Animal bifidobacterium lactis BR061 and its postbiotic for regulating mood and promoting digestion and absorption

Abstract

This invention provides an application of *Bifidobacterium lactis* subspecies BR061 and its postbiotics in regulating mood and promoting digestion and absorption, belonging to the field of microbial and functional development technology. This invention provides *Bifidobacterium lactis* subspecies BR061, with accession number CGMCC No. 37152. This invention further provides the fermentation broth, lyophilized powder, and postbiotics of this *Bifidobacterium lactis* subspecies BR061, as well as their preparation methods, and the application of this *Bifidobacterium lactis* subspecies BR061 and its fermentation broth, lyophilized powder, and postbiotics in regulating mood and promoting digestion and absorption, possessing broad market potential and application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial and functional development technology, specifically relating to the application of Bifidobacterium lactis subspecies BR061 and its postbiotics in regulating mood and promoting digestion and absorption. Background Technology

[0002] With the accelerated pace of modern life, changes in dietary structure, and continuous increase in mental stress, emotional abnormalities (such as anxiety and depressive tendencies) and digestive and absorptive dysfunctions (such as indigestion and intestinal discomfort) have become common health problems affecting the population, and there is a close correlation between the two, easily forming a vicious cycle. Digestive and absorptive dysfunction mainly manifests as symptoms such as bloating, diarrhea, constipation, and loss of appetite. One of its core causes is the imbalance of the gut microbiota—a decrease in the number of beneficial bacteria and an overgrowth of harmful bacteria in the gut, leading to impaired intestinal barrier function, reduced efficiency in the decomposition and absorption of nutrients, and the induction of intestinal inflammatory responses. The mechanisms of emotional abnormalities are complex. In addition to psychological factors, the signal transduction of gut microbiota metabolites through the "microbe-gut-brain axis" is an important regulatory pathway. For example, short-chain fatty acids and indole produced by gut microbiota metabolism can enter the brain through the bloodstream, regulating neuronal activity and the function of mood-related receptors, thus affecting the stability of emotional state.

[0003] Probiotics, as a key means of regulating the balance of the gut microbiota, have been applied to improve digestion and absorption and regulate mood. However, currently available strains have significant limitations: most strains can only exert a single function of promoting digestion and absorption or regulating mood, making it difficult to simultaneously improve the synergistic effects of mood disorders and digestive and absorptive disturbances; some strains have weak gastrointestinal tolerance and low survival rates after passing through the acidic and bile salt environment of the stomach, failing to effectively colonize and exert their effects in the gut; and some strains have mild functional effects, with insufficient improvement in mood regulation and digestion and absorption, making it difficult to meet the actual needs of the population. Therefore, screening probiotics that can simultaneously and effectively regulate mood and significantly promote digestion and absorption is of significant practical importance and application value for improving the balance of the gut microbiota and alleviating mood disorders and digestive and absorptive disturbances, and is also a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0004] To address the problems existing in the prior art, the primary objective of this invention is to provide a *Bifidobacterium lactis* subspecies (…). Bifidobacterium animalis Subsp.lactis) BR061, deposited at: China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, accession number: CGMCC No. 37152, deposit date: December 24, 2025.

[0005] A second objective of this invention is to provide the above-mentioned fermentation broth, freeze-dried powder, postbiotic, and preparation method of Bifidobacterium lactis subsp. BR061.

[0006] A third objective of this invention is to provide applications of the above-mentioned Bifidobacterium lactis subsp. BR061 or its fermentation broth, freeze-dried powder, and postbiotic.

[0007] A fourth objective of this invention is to provide a drug that regulates mood and promotes digestion and absorption.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a subspecies of Bifidobacterium animalis, BR061, wherein the subspecies of Bifidobacterium animalis (BR061) Bifidobacterium animalis The accession number of subsp.lactis)BR061 is CGMCC No.37152.

[0009] This invention provides a method for preparing fermentation broth of Bifidobacterium animalis subsp. lactis BR061, comprising the following steps: inoculating the above-mentioned Bifidobacterium animalis subsp. lactis BR061 into a fermentation medium for fermentation to obtain fermentation broth of Bifidobacterium animalis subsp. lactis BR061.

[0010] Preferably, the fermentation medium is selected from any of the following: (1) Modified MRS medium; (2) A culture medium based on modified MRS medium, with 10% sterile extract of dried tangerine peel, 15% sterile extract of hawthorn and 20% sterile extract of malt added by volume percentage.

[0011] The present invention provides fermentation broth of Bifidobacterium lactis subsp. BR061 prepared by the above preparation method.

[0012] This invention provides a method for preparing freeze-dried Bifidobacterium animalis subsp. lactis BR061 powder, comprising the following steps: centrifuging the above-mentioned fermentation broth of Bifidobacterium animalis subsp. lactis BR061 to obtain Bifidobacterium animalis subsp. lactis BR061 bacterial sludge; mixing the Bifidobacterium animalis subsp. lactis BR061 bacterial sludge with a preservative and freeze-drying it; The protective agent comprises, by weight percentage: 10% skim milk, 8% trehalose, 5% maltodextrin, 2% sucrose, 0.5% tremella polysaccharide, and 0.5% vitamin C.

[0013] This invention provides a freeze-dried powder of Bifidobacterium lactis subsp. BR061 prepared by the above preparation method.

[0014] This invention provides a method for preparing a postbiotic of Bifidobacterium animalis subsp. lactis BR061, comprising the following steps: spray drying the fermentation broth of the above-mentioned Bifidobacterium animalis subsp. lactis BR061.

[0015] This invention provides a postbiotic of Bifidobacterium lactis subsp. BR061 prepared by the above preparation method.

[0016] The present invention provides the application of the above-mentioned Bifidobacterium lactis subsp. BR061 or the above-mentioned fermentation broth of Bifidobacterium lactis subsp. BR061 or the above-mentioned freeze-dried powder of Bifidobacterium lactis subsp. BR061 or the above-mentioned postbiotic of Bifidobacterium lactis subsp. BR061 in any of the following: (1) preparing a medicine for regulating mood; (2) preparing a medicine for treating anxiety; (3) preparing a medicine for inhibiting corticosterone synthesis; (4) preparing a health product or medicine that aids digestion; (5) preparing a medicine that promotes nutrient absorption.

[0017] This invention provides a drug for regulating mood and promoting digestion and absorption, the drug comprising the above-mentioned Bifidobacterium lactis subsp. BR061, or the above-mentioned fermentation broth of Bifidobacterium lactis subsp. BR061, or the above-mentioned freeze-dried powder of Bifidobacterium lactis subsp. BR061, or the above-mentioned postbiotic of Bifidobacterium lactis subsp. BR061.

[0018] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention provides the first-ever synthesis of a strain of Bifidobacterium lactis BR061, whose fermentation metabolites contain substances such as γ-aminobutyric acid, aspartic acid, and anserine. These substances significantly inhibit cortisol synthesis and can significantly reduce cortisol and increase pepsin and trypsin in chronic unpredictable stress symptoms, thus alleviating anxiety. Furthermore, these substances promote digestion and nutrient absorption while exhibiting high safety and broad market potential and application prospects.

[0019] Biological Preservation Instructions Bifidobacterium animalis subsp. lactis BR061, classified as Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis (subsp.lactis), deposited at: China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, accession number: CGMCC No. 37152, deposit date: December 24, 2025. Attached Figure Description

[0020] Figure 1 Effects of fermentation supernatant from different bacterial strains on corticosterone synthesis in mouse adrenocortical cell line Y1.

[0021] Figure 2 Scanning electron microscope image of lyophilized Bifidobacterium animalis subsp. lactis BR061.

[0022] Figure 3 : TIC graph of the experimental test sample.

[0023] Figure 4 : Secondary mass spectrum of goose muscle peptide.

[0024] Figure 5 : Secondary mass spectrum of aspartic acid.

[0025] Figure 6 : Secondary mass spectrum of γ-aminobutyric acid. Detailed Implementation

[0026] This invention provides a subspecies of Bifidobacterium animalis, BR061, wherein the subspecies of Bifidobacterium animalis (BR061) Bifidobacterium animalis The accession number for *Bifidobacterium lactis* subsp. *lactis* BR061 is CGMCC No. 37152. The *Bifidobacterium lactis* subsp. *lactis* BR061 described in this invention was isolated and purified from fecal samples of healthy infants, and identified as belonging to *Bifidobacterium lactis* subsp. *lactis*. Bifidobacterium animalis The subsp.lactis was deposited on December 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0027] This invention provides a method for preparing fermentation broth of *Bifidobacterium animalis* subsp. *lactamase* BR061, comprising the following steps: inoculating the above-mentioned *Bifidobacterium animalis* subsp. *lactamase* BR061 into a fermentation medium for fermentation to obtain fermentation broth of *Bifidobacterium animalis* subsp. *lactamase* BR061. The preferred fermentation conditions are anaerobic static culture at 36-38℃ for 16-26 hours, and the culture time can be selected as 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or 25 hours.

[0028] Preferably, the fermentation medium of the present invention is a modified MRS medium.

[0029] Preferably, the fermentation culture medium of the present invention is a culture medium based on modified MRS medium, with 10% sterile tangerine peel extract, 15% sterile hawthorn extract, and 20% sterile malt extract added by volume percentage. The preparation method of the sterile tangerine peel extract of the present invention includes: pulverizing tangerine peel, soaking it in water, extracting it in a water bath, filtering, centrifuging the filtrate, and sterilizing it to obtain the sterile tangerine peel extract; as an optional embodiment, the present invention pulverizes tangerine peel to 60 mesh, adds distilled water at a material-to-liquid ratio of 1g:10mL, soaks it at room temperature for 30min, extracts it in a 100℃ water bath for 60min, filters it through 4 layers of gauze, centrifuges it at 6000rpm for 5min, collects the supernatant, and filters it through a 0.22μm filter membrane for sterilization to obtain the sterile tangerine peel extract. The preparation method of the sterile malt extract of the present invention includes: pulverizing malt, soaking it in water, extracting it in a water bath, filtering it, centrifuging the filtrate, and sterilizing it to obtain a sterile malt extract; as an optional embodiment, the present invention pulverizes malt to 60 mesh, adds distilled water at a material-to-liquid ratio of 1g:10mL, soaks it at room temperature for 30min, extracts it in a water bath at 100℃ for 60min, filters it through 4 layers of gauze, centrifuges it at 6000rpm for 5min, collects the supernatant, and filters it through a 0.22μm filter membrane to obtain a sterile malt extract. The preparation method of the hawthorn sterile extract of the present invention includes: hawthorn pulverizing and soaking in water, water bath extraction, filtration, centrifugation of the filtrate, and sterilization to obtain hawthorn sterile extract; the present invention pulverizes hawthorn to 60 mesh, adds distilled water at a material-to-liquid ratio of 1g:10mL, soaks at room temperature for 30min, extracts in a 100℃ water bath for 60min, filters through 4 layers of gauze, centrifuges at 6000rpm for 5min to collect the supernatant, and filters through a 0.22μm filter membrane for sterilization to obtain hawthorn sterile extract.

[0030] This invention provides a fermentation broth of *Bifidobacterium animalis* subsp. lactis BR061 prepared by the above-described method. By adding sterile extracts of dried tangerine peel, malt, and hawthorn, this invention significantly enhances the metabolic activity of *Bifidobacterium animalis* subsp. lactis BR061, resulting in a viable cell count of 10⁻⁶ in the fermentation broth. 10 level.

[0031] This invention provides a method for preparing freeze-dried Bifidobacterium animalis subsp. lactis BR061 powder, comprising the following steps: centrifuging the above-mentioned fermentation broth of Bifidobacterium animalis subsp. lactis BR061 to obtain Bifidobacterium animalis subsp. lactis BR061 bacterial sludge; mixing the Bifidobacterium animalis subsp. lactis BR061 bacterial sludge with a protective agent and freeze-drying; preferably, by mass percentage, the protective agent comprises: 10% skim milk, 8% trehalose, 5% maltodextrin, 2% sucrose, 0.5% tremella polysaccharide, and 0.5% vitamin C; the protective agent is prepared with water. The centrifugation conditions of this invention are preferably centrifugation at 8000 rpm for 20 min at 4℃, and the supernatant is discarded after centrifugation to obtain Bifidobacterium animalis subsp. lactis BR061 bacterial sludge. The bacterial sludge and the protective agent are preferably mixed at a mass ratio of 1:1. The freeze-drying conditions of this invention are preferably pre-freezing at -45℃ for 3 h, freeze-drying at -24℃ for 36 h, freeze-drying at 4℃ for 12 h, and desorption drying at 24℃ for 12 h.

[0032] The present invention provides a freeze-dried powder of Bifidobacterium lactis subsp. BR061 prepared by the above preparation method. The cell walls of the freeze-dried powder are intact, no large number of dead cells are observed, and the survival rate of the strain can reach 98.07%.

[0033] This invention provides a method for preparing a postbiotic from *Bifidobacterium animalis* subsp. *lactotrium* BR061, comprising the following steps: spray drying the fermentation broth of *Bifidobacterium animalis* subsp. *lactotrium* BR061. Preferably, the spray drying conditions of this invention are: inlet air temperature 111.3℃, feed rate 161.5 mL / h, outlet air temperature 60℃, and atomizer pressure 0.4 MPa.

[0034] This invention provides a postbiotic of Bifidobacterium lactis subsp. BR061 prepared by the above preparation method, wherein the postbiotic of Bifidobacterium lactis subsp. BR061 includes metabolites such as γ-aminobutyric acid, aspartic acid and anserine.

[0035] The present invention provides the application of the above-mentioned Bifidobacterium lactis subsp. BR061 or the above-mentioned fermentation broth of Bifidobacterium lactis subsp. BR061 or the above-mentioned freeze-dried powder of Bifidobacterium lactis subsp. BR061 or the above-mentioned postbiotic of Bifidobacterium lactis subsp. BR061 in any of the following: (1) preparing a medicine for regulating mood; (2) preparing a medicine for treating anxiety; (3) preparing a medicine for inhibiting corticosterone synthesis; (4) preparing a health product or medicine that aids digestion; (5) preparing a medicine that promotes nutrient absorption.

[0036] This invention demonstrates through experiments that the fermentation supernatant of *Bifidobacterium animalis* subsp. *milk* BR061 can significantly inhibit corticosterone synthesis in adrenal cortical cells, preferably inhibiting adrenocorticotropic hormone (ACTH)-induced corticosterone synthesis. The inhibitory effect of *Bifidobacterium animalis* subsp. *milk* BR061 on corticosterone is superior to existing probiotic strains, such as *Bifidobacterium animalis* subsp. *milk* Bb-12 and *Lactobacillus rhamnosus* LGG strain. Furthermore, the fermentation supernatant, lyophilized powder, and post-biotic of *Bifidobacterium animalis* subsp. *milk* BR061 all exhibit inhibitory effects on corticosterone.

[0037] This invention demonstrates through experiments that the metabolites of *Bifidobacterium lactis* subsp. BR061 are rich in various substances, including γ-aminobutyric acid (GABA), aspartic acid, and anserine. In chronic unpredictable stress symptoms, the metabolites of *Bifidobacterium lactis* subsp. BR061 described in this invention can significantly reduce cortisol, increase pepsin and trypsin, alleviate anxiety, and also promote digestion and nutrient absorption, while exhibiting high safety.

[0038] This invention provides a drug for regulating mood and promoting digestion and absorption, the drug comprising the above-mentioned *Bifidobacterium lactis* subsp. *animal* BR061, or fermentation broth of the above-mentioned *Bifidobacterium lactis* subsp. *animal* BR061, or lyophilized powder of the above-mentioned *Bifidobacterium lactis* subsp. *animal* BR061, or postbiotic of the above-mentioned *Bifidobacterium lactis* subsp. *animal* BR061. The drug of this invention may also include pharmaceutically optional excipients or pharmaceutical carriers.

[0039] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0040] Unless otherwise specified, the following embodiments are all conventional methods.

[0041] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0042] Example 1 1. Isolation of Bifidobacterium animalis subsp. lactis BR061: Twenty fecal samples from healthy infants were collected from Inner Mongolia Autonomous Region and anaerobically refrigerated before being sent to the laboratory for bacterial isolation. Using a commercially available modified MRS solid isolation medium, the samples were serially diluted and anaerobically incubated at 37°C for 72 hours for bacterial isolation. Single colonies were picked and cultured until the strains were purified.

[0043] The purified colonies were subjected to Gram staining, microscopic examination, and H2O2 catalase test. Sixteen Gram-positive strains (SQ1, SQ2, SQ3, SQ4, SQ5, SQ6, SQ7, SQ8, SQ9, SQ10, SQ11, SQ12, SQ13, SQ14, SQ15, and SQ16) that were non-spore-forming and catalase-negative were selected and stored in 15% glycerol at -80°C for later use.

[0044] The preserved strain was inoculated with modified MRS medium at 3% (v / v) and then anaerobically cultured at 37°C for 18 h. The culture product was then inoculated again into modified MRS medium at 3% (v / v) and anaerobically cultured at 37°C for 18 h. After two culture cycles, the strain was activated.

[0045] The activated strain was inoculated at 1% (v / v) into modified MRS medium and anaerobically cultured at 37°C for 24 h. The OD value of the fermentation broth was then measured. 600 The pH value was determined by centrifuging 100 mL of fermentation broth at 8000 rpm for 5 min at 4℃, discarding the supernatant, weighing the bacterial sludge, and selecting well-growing strains. The results are as follows: Table 1. OD value, pH value, and wet cell weight of fermentation broth for different bacterial strains

[0046] The results showed that, under the same culture conditions, strain SQ6 exhibited the best growth, with a high OD value, low pH, and the highest cell count. Therefore, SQ6 was selected as the next research strain.

[0047] 2. Identification of 16S rDNA in strain SQ6 Strain SQ6 was cultured in MRS liquid medium at 37°C for 24 h. 2 mL of the pure culture was sent to Shanghai Sangon Biotech Co., Ltd. for 16S rDNA sequencing. The sequence is shown in SEQ ID No. 1.

[0048] The 16S rDNA fragment of this strain was identified as 1439 bp. Comparison with the NCBI database shows that the taxonomic unit of this species is: Bacteria; Actinomycetota; Actinomycetes; Bifidobacteriales; Bifidobacteriaceae. Bifidobacterium (Bifidobacterium genus); Bifidobacterium animalis subsp. lactis (Bifidobacterium animalis lactis subspecies). The strain SQ6 was named Bifidobacterium animalis lactis subspecies BR061.

[0049] Bifidobacterium animalis subspecies Lactobacillus BR061 was deposited on December 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 37152.

[0050] Experimental Example 1 Effects of Bifidobacterium lactis subsp. BR061 on corticosterone synthesis in mouse adrenal cortex cell line Y1: Bifidobacterium animalis subsp. lactis BR061, Bifidobacterium animalis subsp. lactis Bb-12 (Chr. Hansen (Beijing) Trading Co., Ltd.), and Lactobacillus rhamnosus LGG strain (Chr. Hansen (Beijing) Trading Co., Ltd.) were cultured at 37℃ in modified MRS medium for 24 h. The culture supernatant was obtained by centrifugation (8000 rpm, 5 min) using a microcentrifuge, filtered for sterilization, and used for later use.

[0051] Mouse adrenocortical cell line Y1 was cultured in F12 medium (containing 10% fetal bovine serum, 1.176 g NaHCO3, and 100 U / L penicillin and streptomycin) under humidified conditions of 5% CO2 and 37°C. When the cells reached 80% confluence, they were digested with 0.25% mixed trypsin and passaged from one flask to three flasks. Cells in the logarithmic growth phase were used for experiments.

[0052] Y1 cells in logarithmic growth phase were harvested and their concentration adjusted to 1.0 × 10⁻⁶ cells. 5 The cells / ml were seeded into 6-well plates, and 1 ml of F12 culture medium was added to each well. After culturing for 24 h, the following were added to each experimental group: (1) Blank control group: 2 ml of F12 culture medium; (2) Adrenocorticotropic hormone (ACTH) group: 2 ml of F12 culture medium containing 1 mL of 10 -9mol / L ACTH; (3) BR061 supernatant + ACTH group: 2ml F12 culture medium, containing 200μL BR061 supernatant + 1mL 10 -9 mol / L ACTH; (4) Bb-12 supernatant + ACTH group: 2ml F12 culture medium, containing 200μL Bb-12 supernatant + 1mL 10 -9 mol / L ACTH; (5) LGG supernatant + ACTH group: 2ml F12 culture medium, containing 200μL LGG supernatant + 1mL 10 -9 The concentration of corticosterone in the cell supernatant was mol / L. The experiment was terminated after 24 hours of culture. The cell supernatant was collected in sterile EP container, mixed well, and centrifuged at 2000 rpm for 5 minutes. The supernatant was then collected, and the corticosterone level in the cell supernatant of each group was measured using a radioimmunoassay kit (purchased from Shanghai ELISA kit). Corticosterone levels are expressed in nmol / L. The experiment was repeated three times.

[0053] Experimental results are as follows Figure 1 As shown in the figure, #: P < 0.05, ##: P < 0.01, compared with the blank control group; P < 0.05 P<0.01, compared with the ACTH group. Results showed that Y1 cells treated with ACTH synthesized a large amount of corticosterone, with a significant difference compared to the blank control group (P<0.01). Compared with the ACTH model group, the BR061 supernatant + ACTH group significantly inhibited corticosterone synthesis (P<0.01), and the Bb-12 supernatant + ACTH group and the LGG supernatant + ACTH group also inhibited corticosterone synthesis (P<0.05). The BR061 supernatant + ACTH group showed a significant difference in the level of corticosterone inhibition compared to the Bb-12 supernatant + ACTH group and the LGG supernatant + ACTH group (P<0.05).

[0054] Example 2 After being taken out of the -80℃ freezer, the cryovials of Bifidobacterium animalis subsp. lactis BR061 were thawed at room temperature and inoculated into modified MRS medium at 3% (v / v). After anaerobic culture at 37℃ for 24 h, they were inoculated into modified MRS medium again at 3% (v / v) and anaerobic cultured at 37℃ for 18 h. After two culture cycles, activation was completed, and seed culture of Bifidobacterium animalis subsp. lactis BR061 was obtained.

[0055] The dried tangerine peel, poria cocos, malt, hawthorn, and yam were pulverized to 60 mesh and added to distilled water at a ratio of 1g:10mL. The mixture was soaked at room temperature for 30 minutes, extracted in a 100℃ water bath for 60 minutes, filtered through four layers of gauze, centrifuged at 6000rpm for 5 minutes, and the supernatant was collected. The supernatant was then filtered through a 0.22μm filter membrane for sterilization to obtain a sterile extract of dried tangerine peel, poria cocos, malt, hawthorn, and yam. The extract was stored at -20℃ for later use.

[0056] (1) 10% (v / v) of sterile extracts of dried tangerine peel, poria cocos, malt, hawthorn, and yam were added to the sterilized modified MRS medium to obtain fermentation medium for Bifidobacterium animalis subsp. lactis BR061. In the fermentation medium for Bifidobacterium animalis subsp. lactis BR061, 3% (v / v) of seed culture of Bifidobacterium animalis subsp. lactis BR061 was inoculated. After anaerobic culture at 37℃ for 18 h, the pH and OD values ​​of the fermentation broth were measured. 600 The results for the count of viable bacteria are as follows: Table 2. OD value, pH value and viable cell count of fermentation broth in different culture media

[0057] The results showed that, compared with the modified MRS medium, the fermentation medium with added sterile extracts of dried tangerine peel, malt, and hawthorn significantly promoted the growth of Bifidobacterium animalis subsp. lactis BR061 (P<0.05), while the fermentation medium with added sterile extracts of Poria cocos and Dioscorea opposita had no significant effect on the growth of Bifidobacterium animalis subsp. lactis BR061 (P>0.05).

[0058] (2) Orthogonal experiment to screen the addition ratio of sterile extracts of tangerine peel, malt and hawthorn An orthogonal experiment was conducted to add sterile extracts of dried tangerine peel (5%, 10%, 15%), sterile extracts of malt (10%, 20%, 30%), and sterile extracts of hawthorn (10%, 15%, 20%) to the modified MRS medium according to volume ratios. Different proportions of these extracts were added to the modified MRS medium to obtain the fermentation medium for *Bifidobacterium animalis* subsp. *milk* BR061. Seed culture of *Bifidobacterium animalis* subsp. *milk* BR061 was inoculated into the fermentation medium at 3% (v / v). After anaerobic culture at 37℃ for 18 hours, the viable cell count in the fermentation broth was measured. The results are as follows: Table 3. Results of orthogonal experiments on fermentation of Bifidobacterium animalis subsp. lactis BR061

[0059] In the table, mean 1 represents the mean value under the factors of sterile extract of dried tangerine peel (5%), sterile extract of malt (10%), and sterile extract of hawthorn (10%); mean 2 represents the mean value under the factors of sterile extract of dried tangerine peel (10%), sterile extract of malt (20%), and sterile extract of hawthorn (15%); mean 3 represents the mean value under the factors of sterile extract of dried tangerine peel (15%), sterile extract of malt (30%), and sterile extract of hawthorn (20%).

[0060] The results showed that adding 10% (v / v) sterile extract of dried tangerine peel, 15% (v / v) sterile extract of hawthorn, and 20% (v / v) sterile extract of malt to the modified MRS medium had the greatest promoting effect on the growth of Bifidobacterium animalis subsp. lactis BR061. Range analysis showed that the growth-promoting effect on Bifidobacterium animalis subsp. lactis BR061 was: dried tangerine peel > malt > hawthorn.

[0061] Example 3 Preparation method of fermentation broth of Bifidobacterium animalis subsp. lactis BR061: After being removed from the -80℃ freezer, the cryovials of Bifidobacterium animalis subsp. lactis BR061 were thawed at room temperature and inoculated into modified MRS medium at 3% (v / v). The culture was then anaerobically cultured at 37℃ for 18 hours. The culture product was then inoculated into modified MRS medium again at 3% (v / v) and anaerobically cultured at 37℃ for 18 hours. After two culture cycles, the strain was activated, and the seed culture of Bifidobacterium animalis subsp. lactis BR061 was obtained.

[0062] Dried tangerine peel, malt, and hawthorn were pulverized to 60 mesh and added to distilled water at a ratio of 1g:10mL. The mixture was soaked at room temperature for 30 minutes, extracted in a 100℃ water bath for 60 minutes, filtered through four layers of gauze, centrifuged at 6000rpm for 5 minutes, and the supernatant was collected and sterilized by filtration through a 0.22μm filter membrane to obtain sterile extracts of dried tangerine peel, malt, and hawthorn. In a modified MRS medium, 10% (v / v) of the sterile extract of dried tangerine peel, 15% (v / v) of the sterile extract of hawthorn, and 20% (v / v) of the sterile extract of malt were added to obtain a fermentation medium for *Bifidobacterium animalis* subsp. *lactamella* BR061.

[0063] The seed culture of *Bifidobacterium animalis* subsp. *lactospirum* BR061 was inoculated into the fermentation medium of *Bifidobacterium animalis* subsp. *lactospirum* BR061 at 3% (v / v) and anaerobic static culture was carried out at 37℃ for 24 h to obtain the fermentation broth of *Bifidobacterium animalis* subsp. *lactospirum* BR061. The viable count of the fermentation broth was 1.25 × 10⁻⁶. 10 CFU / mL. It can be seen that after adding 10% (v / v) sterile extract of dried tangerine peel, 15% (v / v) sterile extract of hawthorn, and 20% (v / v) sterile extract of malt, the viable count of Bifidobacterium animalis subsp. lactis BR061 in the fermentation broth can reach 10. 10 level.

[0064] Example 4 1. Screening of preservatives for lyophilized Bifidobacterium animalis subsp. lactis BR061: The fermentation broth of *Bifidobacterium animalis* subsp. *lactamase* BR061 prepared in Example 3 was centrifuged at 8000 rpm for 20 min at 4°C, and the supernatant was discarded to obtain *Bifidobacterium animalis* subsp. *lactamase* BR061 bacterial sludge. The preservative and bacterial sludge were thoroughly mixed at a mass ratio of 1:1 to obtain an emulsion. The preservative was compared with the components in the table below to screen for groups with high preservative performance: Table 4 Formulations of different protective agents

[0065] The viable bacteria count of the emulsion was performed, and the same weight of the emulsion was freeze-dried (pre-frozen at -45℃ for 3 h, freeze-dried at -24℃ for 36 h, freeze-dried at 4℃ for 12 h, and desorption dried at 24℃ for 12 h). The viable bacteria count of the freeze-dried powder was also performed, and the freeze-dried weight was recorded. The results are as follows: Table 5. Viable bacterial counts of different protectants

[0066] The results showed that group C protectant had the best protective effect on bacterial cells, with a survival rate of 98.07%. There was no significant difference in the effectiveness of group B and group A protectants. Group D protectant had the worst protective effect, with the lowest bacterial cell survival rate during freeze-drying. Therefore, group C protectant was selected for preparing freeze-dried Bifidobacterium animalis subsp. lactis BR061 powder.

[0067] 2. Preparation of freeze-dried Bifidobacterium animalis subsp. lactis BR061 powder: The fermentation broth of *Bifidobacterium animalis* subsp. lactis BR061 prepared in Example 3 was centrifuged at 8000 rpm for 20 min at 4°C, and the supernatant was discarded to obtain *Bifidobacterium animalis* subsp. lactis BR061 bacterial sludge. A protectant (10% skim milk, 8% trehalose, 5% maltodextrin, 2% sucrose, 0.5% tremella polysaccharide, and 0.5% vitamin C) was thoroughly mixed with the bacterial sludge at a mass ratio of 1:1, and then freeze-dried (pre-frozen at -45°C for 3 h, freeze-dried at -24°C for 36 h, freeze-dried at 4°C for 12 h, and desorption dried at 24°C for 12 h) to obtain *Bifidobacterium animalis* subsp. lactis BR061 freeze-dried powder.

[0068] Electron microscopy was performed on the lyophilized powder of Bifidobacterium animalis subsp. lactis BR061 to detect cell integrity. The results are as follows: Figure 2 As shown: The cell walls of the freeze-dried Bifidobacterium animalis subsp. lactis BR061 were intact, and no large number of cells died. The protectant played a certain role in protecting the cells during the freeze-drying process.

[0069] Example 5 Preparation of postbiotic from Bifidobacterium animalis subsp. lactis BR061: The fermentation broth of Bifidobacterium animalis subsp. lactis BR061 prepared in Example 3 was treated by spray drying: inlet air temperature 111.3℃, feed rate 161.5mL / h, outlet air temperature 60℃, atomizer pressure 0.4MPa, to obtain post-biotic of Bifidobacterium animalis subsp. lactis BR061.

[0070] Experimental Example 2 Effects of different inoculum agents of Bifidobacterium animalis subsp. lactis BR061 on corticosterone synthesis in mouse adrenal cortex cell line Y1: Preparation of BR061 supernatant: After being removed from the -80℃ freezer, cryovials of *Bifidobacterium animalis* subsp. *lactotrium* BR061 were thawed at room temperature and inoculated into modified MRS medium at 3% (v / v). After anaerobic incubation at 37℃ for 24 h, the supernatant was inoculated again at 3% (v / v) into modified MRS medium and anaerobic incubated at 37℃ for 18 h. After two incubation cycles, activation was complete, yielding the *Bifidobacterium animalis* subsp. *lactotrium* BR061 seed culture. The *Bifidobacterium animalis* subsp. *lactotrium* BR061 seed culture was inoculated into modified MRS medium at 3% (v / v) and anaerobic incubated at 37℃ for 24 h. The fermentation broth (viable count 2.9 × 10⁻⁶) was then fermented. 9 The culture supernatant of the strain (CFU / mL) was obtained by centrifugation (8000 rpm, 5 min) using a microcentrifuge, filtered for sterilization, and set aside for later use.

[0071] Preparation of BR061 optimized supernatant: The fermentation broth of Bifidobacterium animalis subsp. lactis BR061 prepared in Example 3 was centrifuged (8000 rpm, 5 min) using a microcentrifuge to obtain the culture supernatant, which was then filtered to remove bacteria and set aside for later use.

[0072] Preparation of BR061 postbiotic solution: The Bifidobacterium lactis subsp. BR061 postbiotic prepared in Example 5 was dissolved in 1 volume of sterile water, thoroughly mixed, and centrifuged (8000 rpm for 20 min) to remove the precipitate. The supernatant was then used for later use.

[0073] Mouse adrenocortical cell line Y1 was cultured in F12 medium (containing 10% fetal bovine serum, 1.176 g NaHCO3, and 100 U / L penicillin and streptomycin) under humidified conditions of 5% CO2 and 37°C. When the cells reached 80% confluence, they were digested with 0.25% mixed trypsin and passaged from one flask to three flasks. Cells in the logarithmic growth phase were used for experiments.

[0074] Y1 cells in logarithmic growth phase were harvested and their concentration adjusted to 1.0 × 10⁻⁶ cells. 5The cells / ml were seeded into 6-well plates, and 1 ml of F12 culture medium was added to each well. After culturing for 24 h, the following were added to each experimental group: (1) Blank control group: 2 ml of F12 culture medium; (2) Adrenocorticotropic hormone (ACTH) group: 2 ml of F12 culture medium containing 1 mL of 10 -9 mol / L ACTH; (3) BR061 supernatant + ACTH group: 2ml F12 culture medium, containing 200μL BR061 supernatant + 1mL 10 -9 mol / L ACTH; (4) BR061 optimized supernatant + ACTH group: 2ml F12 culture medium, containing 200μL BR061 optimized supernatant + 1mL 10 -9 mol / L ACTH; (5) BR061 post-biotic + ACTH group: 2ml F12 culture medium, containing 0.1mg BR061 post-biotic + 1mL 10 -9 The concentration of corticosterone in the cell supernatant was mol / L. The experiment was terminated after 24 h of culture. The cell supernatant was collected in sterile EP container, mixed well, and centrifuged at 2000 rpm for 5 min. The supernatant was then collected, and the corticosterone level in the cell supernatant of each group was measured using a radioimmunoassay kit (purchased from Shanghai ELISA kit). Corticosterone levels are expressed in nmol / L. The experiment was repeated three times.

[0075] Table 6. Effects of different bacterial agents on corticosterone synthesis in mouse adrenal cortical cell line Y1

[0076] The results showed that Y1 cells treated with ACTH could synthesize a large amount of corticosterone, with a significant difference compared with the blank control group (P<0.01). Compared with the ACTH model group, the BR061 supernatant + ACTH group, the BR061 optimized supernatant + ACTH group, and the BR061 postbiotic + ACTH group could significantly inhibit corticosterone synthesis (P<0.01), and the optimized culture medium of Bifidobacterium animalis subsp. lactis BR061 had a more significant inhibitory effect on corticosterone synthesis.

[0077] Experimental Example 3 Non-target metabolomics assay of fermentation broth of Bifidobacterium animalis subsp. lactis BR061: The fermentation broth of *Bifidobacterium lactis* subsp. BR061 obtained in Example 3 was subjected to UHPLC-QE-MS non-target metabolomics analysis, which was assisted by Sangon Biotech (Shanghai) Co., Ltd. The detection process is as follows: (1) Sample processing Transfer 50 μL of sample to an EP tube, add 200 μL of extraction buffer (methanol:acetonitrile = 1:1 (V / V), containing isotope-labeled internal standard mixture), vortex for 30 s; sonicate for 10 min (ice-water bath); let stand at -40℃ for 1 h; centrifuge the sample at 4℃, 12000 rpm (centrifugal force 13800×g, radius 8.6 cm) for 15 min; collect the supernatant in a sample vial for instrumental analysis.

[0078] (2) On-machine testing The target compounds were separated chromatographically using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph (UPLC) with a Waters ACQUITY UPLC BEH Amide (2.1 mm × 100 mm, 1.7 μm) column. Phase A of the HPLC was aqueous, containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia; Phase B was acetonitrile. The sample pan temperature was 4 °C, and the injection volume was 2 μL.

[0079] The Thermo Q Exactive HFX mass spectrometer can perform primary and secondary mass spectrometry data acquisition under the control of software (Xcalibur, Thermo).

[0080] (3) Data processing After the raw data was converted into mz XML format by Proteo Wizard software, peak identification, peak extraction, peak alignment and integration were performed using a self-developed R program package (with XCMS kernel). Then, it was matched with a self-built secondary mass spectrometry database of Biotree DB (V2.1) for material annotation. The cutoff value of the algorithm score was set to 0.3.

[0081] The experimental test sample TIC graph is shown below. Figure 3 As shown.

[0082] The secondary mass spectrum of goose muscle peptide is shown below. Figure 4 As shown, Figure 4 The image shows a secondary mass spectrum acquired in positive ion mode at a collision energy of 10.0 eV. The mass-to-charge ratio of the secondary mass spectrum is in the range of 40–1250 Da. Fragmentation analysis was performed on the parent ion with a mass-to-charge ratio of 241.1 Da at a retention time of 10.472 min.

[0083] The secondary mass spectrum of aspartic acid is shown below. Figure 5 As shown, Figure 5 The image shows a secondary mass spectrum acquired in positive ion mode at a collision energy of 10.0 eV. The mass-to-charge ratio of the secondary mass spectrum is in the range of 40–1250 Da. Fragmentation analysis was performed on the parent ion with a mass-to-charge ratio of 134.0 Da at a retention time of 9.779 min.

[0084] The secondary mass spectrum of γ-aminobutyric acid is as follows: Figure 6 As shown, Figure 6 The image shows a secondary mass spectrum acquired in positive ion mode at a collision energy of 25.0 eV. The mass-to-charge ratio of the secondary mass spectrum is in the range of 50–1000 Da. Fragmentation analysis was performed on the parent ion with a mass-to-charge ratio of 104.1 Da at a retention time of 4.565 min.

[0085] The results showed that the metabolites of Bifidobacterium animalis subsp. lactis BR061 contained γ-aminobutyric acid, aspartic acid, and anserine.

[0086] Test Example 4 The lyophilized powder of *Bifidobacterium lactis* subsp. *Lactobacillus* BR061 prepared in Example 4 and the postbiotic of *Bifidobacterium lactis* subsp. *Lactobacillus* BR061 prepared in Example 5 were used to conduct an experiment on the intervention of *Bifidobacterium lactis* subsp. *Lactobacillus* BR061 in mice to induce depression. Eighty 5-week-old male SPF-grade ICR mice were used in this experiment, with 10 mice in each group, weighing 18-22g. The mice were acclimatized for 7 days to establish a chronic unpredictable stress (CUMS) mouse model for a total of 28 days. The stressors used to construct the CUMS mouse model included: (1) water deprivation for 24 hours; (2) restraint in a restraint container for 2 hours; (3) tail suspension for 1 minute; (4) shaking the cage for 10 minutes; (5) cage tilting at 45˚ for 24 hours; (6) fasting for 24 hours; and (7) keeping the cage moist overnight (200 ml / cage). The mice were subjected to stress treatment with stressors (1) to (7) in sequence (one type per day, 7 days as one round) to construct the CUMS mouse model. The average weight of the control group mice was 27.52g on day 28 of modeling, while the average weight of the CUMS model mice was 24.18g on day 28. The average weight of the model group mice was significantly lower than that of the control group mice, which is consistent with the characteristics of the CUMS mouse model.

[0087] After successful modeling, experiments were conducted. Based on the recommended oral dosage for humans, the daily dose of lyophilized Bifidobacterium animalis subsp. lactis BR061 powder was 5.0 × 10⁻⁶. 9 CFU / day·60kg, and Bifidobacterium animalis subsp. lactis BR061 postbiotic 0.85g / day·60kg. Let the low, medium, and high doses of lyophilized Bifidobacterium animalis subsp. lactis BR061 be 8.35 × 10⁻⁶ CFU / day·60kg, respectively. 6 cfu / animal, 1.67×10 7 cfu / animal and 5.0×10 7CFU / animal, with low, medium, and high doses of Bifidobacterium animalis subsp. lactis BR061 postbiotics at 0.1 g / (kg·bw), 0.2 g / (kg·bw), and 0.6 g / (kg·bw), respectively, equivalent to 5, 10, and 30 times the recommended human dose. The gavage method is as follows: Table 7. Gavage methods for each group of mice

[0088] After 28 days of gavage treatment, the following experiment was conducted: (1) Forced swimming test: The forced swimming test is a classic depression model. The time a mouse remains motionless while floating on the water surface can be used to detect depressive behavior. Before the forced swimming test, mice were placed in a forced swimming tank (water depth 10cm, diameter 16cm, water temperature maintained at 25℃) for 15 minutes of pre-acclimatization swimming. After being dried with a towel, the mice were forced to swim for 6 minutes 24 hours later. The cumulative time the mice remained motionless while floating on the water surface in the last 4 minutes was calculated. Criteria for judging motionless time: The mouse is considered motionless when it stops struggling or is in a floating state and its limbs make slight movements to keep it floating on the water surface.

[0089] (2) Dark and light box experiment: In a quiet environment, the animal is placed in a box with a length, width and height of 80cm, 80cm and 40cm respectively (half of the box is covered as a dark box and half of the box is open as a light box). After the animal is placed in the light box, the measurement begins. Each time is timed for 10 minutes and the total time the animal stays in the light box is recorded. The longer the mouse stays in the light box, the less anxious the mouse is.

[0090] (3) Elevated cross maze experiment: The experimental setup consists of two open arms and two closed arms. The open arms are composed of two platforms with a side length of 100cm each, without guardrails; the closed arms are composed of two platforms with a side length of 100cm each, with 20cm guardrails at the edges. During the test, the test mice are placed in the central area of ​​the cross, and the time the mice spend in the open arms is recorded and observed. The test lasts for 5 minutes.

[0091] Table 8 Results of the forced swimming test, light-dark box test, and elevated cross maze test for each group of mice.

[0092] Note: #: P<0.05, ##: P<0.01, compared with the control group; P < 0.05 P < 0.01, compared with the model group.

[0093] The results showed that in the forced swimming test, the light-dark chamber test, and the elevated cross maze test, the model group and the control group showed significant differences (P<0.01). Compared with the model group, the medium and high dose groups of Bifidobacterium animalis subsp. lactis BR061 lyophilized powder and the medium and high dose groups of postbiotics showed significant differences (P<0.01). The low dose group of Bifidobacterium animalis subsp. lactis BR061 postbiotics showed a difference from the model group (P<0.05). The low dose group of Bifidobacterium animalis subsp. lactis BR061 lyophilized powder only showed a difference in the light-dark chamber test (P<0.05). These three experiments indicate that Bifidobacterium animalis subsp. lactis BR061 has a relieving effect on anxiety in mice.

[0094] (4) One day after all behavioral tests were completed, blood was collected from the eyes of mice 30 minutes after administration. The blood collected from the eyes of mice was left to stand at 4°C for 4 hours and then the serum was separated (speed: 3500r / min, 10min). The serum was aliquoted into 0.5mL centrifuge tubes and stored at -80°C for later use. The cortisol content in the serum was detected using an ELISA kit.

[0095] (5) The mice were euthanized and the stomach and small intestine were immediately dissected. The contents were collected into sterile EP tubes. The digestive enzyme activities in serum, gastric contents and small intestinal contents were measured according to the instructions of the pepsin and trypsin assay kit (purchased from Nanjing Jiancheng Biotechnology Institute).

[0096] Table 9. Results of cortisol, pepsin, and trypsin levels in mice of each group.

[0097] Note: #: P<0.05, ##: P<0.01, compared with the control group; P < 0.05 P < 0.01, compared with the model group.

[0098] The results showed that, compared with the control group, the serum cortisol level in the model group mice was significantly increased (P<0.01), while the pepsin and trypsin levels were significantly decreased (P<0.01). Compared with the model group, the medium and high doses of Bifidobacterium animalis subsp. lactis BR061 lyophilized powder and the medium and high doses of postbiotics significantly reduced the serum cortisol level and significantly increased the pepsin and trypsin levels in mice (P<0.05, P<0.01). During chronic unpredictable stress in mice, cortisol increased and pepsin and trypsin decreased, resulting in a significant difference in body weight between the mice and the control group (P<0.05). After intervention with Bifidobacterium animalis subsp. lactis BR061, the cortisol level in mice decreased, while the pepsin and trypsin levels increased, indicating that this strain can promote the digestive function and nutrient absorption of mice.

[0099] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A subspecies of Bifidobacterium lactis, BR061, characterized in that, The animal Bifidobacterium lactis subspecies ( Bifidobacterium animalis The accession number of subsp.lactis)BR061 is CGMCC No.37152.

2. A method for preparing fermentation broth of Bifidobacterium animalis subsp. lactis BR061, characterized in that, The process includes the following steps: inoculating the animal Bifidobacterium lactis subsp. BR061 as described in claim 1 into a fermentation medium for fermentation to obtain animal Bifidobacterium lactis subsp. BR061 fermentation broth.

3. The preparation method according to claim 2, characterized in that, The fermentation medium is selected from any of the following: (1) Modified MRS medium; (2) A culture medium based on modified MRS medium, with 10% sterile extract of dried tangerine peel, 15% sterile extract of hawthorn and 20% sterile extract of malt added by volume percentage.

4. The fermentation broth of Bifidobacterium lactis subsp. BR061 prepared by the preparation method according to any one of claims 2 to 3.

5. A method for preparing lyophilized powder of Bifidobacterium animalis subsp. lactis BR061, characterized in that, The process includes the following steps: centrifuging the fermentation broth of Bifidobacterium animalis subsp. lactis BR061 as described in claim 4 to obtain Bifidobacterium animalis subsp. lactis BR061 bacterial sludge; The Bifidobacterium lactis subsp. animalis BR061 bacterial sludge was mixed with a preservative and then freeze-dried. The protective agent comprises, by weight percentage: 10% skim milk, 8% trehalose, 5% maltodextrin, 2% sucrose, 0.5% tremella polysaccharide, and 0.5% vitamin C.

6. The freeze-dried powder of Bifidobacterium lactis subsp. BR061 prepared by the preparation method according to claim 5.

7. A method for preparing a postbiotic from Bifidobacterium lactis subspecies BR061, characterized in that, The process includes the following steps: spray drying the fermentation broth of Bifidobacterium lactis subsp. BR061 as described in claim 4.

8. The animal Bifidobacterium lactis subsp. BR061 postbiotic prepared by the preparation method according to claim 7.

9. The use of *Bifidobacterium lactis* subsp. *br061* according to claim 1, or the fermentation broth of *Bifidobacterium lactis* subsp. *br061* according to claim 4, or the freeze-dried powder of *Bifidobacterium lactis* subsp. *br061* according to claim 6, or the postbiotic of *Bifidobacterium lactis* subsp. *br061* according to claim 8, in any of the following, characterized in that, (1) To prepare medicines for regulating mood; (2) To prepare medicines for treating anxiety disorders; (3) Preparation of drugs that inhibit corticosterone synthesis; (4) Prepare health products or medicines that aid digestion; (5) Prepare medicines that promote nutrient absorption.

10. A drug for regulating mood and promoting digestion and absorption, characterized in that, The drug comprises Bifidobacterium lactis subsp. BR061 as described in claim 1, or fermentation broth of Bifidobacterium lactis subsp. BR061 as described in claim 4, or freeze-dried powder of Bifidobacterium lactis subsp. BR061 as described in claim 6, or postbiotic of Bifidobacterium lactis subsp. BR061 as described in claim 8.

Images