A bifidobacterium breve-containing medicinal and edible composite composition and application thereof

Abstract

The present application relates to the field of biotechnology, traditional Chinese medicine and nutraceuticals, and specifically relates to a kind of homologous composition of medicine and food containing Bifidobacterium breve and application thereof in preparation of product for preventing or treating anxiety and depression, improving sleep disorders and assisting weight management.The composite composition is Bifidobacterium breve and homologous composition of medicine and food, wherein the proportion of Bifidobacterium breve and homologous composition of medicine and food is 1 x 10 10 CFU Bifidobacterium breve is mixed with 60 g of homologous composition of medicine and food of crude medicinal material dose.The composition of the present application has significant synergistic effect, can effectively prevent or relieve anxiety, depression symptoms, improve sleep quality, and can assist in reducing body weight and body fat.The mechanism of action involves double targets of "gut-brain-metabolic axis", which complement each other to promote neuro-immune-endocrine and metabolic balance.The composition of the present application can be used for preparing medicine, functional food or health product, and provides a safe and effective new choice for the intervention of multiple health problems (emotion, sleep, metabolic disorder).

Description

Technical Field

[0001] This invention relates to the fields of biotechnology, traditional Chinese medicine, and nutritional health products, specifically to a food-medicine homologous compound containing Bifidobacterium breve and its application in the preparation of products for the prevention or treatment of anxiety and depression, improvement of sleep disorders, and assistance in weight management. Background Technology

[0002] With the accelerated pace of modern life and increased work pressure, anxiety, depression, and sleep disorders have become major public health problems affecting global public health. Epidemiological data show that the incidence of these mental and emotional disorders is increasing year by year. At the same time, both clinical observations and basic research indicate that these problems are often accompanied by metabolic disorders, such as stress-induced weight gain, central obesity, and insulin resistance, forming a vicious cycle of mental-metabolic abnormalities.

[0003] Currently, first-line treatments for anxiety, depression, and sleep disorders primarily rely on chemical drugs, such as selective serotonin reuptake inhibitors (SSRIs) or benzodiazepine sedative-hypnotics. While these drugs can control symptoms in the acute phase, their limitations are becoming increasingly apparent: they generally have slow onset of action, significant individual variability in treatment response rates, require long-term use, and are prone to causing drug dependence. Furthermore, side effects such as drowsiness, dizziness, gastrointestinal discomfort, sexual dysfunction, and potential negative metabolic effects severely impact patient medication adherence and quality of life, making it difficult to meet the needs for long-term, safe health interventions. Therefore, developing novel intervention strategies that are safe, effective, multi-target synergistic, and capable of simultaneously improving mental and metabolic problems, based on new targets and theoretical perspectives, has significant social and clinical value.

[0004] In recent years, with the deepening research on the "gut-brain axis" theory, the complex bidirectional communication between the gut microbiota and the central nervous system has been gradually revealed. The gut microbiota can regulate neurotransmitter synthesis, neuroinflammatory responses, and hypothalamic-pituitary-adrenal (HPA) axis function through immune, neuroendocrine, and vagus nerve pathways, thereby profoundly influencing the host's mood, cognition, and behavior. Based on this theory, "mental probiotics"—that is, a class of live microorganisms that, when ingested in sufficient quantities, can produce health benefits for patients with mental illness—have become a new research hotspot in the field of mental health. *Bifidobacterium breve* (… Bifidobacterium breveAs important symbiotic bacteria in the human gut, probiotics have been proven by numerous studies to have the potential to improve anxiety and depression-like behaviors and regulate stress responses. However, existing research has largely focused on validating the effects of single strains, with significant strain specificity and substantial differences in intervention responses among individuals. More importantly, the intervention pathways of single probiotic strains are relatively limited, mainly exerting their effects through regulating the gut microbiota and related neuroimmune pathways. For systemic problems caused by complex TCM pathogenesis (such as liver qi stagnation and heart and spleen deficiency) due to emotional disorders and organ dysfunction, the breadth and depth of intervention are still insufficient, making it difficult to achieve the ideal effect of overall regulation.

[0005] Furthermore, traditional Chinese medicine has accumulated thousands of years of valuable experience in regulating emotions, calming the mind, and promoting sleep, forming a unique theoretical system and principles of medication. Its advantages lie in its "holistic view" and "syndrome differentiation and treatment," systematically regulating the body's balance through the synergistic effects of multiple components and targets. Currently, integrating modern biotechnology with traditional Chinese medicine theory has become an important trend in new drug development and functional food development. However, existing intervention strategies still have significant shortcomings. On the one hand, single probiotic or traditional Chinese medicine interventions often have limitations. As mentioned earlier, although probiotics can target the gut-brain axis, their regulatory effect on the core pathogenesis of traditional Chinese medicine (such as liver stagnation and spleen deficiency, phlegm and dampness accumulation) is limited. On the other hand, although traditional Chinese medicine compound formulas can provide holistic regulation, their mechanisms of action are often unclear, especially lacking modern scientific evidence on their direct or indirect regulation of gut microbiota structure and specific neurotransmitter metabolic pathways, which limits their standardized and precise application.

[0006] Therefore, developing a novel compound preparation that combines the targeted nature of probiotics with the holistic regulatory advantages of traditional Chinese medicine, and through synergistic effects, simultaneously intervenes in the modern biological pathways of the gut-brain axis and core TCM pathogenesis such as "liver-spleen disharmony and phlegm-dampness obstruction," to address anxiety, depression, sleep disorders, and their accompanying metabolic disturbances in a safer and more comprehensive manner, has become an urgent technical challenge and an important direction for development in this field. Summary of the Invention

[0007] The purpose of this invention is to overcome the limitations of existing technologies in regulating multiple symptoms (emotions, sleep, metabolic disorders) with single probiotics or traditional Chinese medicine interventions, and to provide a safe, efficient, multi-target synergistic compound composition of food and medicine containing Bifidobacterium breve and its application.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A medicinal and edible compound composition containing Bifidobacterium breve, wherein the compound composition comprises Bifidobacterium breve and a medicinal and edible compound, wherein the ratio of Bifidobacterium breve to the medicinal and edible compound is 1×10. 10CFU-Bifidobacterium breve is mixed with 60 g of raw medicinal material in a food-medicine homology composition.

[0009] The *Bifidobacterium breve* BKR006 was deposited at the China General Microbiological Culture Collection Center (CGMCC, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing) and classified as *Bifidobacterium breve*. Bifidobacterium breve, at The accession number is CGMCC No. 36096, dated September 26, 2025.

[0010] The *Bifidobacterium breve* is one or more of the following: live cells, inactivated cells, cell components, or metabolites of the strain; wherein, the live cells are obtained by inoculating strain BKR006, preserved in glycerol tubes at -80℃, into modified MRS liquid medium and culturing at 37℃ under anaerobic conditions for 18-24 hours until the end of the logarithmic growth phase (OD600>1.5). The fermentation broth after culture is centrifuged at 4℃ and 5000 rpm for 10 minutes, and the supernatant is discarded to obtain the cells.

[0011] The medicinal and edible composition comprises, by weight, 1-6 parts Buddha's Hand, 0.5-4 parts Rose, 1-6 parts Codonopsis, 1-6 parts Poria, 1-6 parts Fried Coix Seed, 0.5-4 parts Tangerine Peel, 1-6 parts Fried Jujube Seed, and 0.5-2 parts Prepared Licorice.

[0012] The application of the aforementioned food-medicine composite composition containing Bifidobacterium breve, specifically its application in regulating serum corticosterone (CORT) levels in chronic social defeat stress (CSDS).

[0013] The composite composition is used in the preparation of products for regulating mood, improving sleep, and / or assisting in weight management.

[0014] A functional product comprising the aforementioned composite composition in its formulation.

[0015] The compound composition contains Bifidobacterium breve (Bifidobacterium breve). Bifidobacterium breve BKR006 includes live bacteria, inactivated bacteria, bacterial components or their metabolites; the food-medicine homology composition is an extract, concentrate or powder of the food-medicine homology composition.

[0016] The dosage form of the product formulation is selected from capsules, tablets, powders, granules, pills, or liquid dosage forms.

[0017] An application of the aforementioned functional product, wherein the functional product is used as one or more products for regulating mood, improving sleep, and assisting in weight management.

[0018] The mood regulation includes preventing, alleviating, or treating anxiety or depression and their related symptoms; the sleep improvement includes shortening sleep latency, prolonging total sleep time, or improving sleep efficiency; the weight management assistance includes reducing weight, body fat percentage, or reducing fat accumulation (this function is based on the composition's mechanism of strengthening the spleen and removing dampness).

[0019] The two systems in the composite composition or functional product exert their effects through a dual synergistic mechanism, which includes: a. Gut-brain axis regulation: inhibiting neuroinflammation (e.g., reducing the levels of IL-1β, IL-6 and / or TNF-α) and / or restoring the expression of brain-derived neurotrophic factor (BDNF); b. Liver-spleen regulation: soothing the liver and regulating qi, strengthening the spleen and eliminating dampness and / or resolving dampness and harmonizing the middle jiao.

[0020] The beneficial effects of the compound composition of the present invention are mainly reflected in the following aspects: 1. Synergistic and synergistic regulatory mechanisms: This composition cleverly combines the precise intervention of modern probiotics targeting the "gut-brain axis" with the holistic regulation of liver and spleen metabolism by traditional Chinese medicine.

[0021] Bifidobacterium breve BKR006 improves mood and cognitive function by regulating gut microbiota and metabolites (such as butyrate), repairing damaged intestinal barrier function, inhibiting the entry of endotoxins such as lipopolysaccharide (LPS) into the blood, thereby reducing the level of neuroinflammation in the central nervous system (such as IL-1β), restoring the expression of brain-derived neurotrophic factor (BDNF).

[0022] The food-medicine homology composition (ZY-001) follows the traditional Chinese medicine theory of "soothing the liver and strengthening the spleen, eliminating dampness and calming the mind". It improves the pathogenesis of liver stagnation and spleen deficiency with internal disturbance of phlegm and dampness by regulating the flow of qi, strengthening the spleen and stomach, removing dampness and turbidity, and calming the mind.

[0023] The combination of these two ingredients in a specific ratio was used to regulate stress hormones (serum corticosterone CORT) and reduce visceral fat (epididymal fat). While neither Bifidobacterium breve BKR006 nor the food-medicine homologous composition ZY-001 showed significant statistical differences when used alone, their combined use produced a highly significant improvement. This breaks the conventional understanding of an "additive effect" and reveals a deep coupling mechanism within the gut-brain-metabolism axis. Particularly in the regulation of the HPA axis (stress axis), a single component cannot counteract the elevated corticosterone levels caused by intense chronic social defeat stress (CSDS). Only when "intestinal barrier repair" and "liver-regulating qi" are performed simultaneously can the excessive stress response be effectively suppressed.

[0024] 2. Broad range of health benefits and safety: Anti-anxiety and anti-depression: In the face of severe chronic stress, single probiotics or traditional Chinese medicine cannot effectively reduce serum corticosterone (CORT) levels, while compound combinations significantly reduce CORT levels, blocking the vicious cycle of anxiety and depression from the endocrine source.

[0025] Improved sleep: This compound composition significantly shortens the sleep latency, prolongs the total sleep time, and at the same time reduces central inflammation and increases GABA levels, which has a good effect on improving primary insomnia and insomnia accompanied by anxiety.

[0026] Assisted weight management: In a high-fat diet-induced obesity model, a single component failed to significantly reduce visceral fat (epididymal fat) weight. However, this compound composition, through strengthening the spleen and removing dampness, improving metabolism (reducing TG, TC, and ALT), and repairing the intestinal barrier (reducing permeability and inhibiting TNF-α inflammation in adipose tissue), synergistically inhibits weight gain and fat accumulation caused by a high-fat diet, providing a new approach for the adjunctive intervention of obesity.

[0027] High safety: Bifidobacterium breve BKR006 has excellent probiotic properties and food-grade safety, while all components in the food-medicine homology composition are nationally approved food-medicine homology substances with no toxic side effects and can be taken for a long time.

[0028] In summary, the compound composition provided by this invention overcomes the shortcomings of the prior art. With its unique "gut-brain-metabolic axis" synergistic intervention strategy, it exhibits extremely high biological activity and synergistic effects in regulating mood, improving sleep, and assisting in weight management, and has great application value and broad market prospects. Detailed Implementation

[0029] The specific embodiments of the present invention will be further described below with reference to examples.

[0030] The compound composition proposed in this invention (containing Bifidobacterium breve BKR006 and a traditional Chinese medicine composition derived from food and medicine) aims to synergistically intervene in anxiety, depression, sleep disorders, and metabolic disorders (obesity). Its core mechanism lies in the targeted regulation of the gut-brain axis by BKR006 (inhibiting neuroinflammation, repairing the intestinal barrier, regulating SCFA, and restoring BDNF), while the traditional Chinese medicine composition derived from food and medicine (hereinafter referred to as ZY-001) regulates liver and spleen metabolism (regulating Qi, eliminating dampness and resolving phlegm) through the TCM theory of "soothing the liver and strengthening the spleen, resolving dampness and calming the mind." The combination of these two components achieves multi-target synergistic intervention on the gut-brain-metabolic axis. The compound composition contains Bifidobacterium breve BKR006, which exerts its effects through multiple gut-brain axis pathways, including inhibiting neuroinflammation, improving intestinal barrier function, regulating short-chain fatty acid levels, and restoring brain-derived neurotrophic factor (BDNF) expression. Its mechanisms are well-defined and its effects are significant. Simultaneously, the composition, containing ingredients that are both food and medicine, is formulated based on the therapeutic principles of "soothing the liver and strengthening the spleen, resolving dampness and calming the mind." It targets the common pathogenesis of liver stagnation and spleen deficiency, heart-spirit malnourishment, and phlegm-dampness disturbance in modern people, effectively improving anxiety and insomnia, and assisting in regulating weight and body fat through strengthening the spleen and eliminating dampness. Its food-medicine homology nature gives it the advantages of high safety and long-term use.

[0031] The strain BKR006 involved in the following examples is classified as Bifidobacterium breve (Bifidobacterium breve). Bifidobacterium breve This strain was deposited in 2025 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 36096.

[0032] The culture medium used in this embodiment of the invention is a modified MRS medium. This formulation has been optimized for the growth characteristics of *Bifidobacterium breve*, making it more suitable for its efficient proliferation. The formulation includes: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, diammonium hydrogen citrate 2 g / L, dipotassium hydrogen phosphate (K₂HPO₄) 2 g / L, sodium acetate 5 g / L, magnesium sulfate (MgSO₄·7H₂O) 0.1 g / L, manganese sulfate (MnSO₄·H₂O) 0.05 g / L, Tween-80 1 mL / L, and L-cysteine ​​hydrochloride 0.5 g / L. This medium provides abundant carbon and nitrogen sources and growth factors, effectively supporting the growth of *Bifidobacterium breve* BKR006.

[0033] The following examples illustrate the method for preparing probiotic suspensions: BKR006 strain, preserved in glycerol tubes at -80℃, was inoculated into modified MRS liquid medium and cultured at 37℃ under anaerobic conditions for 18–24 hours until the end of the logarithmic growth phase (OD600 > 1.5). The fermentation broth was then centrifuged at 4℃ and 5000 rpm for 10 minutes, and the supernatant was discarded to recover the bacterial cells. The bacterial cells were resuspended and washed twice with sterile physiological saline, and the final bacterial concentration was adjusted to, for example, 1 × 10⁻⁶. 11 CFU / mL, store at 4℃ for later use.

[0034] The preparation method of the medicinal and edible herbal composition (code ZY-001) involved in the following examples is as follows: Weigh each group of herbs according to the preferred weight ratio (3 parts Buddha's Hand, 2 parts Rose, 3 parts Codonopsis, 3 parts Poria, 3 parts stir-fried Coix Seed, 2 parts Tangerine Peel, 3 parts stir-fried Ziziphus Jujuba Seed, 1 part prepared Licorice Root), mix them, add water and decoct twice, 1 hour each time. Combine the decoctions, filter, and concentrate to 0.5g solids per mL for later use. For animal experiments, administer the mixture by gavage at a dose of X g / kg (based on the original herbal material) dissolved in physiological saline.

[0035] The experimental results were statistically analyzed using GraphPad Prism 9.5. Data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA combined with Tukey's multiple comparison test was used for comparisons between groups. This indicates a highly significant difference compared to the control group (p < 0.001). This indicates a highly significant difference compared to the control group (p < 0.01). , indicates a significant difference compared to the control group (p<0.05). ###p<0.001 indicates a highly significant difference compared to the model group (p<0.001); ##p<0.01 indicates a highly significant difference compared to the model group (p<0.01); #p<0.05 indicates a significant difference compared to the model group (p<0.05). $$$p<0.001, $$p<0.01, $p<0.05 indicate a significant synergistic effect between the combination group and the single intervention group (BKR or TCM).

[0036] Example 1: Study on the synergistic effect and mechanism of compound composition in anti-anxiety and antidepressant effects in CSDS chronic stress model Experimental Objective: This embodiment aims to evaluate the synergistic intervention effect of the compound Bifidobacterium breve BKR006 and ZY-001 on anxiety-like and depression-like behaviors in a chronic social frustration stress (CSDS) model. To highlight the specificity and superiority of the composition, the classic liver-soothing and mood-regulating formula "Xiaoyao Wan" was introduced as a positive control. The efficacy differences between the composition of this invention and Xiaoyao Wan alone or in combination with BKR006 strain were compared, with a focus on exploring its synergistic regulatory effect on the key stress hormone (corticosterone CORT).

[0037] Test method: 1. Animals and Model Establishment: Male C57BL / 6J mice were used as experimental mice, and male CD-1 (ICR) mice were used as aggressor mice. A CSDS model was used: C57BL / 6J mice were placed in the cage of the aggressor mice and subjected to physical aggression for 10 minutes, once daily for 10 consecutive days. A different aggressor mouse was used each day. After failure, the mice were placed in the same cage and kept in close proximity to the aggressor mice for 24 hours through a transparent partition (sensory stress).

[0038] 2. Grouping and drug administration: Mice were randomly divided into 7 groups (n=10 per group). (1) Control group: Not subjected to CSDS stress, administered physiological saline by gavage; (2) CSDS model group (CSDS): subjected to CSDS stress and administered physiological saline by gavage; (3) BKR006 group (CSDS+BKR): subjected to CSDS stress, and administered BKR006 live bacterial suspension (1×10) by gavage. 10 CFU / kg); (4) ZY-001 Traditional Chinese Medicine Group (CSDS+TCM): Patients suffering from CSDS stress were given daily oral administration of the traditional Chinese medicine group ZY-001 of this invention (dosage equivalent to 3 g / kg of raw medicinal materials).

[0039] (5) Xiaoyao Pill Group (CSDS+XYW): Suffering from CSDS stress, the patient was given a daily oral gavage of commercially available Xiaoyao Pill concentrated extract (dose equivalent to 3 g / kg of raw medicinal material).

[0040] (6) Xiaoyao Pill + Bacterial Group (CSDS+XYW+BKR): Patients suffering from CSDS stress were given Xiaoyao Pill (3 g / kg) and BKR006 live bacterial suspension (1×10⁻⁶) by gavage daily. 10 CFU / kg).

[0041] (7) Combination group (CSDS+Combo): Patients suffering from CSDS stress were given daily oral administration of BKR006 (1×10) of this invention. 10CFU / kg and ZY-001 (3 g / kg) were administered via daily gavage starting 2 weeks before CSDS modeling and continuing until the end of CSDS modeling (10 days in total) and the subsequent recovery period of 14 days, for a total of 38 days of administration.

[0042] 3. Behavioral assessment: On day 14 after the last stress, the following assessments were conducted: (1) Social Interaction Test (SIT): The Social Interaction Ratio (SI Ratio) was calculated. (2) Forced Swimming Test (FST): The immobility time within 6 minutes was recorded. (3) Suspended Tail Test (TST): The immobility time within 6 minutes was recorded.

[0043] 4. Mechanistic indicator detection: After the behavioral test was completed, the mice (n=10 / group) were sacrificed and samples were collected.

[0044] (1) Intestinal-brain axis indicators (BKR006 mechanism): ELISA was used to detect the levels of IL-1β and BDNF in the PFC (prefrontal cortex); Evans Blue method was used to detect intestinal permeability (Evans Blue dye that has penetrated into the tissue was extracted with formamide organic solvent, the absorbance of the extract was measured by spectrophotometer, and it was converted into the actual mass of dye (μg) according to the standard curve, and finally the ratio of dye mass to tissue wet weight (μg / g) was calculated); GC method was used to detect the level of butyrate in cecal contents.

[0045] (2) Liver and spleen metabolic indicators (TCM mechanism): Serum corticosterone (CORT) level was detected by ELISA; serum ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels were detected by kit to assess liver stagnation and metabolic function.

[0046] Experimental results: The compound composition synergistically improves CSDS-induced social avoidance and depression-like behaviors. As shown in Table 1, the SI Ratio of the CSDS model group mice was significantly lower than that of the control group. The immobility time of FST and TST increased significantly. This indicates that CSDS leads to persistent social avoidance and depressive-like behavior.

[0047] Compared with the CSDS model group, both the CSDS+BKR group and the CSDS+TCM group showed significant improvement (#p<0.05, ##p<0.01). Xiaoyao Wan intervention alone (CSDS+XYW group) failed to significantly improve any behavioral indicators. The combination of Xiaoyao Wan and BKR006 (CSDS+XYW+BKR group) showed a slight improvement in behavioral indicators (#p<0.05), but its effect was significantly weaker than any single component of this invention.

[0048] However, most notably, the behavioral indicators of the CSDS+Combo (compound group) of this invention showed the greatest improvement, which was significantly better than the model group (###p<0.001) and significantly better than the two single intervention groups ($p<0.05 or $$$p<0.01), demonstrating a clear synergistic effect.

[0049] Table 1 Comparison of behavioral indicators of mice in each group

[0050] Note: Compared to the Control group, Compared with the CSDS group, #p<0.05, ##p<0.01, ###p<0.001; compared with the CSDS+BKR and CSDS+TCM groups, $p<0.05, $$p<0.01. (n=10)

[0051] The compound composition synergistically regulates the gut-brain axis and liver-spleen metabolism. As shown in Tables 2 and 3, the gut-brain axis and liver-spleen metabolic indicators in the CSDS model group were all disordered. Regarding gut-brain axis related indicators (Table 2), the CSDS+BKR group showed a significant regulatory advantage (##p<0.01), while the CSDS+TCM group had a weaker effect. Notably, the Xiaoyao Wan group (CSDS+XYW) and the Xiaoyao Wan+bacteria group (CSDS+XYW+BKR) did not show statistically significant improvements in PFC IL-1β, BDNF, intestinal permeability, and cecal butyrate. In contrast, the CSDS+Combo group of this invention restored almost all gut-brain axis disorder indicators to normal control levels (###p<0.001), and was significantly superior to all other intervention groups ($$p<0.01).

[0052] Regarding liver and spleen metabolism and stress hormone indicators (Table 3), the most crucial finding lies in serum corticosterone (CORT) levels. Using BKR006, ZY-001 alone, or even Xiaoyao Wan or a combination of Xiaoyao Wan and BKR006, showed no statistically significant difference in serum CORT levels compared to the CSDS model group. This indicates that under intense chronic stress, single interventions or non-specific combinations (such as Xiaoyao Wan + BKR006) are insufficient to effectively inhibit excessive activation of the HPA axis.

[0053] However, the CSDS+Combo (compound group) of this invention significantly reduced serum CORT levels (###p<0.001), restoring them to near-normal levels, and was significantly superior to all single intervention groups and the Xiaoyao Wan control group ($$$p<0.001). This strongly demonstrates that only through the precise synergy between the specific strain BKR006 and the specific traditional Chinese medicine formula ZY-001 in this invention can the core pathway of stress signaling be effectively blocked.

[0054] Table 2 Comparison of key indicators of the gut-brain axis in mice of different groups

[0055] Table 3 Comparison of key liver and spleen related indicators among different groups of mice

[0056] Note: Compared to the Control group, Compared with the CSDS model group, #p<0.05, ##p<0.01, ###p<0.001; compared with a single group, $$p<0.01, $$$p<0.001. (n=10) Conclusion: The results of this embodiment confirm that the compound composition of Bifidobacterium breve BKR006 and ZY-001 can synergistically alleviate anxiety and depression-like behaviors caused by chronic stress. The mechanism lies in the precise targeting of BKR006 (probiotic) to the gut-brain axis, inhibiting neuroinflammation, restoring BDNF, repairing the intestinal barrier, and increasing butyrate production; while ZY-001 (traditional Chinese medicine) targets the liver to relieve depression and strengthen the spleen to resolve dampness (improving ALT / AST). More importantly, through comparison with the classic formula Xiaoyao Wan, this study reveals the breakthrough of this compound composition in the unique synergistic effect produced by its precise formulation. Furthermore, the compound composition demonstrates a breakthrough in regulating the core mechanism of stress: neither a single component nor a simple combination of Xiaoyao Wan and this strain can reduce CSDS-induced high levels of corticosterone (CORT), while the compound composition of this invention, combining a specific strain with a specific traditional Chinese medicine formula, successfully reverses this core pathological change, achieving a powerful inhibitory effect on the HPA axis and fundamentally blocking the stress response. This indicates that the effect of the present invention is not a simple combination of "probiotics" and "liver-soothing traditional Chinese medicine", but rather stems from the unique and scientific compatibility between BKR006 and ZY-001, thereby achieving an unprecedented comprehensive intervention in the integrated regulatory network of "gut-brain-metabolism".

[0057] Example 2: Study on the synergistic effect of the compound composition on pentobarbital sodium-induced sleep in mice Experimental Objective: This experiment aims to evaluate the synergistic effect of the combination of Bifidobacterium breve BKR006 and ZY-001 on sleep in mice and to explore its effects on central nervous system inflammation and the GABAergic system.

[0058] Test method: 1. Animals and grouping: Male Kunming mice weighing 18–22 g were selected. They were randomly divided into 5 groups of 10 mice each: (1) Control group: administered physiological saline by gavage; (2) BKR006 group: administered BKR006 live bacterial suspension (1×10⁻⁶) by gavage. 10 (3) Traditional Chinese Medicine Group (TCM): ZY-001 (3 g / kg raw herb) administered by gavage; (4) Combo Group: BKR006 (1×10 CFU / kg) administered by gavage. 10 (5) Positive drug group (Dia): Diazepam (2 mg / kg) administered by gavage.

[0059] 2. Dosage regimen: Administer once daily by gavage for 10 consecutive days.

[0060] 3. Sleep test: 30 minutes after the last administration, mice in each group were intraperitoneally injected with sodium pentobarbital (42 mg / kg, calculated based on body weight). The sleep latency (time for the righting reflex to disappear) and sleep duration (time for the righting reflex to recover) were recorded.

[0061] 4. Mechanism indicator detection: Another batch of mice (n=6 / group) were taken and the administration regimen was the same as above. The mice were sacrificed 30 min after the last administration and PFC (prefrontal cortex) and hippocampus tissues were collected. (1) Gut-brain axis indicator (BKR006 mechanism): IL-1β level in PFC was detected by ELISA. (2) Tranquilizing and calming indicator (TCM mechanism): GABA (γ-aminobutyric acid) level in hippocampus was detected by HPLC.

[0062] Experimental results: The compound composition synergistically prolongs sleep time As shown in Table 4, there were no significant differences in sleep latency and sleep duration between the BKR group and the control group. The TCM group (traditional Chinese medicine group) significantly shortened the sleep latency (##p<0.01) and prolonged the sleep duration (##p<0.01), consistent with the calming effect of Ziziphus jujuba seed in ZY-001. Crucially, the Combo group (compound group) had the shortest sleep latency and the longest sleep duration (###p<0.001), significantly better than the TCM group ($$p<0.01), indicating a strong synergistic effect between BKR006 and ZY-001 in improving sleep.

[0063] Table 4. Effects of the compound composition on the sleep-inducing effect of sodium pentobarbital.

[0064] Note: Compared with the Control group, ##p<0.01, ###p<0.001; compared with the TCM group, $$p<0.01. (n=10) The compound composition synergistically regulates central inflammation and GABA levels. As shown in Table 5, the BKR group significantly reduced IL-1β levels in the PFC (##p<0.01). The TCM group significantly increased GABA levels in the hippocampus (##p<0.01). The Combo group achieved both objectives simultaneously: significantly reduced IL-1β (###p<0.001) and significantly increased GABA levels (###p<0.001), with both effects superior to the single groups ($p<0.05).

[0065] Table 5 Comparison of key mechanism indicators in the brains of mice in each group

[0066] Note: Compared with the Control group, ##p<0.01, ###p<0.001; compared with the BKR and TCM groups, $p<0.05. (n=6) in conclusion: The results of this embodiment confirm that the compound composition of BKR006 and ZY-001 has a synergistic sedative-hypnotic effect. The mechanism lies in the fact that BKR006 reduces central nervous system inflammation (IL-1β) through the gut-brain axis pathway, creating a low-inflammatory "basal environment" for sleep; while ZY-001 (a formula derived from both food and medicine) exerts a calming and soothing effect, upregulating the GABAergic system. The combination of the two synergistically improves sleep quality.

[0067] Example 3: Synergistic intervention and mechanism study of compound composition on obesity induced by high-fat diet (HFD) Experimental Objective: This study aims to evaluate the synergistic intervention effect of the compound combination of Bifidobacterium breve BKR006 and ZY-001 on body weight, body fat and metabolic disorders induced by high-fat diet (HFD) in obese mice, and to explore its synergistic effect of "strengthening the spleen and removing dampness" (TCM mechanism) and "intestinal barrier-metabolism" (BKR006 mechanism).

[0068] Test method: 1. Animals and model establishment: Male C57BL / 6J mice were randomly divided into two groups: the control group (n=10) was fed a standard diet; the HFD group (n=40) was fed a high-fat diet (D12492, 60% kcal fat).

[0069] 2. Grouping and administration: After 8 weeks of HFD feeding, the HFD group mice were randomly divided into 4 groups (n=10 in each group), while the Control group remained unchanged. (1) Control group: standard diet + oral administration of physiological saline; (2) HFD model group: HFD + oral administration of physiological saline; (3) BKR006 group (HFD+BKR): HFD + oral administration of BKR006 (1×10 10 (4) Traditional Chinese Medicine Group (HFD+TCM): HFD + ZY-001 (3 g / kg raw herbal material) administered by gavage; (5) Compound Group (HFD+Combo): HFD + BKR006 + ZY-001 administered by gavage. The intervention (gavage) and HFD feeding continued for 4 weeks.

[0070] 3. Phenotypic index detection: Record weight gain and epididymal fat (eWAT) weight after 4 weeks of intervention.

[0071] 4. Mechanism indicators: (1) Spleen-strengthening and dampness-removing / metabolic indicators (TCM mechanism): Detect serum triglyceride (TG), total cholesterol (TC) and ALT levels. (2) Intestinal barrier-inflammatory indicators (BKR006 mechanism): Evans Blue method to detect intestinal permeability; ELISA method to detect TNF-α level in epididymal adipose tissue (eWAT) (reflecting low-grade inflammation caused by obesity).

[0072] Experimental results: The compound composition synergistically inhibits HFD-induced weight gain and fat accumulation. As shown in Table 6, the body weight gain and epididymal fat weight in the HFD model group were significantly higher than those in the Control group. The HFD+BKR group could inhibit weight gain, the HFD+TCM group could reduce weight but not significantly, while the HFD+Combo group had the most significant effect (###p<0.001).

[0073] Using BKR006 alone (HFD+BKR group) or the traditional Chinese medicine combination alone (HFD+TCM group) did not significantly differ in eWAT weight compared to the HFD model group, indicating that single interventions are insufficient to reverse existing visceral fat accumulation. In contrast, the eWAT weight of the HFD+Combo group was significantly reduced (###p<0.001) and significantly lower than both single intervention groups ($$$p<0.001). This confirms that the compound combination possesses unique biological activity in fat reduction (especially visceral fat) that is not present in single components.

[0074] Table 6 Comparison of body weight and adipose phenotype among different groups of mice

[0075] Compared to the Control group, Compared with the HFD group, #p<0.05, ###p<0.001; compared with the HFD+BKR and HFD+TCM groups, $$$ p<0.001. (n=10) The compound composition synergistically improves metabolic disorders, intestinal barrier function, and low-grade inflammation. As shown in Table 7, the HFD model group exhibited severe metabolic disorders (elevated TG, TC, and ALT), intestinal barrier disruption (elevated permeability), and lipid inflammation (elevated eWAT and TNF-α). The HFD+BKR group showed significant effects in improving intestinal permeability and lipid inflammation (##p<0.01). The HFD+TCM group showed significant effects in improving metabolic indicators (TG, TC, ALT) (##p<0.01), consistent with its positioning of "strengthening the spleen and removing dampness". The HFD+Combo (compound group) synergistically combined the advantages of both, achieving the best improvement effect on all indicators (###p<0.001), and was significantly better than the single intervention group ($p<0.05 or $$$p<0.01).

[0076] Table 7 Comparison of metabolic, intestinal, and inflammatory markers in mice of different groups

[0077] Note: Statistical identifiers are the same as in Table 6. (n=6) Conclusion: The results of this embodiment confirm that the compound composition of BKR006 and ZY-001 synergistically inhibits HFD-induced obesity and metabolic disorders. The mechanism lies in the fact that ZY-001 exerts its "spleen-strengthening and dampness-removing" effect, targeting and improving blood lipids and liver function (TG, TC, ALT); while BKR006 repairs the "intestinal barrier" and reduces HFD-induced low-grade inflammation of adipose tissue (eWATTNF-α). Single probiotic or traditional Chinese medicine interventions cannot effectively reduce HFD-induced visceral fat (epididymal fat) accumulation, but when used in combination, the synergistic effect across multiple pathways (intestinal-metabolic-inflammatory) achieves superior weight management results, fully demonstrating the unique value and inventiveness of the compound composition of this invention in the field of assisted weight management.

Claims

1. A medicinal and edible compound composition containing Bifidobacterium breve, characterized in that: The compound composition is Bifidobacterium breve ( Bifidobacterium breve The sample contains BKR006 and a food-medicine homology composition, wherein the preservation number of *Bifidobacterium breve* is CGMCC No. 36096; the ratio of *Bifidobacterium breve* and the food-medicine homology composition is 1×10⁻⁶. 10 CFU of Bifidobacterium breve is mixed with a 60 g dose of the original medicinal material in a food-medicine homology composition. The medicinal and edible composition comprises, by weight, 3 parts Buddha's Hand, 2 parts Rose, 3 parts Codonopsis, 3 parts Poria, 3 parts stir-fried Coix Seed, 2 parts Tangerine Peel, 3 parts stir-fried Jujube Seed, and 1 part prepared Licorice.

2. The medicinal and edible compound composition containing Bifidobacterium breve according to claim 1, characterized in that: The *Bifidobacterium breve* is one or more of the following: live cells, inactivated cells, cell components, or metabolites of the strain; wherein, the live cells are obtained by inoculating strain BKR006, preserved in glycerol tubes at -80℃, into modified MRS liquid medium and culturing at 37℃ under anaerobic conditions for 18-24 hours until the end of the logarithmic growth phase, with OD... 600 > 1.5; After cultivation, the fermentation broth was centrifuged at 4℃ and 5000 rpm for 10 minutes, and the supernatant was discarded to obtain the bacterial cells.

3. The application of the medicinal and edible compound composition containing Bifidobacterium breve as described in claim 1, characterized in that: The composite composition is used in the preparation of drugs for the prevention, relief or treatment of anxiety or depression, the preparation of drugs for improving sleep, and the preparation of drugs for assisting in weight management.

4. A functional product, characterized in that: The composite composition according to claim 1.

5. The functional product according to claim 4, characterized in that: The compound composition contains Bifidobacterium breve as live cells, inactivated cells, cell components or metabolites of Bifidobacterium breve BKR006; the food-medicine homology composition is an extract, concentrate or powder of a food-medicine homology composition.

6. The functional product according to claim 4, characterized in that: The dosage form of the product formulation is selected from capsules, tablets, powders, granules, pills, or liquid dosage forms.

7. An application of the functional product according to claim 4, characterized in that: The functional products are used in the preparation of drugs for the prevention, relief or treatment of anxiety or depression, improvement of sleep, and assistance in weight management.

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