gavage period, all mice continued to be fed the corresponding diet and had free access to food and water for eight weeks.

[0065] During the experiment, mouse weight and food intake were recorded weekly. After the experiment, mice were fasted for 12 hours but allowed free access to water. Blood was collected from the eye, serum was separated, and serum lipid parameters, including total cholesterol, triglycerides, LDL cholesterol, HDL cholesterol, fasting blood glucose, and fasting insulin levels, were measured. After euthanasia, the epididymal fat pad and perirenal fat pad were dissected, and wet weight was measured to calculate body fat percentage. Colonic contents were collected to analyze intestinal flora structure and short-chain fatty acid content.

[0066] Specifically, during the experiment, there was no significant difference in the average daily food intake among the groups of mice, thus excluding the influence of differences in food intake on the experimental results. After the experiment, the body weight of the mice in the model control group was significantly higher than that of the basal diet group, the body fat percentage was significantly increased, and the serum lipid indicators and blood glucose and insulin levels were significantly abnormal, proving that the obesity model was stable.

[0067] Compared with the model control group, mice in the medium-dose and high-dose groups had reduced body weight by 12.3% and 15.7%, respectively, and reduced body fat percentage by 18.5% and 22.4%, respectively. The wet weight of the epididymal fat pad and perirenal fat pad were significantly reduced. Regarding serum parameters, mice in the medium-dose and high-dose groups showed reductions in serum total cholesterol by 21.2% and 25.6%, triglycerides by 18.7% and 22.3%, low-density lipoprotein cholesterol by 24.5% and 28.9%, respectively, and increases in high-density lipoprotein cholesterol by 15.3% and 18.2%, respectively. Fasting blood glucose and insulin resistance index were also significantly improved.

[0068] Regarding the intestinal metabolic environment, compared with the model control group, the total amount of short-chain fatty acids in the colon contents of mice in the medium-dose group and the high-dose group increased by 58% and 72%, respectively, with significant increases in the contents of acetic acid, propionic acid, and butyric acid. Intestinal flora structure analysis showed that the ratio of Firmicutes to Bacteroidetes in the intestines of mice in the combined gavage group was significantly reduced, the abundance of beneficial bacteria such as Bifidobacterium and Lactobacillus was significantly increased, and the abundance of harmful bacteria such as Desulfovibrio was significantly reduced, indicating a significant improvement in the intestinal flora structure.

[0069] It is understood that the experimental results of this embodiment demonstrate that the composition prepared in this invention can effectively improve the weight and body fat level of obese mice induced by a high-fat diet, regulate blood lipid and blood glucose metabolism, improve the intestinal flora structure and metabolic environment, and has clear effects on weight management and metabolic regulation.

[0070] This embodiment objectively verifies the weight management and metabolic regulation efficacy of the composition of the present invention through standardized animal experiments, clarifying the correspondence between the dosage and effect of the composition. The experimental results of this embodiment provide reliable experimental support for the practical application of the composition, and at the same time prove that the composition of the present invention can achieve safe and effective weight management by regulating the intestinal metabolic environment, solving the technical problems of weak synergistic effect and unstable efficacy of multi-strain compositions in the prior art.

[0071] Comparative Example This comparative example employs a conventional method for preparing multi-strain probiotic compositions, involving a simple physical mixing of the three target strains without incubation with the cell-free supernatant metabolic filtrate of Bacillus coagulans. This method is used to compare and verify the advancement and beneficial effects of the present invention. The viable cell ratio and total viable cell count of the three strains in this comparative example are consistent with those in Example 1, ensuring the uniqueness of the experimental variables.

[0072] Specifically, the strains and culture medium formulations used in this comparative example are completely consistent with those in Example 1. Bacillus coagulans spore powder, Bifidobacterium animalis spore powder, and Lactobacillus plantarum spore powder were prepared separately. The preparation method of the Bacillus coagulans spore powder is completely consistent with that in Example 1, with a viable count of not less than 2.0 × 10^11 CFU / g and a spore rate of not less than 98%.

[0073] The preparation of Bifidobacterium animalis powder involved centrifuging the fermentation broth of Bifidobacterium animalis after fermentation to collect the bacterial sludge, washing it twice with sterile physiological saline, and then directly performing vacuum freeze drying. The pre-freezing temperature was -40℃, the pre-freezing time was 12h, the cold trap temperature was -55℃, the vacuum degree was 10Pa, and the drying time was 36h to obtain Bifidobacterium animalis single-strain powder with a viable count of not less than 1.0×10^11 CFU / g.

[0074] To prepare Lactobacillus plantarum powder, the fermented Lactobacillus plantarum broth was centrifuged to collect the bacterial sludge, washed twice with sterile physiological saline, and then directly subjected to vacuum freeze drying. The pre-freezing temperature was -40℃, the pre-freezing time was 12h, the cold trap temperature was -55℃, the vacuum degree was 10Pa, and the drying time was 36h to obtain Lactobacillus plantarum single-strain powder with a viable count of not less than 1.0×10^11 CFU / g.

[0075] The *Bacillus coagulans* spore powder, *Bifidobacterium animalis* single-strain powder, and *Lactobacillus plantarum* single-strain powder prepared above were physically mixed in a mass ratio of 2:1:1. The mixture was then mixed for 30 minutes in a three-dimensional motion mixer at a speed of 15 rpm in a room temperature, dry, and sterile environment to obtain a comparative probiotic composition. Viable cell count testing showed that the final composition had a viable cell ratio of *Bacillus coagulans*, *Bifidobacterium animalis*, and *Lactobacillus plantarum* of 1:1:1, with a total viable cell count of not less than 1.0 × 10^11 CFU / g, consistent with the composition of Example 1.

[0076] Specifically, the composition prepared in this comparative example was compared with the composition of Example 1 in parallel tests, including storage stability test, mouse intestinal colonization efficiency test, and weight management efficacy test. The test methods were completely consistent with those of Examples 1, 2, and 3.

[0077] Storage stability test results showed that after the composition of this comparative example was stored at 25°C and 60% relative humidity for 6 months, the total viable count retention rate was only 52%, and the viable count retention rate of Bifidobacterium animalis was only 38%, which was significantly lower than that of the composition of Example 1.

[0078] The colonization efficiency test results in mice showed that after gavage administration of the composition in this comparative example, the highest abundance of Bifidobacterium animalis in the distal colon was only 2.1 × 10^6 CFU / g content, which was only 22.8% of the abundance corresponding to the composition in Example 2, and the abundance dropped below the detection limit after 48 hours, failing to achieve stable colonization. The highest abundance of Lactobacillus plantarum in the mid-small intestine was only 3.2 × 10^6 CFU / g content, which was only 37.6% of the abundance corresponding to the composition in Example 2.

[0079] The results of the weight management efficacy test showed that after 8 weeks of gavage administration of the composition in this comparative example at a medium dose, the weight of obese mice decreased by only 4.2% and the body fat percentage decreased by only 6.7%. The improvement in serum lipid indicators and intestinal metabolic environment was significantly lower than that of the composition in Example 3.

[0080] This comparative example uses a conventional physical mixing method of multiple strains to prepare a probiotic composition. Compared with the technical solution of this invention, the storage stability of the composition is significantly reduced, the colonization efficiency of the strains in the intestine is greatly decreased, and the effects of weight management and metabolic regulation are significantly weakened. The experimental results of this comparative example conversely demonstrate that the technical solution of this invention, which disperses Bifidobacterium animalis and Lactobacillus plantarum in Bacillus coagulans metabolic filtrate for incubation, and then mixes it with Bacillus coagulans spore powder, can significantly improve the storage stability and intestinal colonization efficiency of the strains, achieve synergistic effects between strains, solve the core technical problems existing in the prior art, and has outstanding substantive features and significant progress.

[0081] To clearly demonstrate the differences in core technical solutions and performance indicators between the embodiments of the present invention and the comparative examples, the core parameters and test results of the three embodiments and one comparative example are summarized in the following comparison table.

[0082]

[0083] The above comparative results clearly demonstrate that the composition prepared in the embodiments of the present invention is significantly superior to the comparative composition prepared by conventional methods in the prior art in terms of strain storage stability, intestinal colonization efficiency, and weight management efficacy. The present invention, through a technical solution of metabolic filtrate incubation and sequential synergistic effects of strains, effectively solves the core technical problems of low survival rate, poor colonization efficiency, and weak synergistic effect of anaerobic strains in the prior art, achieving a comprehensive improvement in the performance of the composition and providing a stable, effective, and scalable probiotic composition technical solution for the field of weight management.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A Bacillus coagulans weight management composition for regulating the metabolic environment, characterized in that, The composition comprises: Bacillus coagulans; Animal Bifidobacterium; Lactobacillus plantarum; And the cell-free supernatant metabolic filtrate produced by fermentation of the aforementioned Bacillus coagulans; The animal bifidobacteria and the plant lactobacillus are dispersed in the cell-free supernatant metabolic filtrate produced by the fermentation of Bacillus coagulans to form a mixture.

2. The Bacillus coagulans weight management composition for regulating the metabolic environment according to claim 1, characterized in that, The mixture is obtained by dispersing the bacterial sludge of Bifidobacterium animalis and the bacterial sludge of Lactobacillus plantarum together in the cell-free supernatant metabolic filtrate produced by the fermentation of Bacillus coagulans, followed by incubation and drying.

3. The Bacillus coagulans weight management composition for regulating the metabolic environment according to claim 1, characterized in that, The composition is formed by physically mixing bacterial powder containing Bifidobacterium animalis and Lactobacillus plantarum obtained after incubation and drying with spore powder of Bacillus coagulans.

4. The Bacillus coagulans weight management composition for regulating the metabolic environment according to claim 1, characterized in that, The cell-free supernatant metabolic filtrate is obtained by centrifuging or filtering Bacillus coagulans after fermentation culture to remove bacterial cells.

5. The Bacillus coagulans weight management composition for regulating the metabolic environment according to claim 1, characterized in that, In the composition, Bacillus coagulans germinates at the anterior end of the intestine, consuming free oxygen and providing an anaerobic environment for the colonization of Bifidobacterium animalis and Lactobacillus plantarum. Lactobacillus plantarum colonizes in the middle of the small intestine, while Bifidobacterium animalis colonizes in the distal colon.

6. The Bacillus coagulans weight management composition for regulating the metabolic environment according to claim 1, characterized in that, The cell-free supernatant metabolic filtrate produced by the fermentation of Bacillus coagulans contains short-chain fatty acids, catalase, and amino acids.

7. The Bacillus coagulans weight management composition for regulating the metabolic environment according to claim 1, characterized in that, The composition contains Bacillus coagulans, Bifidobacterium animalis, and Lactobacillus plantarum in a live bacteria ratio of 1:1:

1.

8. The Bacillus coagulans weight management composition for regulating the metabolic environment according to claim 1, characterized in that, The *Lactobacillus plantarum* strain is a strain that secretes anti-inflammatory factors.

9. The Bacillus coagulans weight management composition for regulating the metabolic environment according to claim 1, characterized in that, The animal bifidobacteria mentioned are strains that regulate energy absorption.

10. The Bacillus coagulans weight management composition for regulating the metabolic environment according to claim 1, characterized in that, The Bacillus coagulans is a strain with a spore structure and is resistant to gastric acid.

Images