Bifidobacterium animalis subsp. Lactis B7 as well as composition and application thereof

Abstract

The invention relates to a bifidobacterium animalis subsp. Lactis B7 as well as a composition and application thereof, and belongs to the technical field of microorganisms, the bifidobacterium animalis subsp. Lactis B7 is preserved in the China Center for Type Culture Collection on April 19, 2024, and the preservation number is CCTCC NO: M2024734. The bifidobacterium animalis subsp. Lactis B7 is named as Bifidobacterium animalis subsp. Lactis B7, the composition is named as Bifidobacterium animalis subsp. Lactis B7, and the composition is named as Bifidobacterium animalis subsp. Lactis B7. The invention also discloses application of the B7 fermentation composition in preparation of medicines for preventing and treating ulcerative colitis, constipation and constipation-type ulcerative colitis. According to the bifidobacterium animalis subsp. Lactis B7, high-yield short-chain fatty acid and indolepropionic acid are achieved, sulforaphane is generated through glucoraphanin, intestinal tract movement is improved, ulcerative colitis or inflammatory bowel disease activity index DAI and weight loss caused by the disease activity index DAI are effectively relieved, mouse colon injury is remarkably relieved, and mucosa layers are protected; the effects of resisting inflammation, inhibiting pathogenic bacteria and improving the intestinal barrier function are achieved.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a strain of Bifidobacterium animalis subsp. Lactobacillus B7, its composition, and its application. Background Technology

[0002] In recent years, with changes in lifestyle and dietary habits, the prevalence of inflammatory bowel disease and constipation has been increasing year by year. Inflammatory bowel disease is an inflammatory gastrointestinal disease characterized by chronicity and relapse. It can be divided into ulcerative colitis (UC) and Crohn's disease (CD). Diarrhea and mucus bloody stools are the main clinical manifestations in the diagnostic criteria for ulcerative colitis both domestically and internationally. A large number of epidemiological and clinical data show that although constipation-type UC is rare, with the increase in the global prevalence of UC, the number of patients with constipation symptoms related to UC has also increased year by year. In a study on bowel-guided pelvic floor behavioral therapy for fecal incontinence and constipation in patients with inflammatory bowel disease, 55% of the 40 patients had constipation, while 45% had fecal incontinence, which is much higher than that of the general population (SPERBERAD, et al. Worldwide Prevalence and Burden of Functional Gastrointestinal Disorders, Results of Rome Foundation Global Study [J]. Gastroenterology, 2021, 160(1): 99-114).

[0003] Recent studies have shown that the pathogenesis of constipation-predominant ulcerative colitis involves genetic susceptibility, gut microbiota dysbiosis, intestinal barrier defects, anorectal dysfunction, individual immunity, environmental factors, and lifestyle habits. In current technologies, drug therapy, such as traditional Chinese medicine, is generally used to treat constipation-predominant ulcerative colitis. CN118717861A discloses a traditional Chinese medicine enema decoction for treating constipation-predominant ulcerative colitis, made from radish seed, immature bitter orange peel, aster root, coptis root, scutellaria root, rehmannia root, and angelica root. This enema decoction can effectively solve the medication problem in treating constipation-predominant ulcerative colitis. In addition, guanidinoyl cyclase C agonists, purcanatae and docanatae, are also used to treat constipation-predominant ulcerative colitis, but these biological agents are expensive, and long-term use can easily cause many adverse reactions in patients.

[0004] In recent years, an increasing number of studies have found that severe dysbiosis of the gut microbiota in patients with constipation-predominant ulcerative colitis can worsen their condition. Furthermore, the use of related medications can disrupt the balance of gut microbiota. Probiotic therapy can directly or indirectly regulate the gut microbiota or exert therapeutic effects through its metabolites. For example, CN119331785A discloses a breast milk-derived *Bifidobacterium lactis* subsp. *mammary* and its applications in relieving ulcerative colitis and constipation. Bifidobacterium animalis subsp.lactisLIHUO 01, this strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 20, 2024, with accession number GDMCC No:65512. This *Bifidobacterium animalis* subspecies *Lactobacillus* LIHUO01 exhibits good in vitro probiotic properties, showing effects in relieving ulcerative colitis, improving constipation, and regulating intestinal flora. It has great application potential in the preparation of foods, health products, or pharmaceuticals for relieving ulcerative colitis, improving constipation, or regulating intestinal flora. However, in existing publicly available technologies, the efficacy targets are unclear, and research focuses solely on constipation or ulcerative colitis. Therefore, developing specific strains or metabiotics of specific strains that can improve constipation-predominant ulcerative colitis is of great significance for the prevention and control of this intestinal disease. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the shortcomings of the prior art and provide a strain of Bifidobacterium animalis subsp. lactis B7 that can produce high levels of sulforaphane and indolepropionic acid, as well as new applications of fermentation compositions of Bifidobacterium animalis subsp. lactis B7. Specifically, it is used in probiotic preparations or drugs for the preparation of sulforaphane or tryptophan metabolites, or for regulating intestinal motility and alleviating ulcerative colitis or inflammatory bowel disease. In particular, it utilizes the Bifidobacterium animalis subsp. lactis B7 strain and its metabolites, including but not limited to sulforaphane and tryptophan metabolites, to improve intestinal barrier function, increase intestinal motility, and prevent and treat ulcerative colitis, especially constipation-type ulcerative colitis.

[0006] One of the technical solutions adopted by this invention to solve its technical problem is: A strain of Bifidobacterium animalis subsp. lactis B7, wherein Bifidobacterium animalis subsp. lactis B7 ( Bifidobacterium animalis subsp.lactis B7 was deposited at the China Center for Type Culture Collection on April 19, 2024, with accession number CCTCC NO:M2024734.

[0007] Bifidobacterium animalis subsp. lactis B7 was isolated from the intestines of healthy infants in Chengdu, Sichuan. The purified strain was identified by 16S rRNA, and the 16S rRNA sequence is shown in SEQ ID NO: 1. The obtained 16S rRNA sequence was compared with that of Bifidobacterium animalis subsp. lactis in Genebank. Bifidobacterium animalis subsp. Lactis With a similarity greater than 99%, this strain can be identified as *Bifidobacterium animalis* subsp. *lactam*. Bifidobacterium animalis subsp.lactis Based on the 16S rRNA gene sequence alignment results Scardovia inopinataA Neighbor-Joining phylogenetic tree constructed using DSM10107 (AB029087) as an outer branch was named Bifidobacterium animalis subsp. B7 (…). Bifidobacterium animalis subsp.lactis B7).

[0008] The colonies formed by *Bifidobacterium lactis* subsp. B7 of this invention on MRS medium are smooth, with intact edges, milky white in color, shiny, and soft in texture, and appear rod-shaped under a microscope. Its optimal growth temperature is 37–46℃, and its optimal growth pH is 3.0–7.0.

[0009] Preferably, the animal Bifidobacterium lactis subspecies B7 produces short-chain fatty acids through fermentation, and uses glucosinolates to synthesize sulforaphane and tryptophan to synthesize indole-3-propionic acid (also known as indolepropionic acid, abbreviated as IPA) and other tryptophan metabolites, thereby increasing intestinal peristalsis and relieving or treating constipation-type ulcerative colitis or inflammatory bowel disease.

[0010] One of the technical solutions adopted by the present invention to further solve its technical problem is: A fermentation composition is prepared by fermentation of the above-mentioned Bifidobacterium animalis subsp. Lactobacillus B7.

[0011] The fermentation composition comprises live and inactivated Bifidobacterium lactis B7, fermentation broth supernatant, fermentation precipitate, and Bifidobacterium lactis B7 post-biotic or spray-dried / lyophilized powder; the fermentation composition is prepared by fermentation of Bifidobacterium lactis B7 in MRS medium, or MRS-GRP medium supplemented with broccoli seed water extract or sulforaphane, or MRS medium supplemented with tryptophan.

[0012] Preferably, the fermentation supernatant of Bifidobacterium animalis subsp. lactis B7 is prepared by fermenting Bifidobacterium animalis subsp. lactis B7 in MRS medium at 37°C for 24 hours, activating it twice, transferring it to fresh MRS medium at a 5% inoculum, culturing it under the same conditions for 12 hours, collecting the bacterial cells by centrifugation, and then inoculating it into MRS medium, or MRS-GRP medium supplemented with broccoli seed water extract or sulforaphane, or MRS medium supplemented with tryptophan at a 5% inoculum, culturing it at 37°C for 24 hours, collecting the fermented milk supernatant by low-temperature centrifugation (10000 r / min, 10 min, 4°C), and preparing the supernatant by high-temperature inactivation (121°C, 15 min), concentration, and spray drying. This metabolite contains active ingredients such as sulforaphane and tryptophan metabolites. MRS medium supplemented with broccoli seed water extract or sulforaphane can promote sulforaphane synthesis during the fermentation of Bifidobacterium lactis B7. MRS medium supplemented with tryptophan produces a large amount of indolepropionic acid during the fermentation of Bifidobacterium lactis B7, thereby further improving intestinal motility, increasing the level of intestinal anti-inflammatory factors, and enhancing anti-inflammatory efficacy.

[0013] Preferably, the post-biotic of Bifidobacterium animalis subsp. lactis B7 is a solid powder containing inactivated Bifidobacterium animalis subsp. lactis B7 cells and its metabolites, which is produced by cell lysis, inactivation, concentration, and spray drying after fermentation culture of Bifidobacterium animalis subsp. lactis B7 in MRS medium, or MRS-GRP medium supplemented with broccoli seed water extract or glucosinolate, or MRS medium supplemented with tryptophan, including dead cells, cell lysates and / or fermentation broth.

[0014] Another technical solution adopted by the present invention to further solve its technical problem is: A probiotic preparation comprising the aforementioned Bifidobacterium animalis subsp. lactis B7 or a fermented composition, wherein the content of Bifidobacterium animalis subsp. lactis B7 in the probiotic preparation is ≥2×10⁻⁶. 7 CFU / g or 2×10 7 CFU / ml.

[0015] Preferably, the probiotic preparation further includes glucosinolates or broccoli seed water extract or indole-3-propionic acid.

[0016] Another technical solution adopted by the present invention to further solve its technical problem is: Application of Bifidobacterium animalis subsp. lactis B7 in the production of sulforaphane and / or tryptophan metabolites. In one exemplary embodiment, the tryptophan metabolite is indole-3-propionic acid.

[0017] The use of Bifidobacterium animalis subsp. lactis B7 or fermentation compositions of Bifidobacterium animalis subsp. lactis B7 in the preparation of products that regulate intestinal flora, promote intestinal peristalsis, enhance immunity, or have anti-inflammatory, antibacterial, or constipation-relieving effects.

[0018] The use of Bifidobacterium animalis subsp. lactis B7 and / or the above-mentioned fermentation composition in the preparation of medicaments for the prevention, relief or treatment of ulcerative colitis, inflammatory bowel disease, constipation and constipation-predominant ulcerative colitis.

[0019] Application of Bifidobacterium animalis subsp. lactis B7 as a starter culture in the preparation of fermented foods such as kimchi and sauerkraut.

[0020] In this application, the amount of live Bifidobacterium animalis subsp. lactis B7 in the composition is 5 × 10⁻⁶. 9 ~2×10 11 CFU / day, the dosage of inactivated Bifidobacterium animalis subsp. lactis B7 is 1×10⁻⁶ CFU / day. 10 ~2×10 11 The daily metabolic rate of *Bifidobacterium animalis* subsp. *lactamase* B7 is 100-1000 mg, and the postbiotic rate is 5.0 × 10⁻⁶ mg / day. 9~1×10 11 Items / day.

[0021] The present invention provides a medicament for the prevention and / or treatment of ulcerative colitis, inflammatory bowel disease, constipation and constipation-predominant ulcerative colitis, the medicament comprising the above-mentioned Bifidobacterium lactis subsp. B7 and / or the above-mentioned fermentation composition.

[0022] The drug achieves its preventive and therapeutic effects on ulcerative colitis, especially constipation-predominant ulcerative colitis, through at least one of the following functions: a) High production of short-chain fatty acids acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and hexanoic acid, improving the level of short-chain fatty acids in metabolites; b) Increase intestinal peristalsis; c) It can alleviate weight loss caused by ulcerative colitis or inflammatory bowel disease, and at the same time alleviate the disease activity index (DAI) of ulcerative colitis or inflammatory bowel disease; d) Significantly reduces colonic damage and protects the mucosal layer; reduces the levels of pro-inflammatory factors IL-6, TNF-α, IL-17, and IL-22 in the serum of patients with colitis, increases the level of anti-inflammatory factor IL-10, and alleviates intestinal inflammation; e) It can significantly reduce LPS and D-LA (D-lactic acid) levels, improve intestinal barrier function, and reduce intestinal permeability; f) It can significantly increase IPA content and increase intestinal homeostasis.

[0023] The drug also includes pharmaceutically acceptable carriers and / or pharmaceutical excipients.

[0024] In one embodiment, the pharmaceutically acceptable carrier includes, but is not limited to, one or more of the following: fillers, binders, wetting agents, disintegrants, or lubricants.

[0025] Preferably, the drug is an oral medication.

[0026] In one embodiment, the filler is one or more of trehalose, chitosan, starch, or dextrin; the binder is one or more of liquid glucose, starch paste, or syrup; the wetting agent is one or more of glycerol or ethanol; the disintegrant is one or more of crospovidone, sodium carboxymethyl starch, or crospovidone carboxymethyl starch; and the lubricant is one or more of magnesium silicate stearate or sodium fumarate stearate.

[0027] The beneficial effects of this invention on Bifidobacterium lactis subspecies B7: This invention relates to *Bifidobacterium lactis* subsp. B7, which exhibits a wide growth temperature range, growing well within 37-46℃ and demonstrating good oxygen tolerance. Under long-term storage at room temperature and accelerated storage at 37℃ for 5 weeks, the survival rate is 52.4%, and the spray-dried bacterial powder prepared from its fermentation broth shows good stability. *Bifidobacterium lactis* subsp. B7 produces high levels of short-chain fatty acids, converting glucosinolates into sulforaphane, increasing intestinal peristalsis. Simultaneously, *Bifidobacterium lactis* subsp. B7 can metabolize tryptophan or broccoli seed water extract to produce high levels of indole-3-propionic acid (IPA), which can alleviate weight loss caused by ulcerative colitis or inflammatory bowel disease, while also reducing the disease activity index (DAI) of ulcerative colitis or inflammatory bowel disease, significantly reducing colonic damage in mice, and protecting the mucosal layer. It also reduces the levels of pro-inflammatory factors IL-6, TNF-α, IL-17, and IL-22 in serum with colitis, while increasing the level of the anti-inflammatory factor IL-10, thus exerting an anti-inflammatory effect. Meanwhile, B7 intervention can significantly reduce LPS and D-LA levels, improve intestinal barrier function, and reduce intestinal permeability, showing great promise in the preparation of drugs for the prevention and / or relief of constipation-predominant ulcerative colitis or inflammatory bowel disease.

[0028] After 12 hours of fermentation, the acid content reaches as high as 0.82g / 100ml, which has the advantages of rapid and sustained acid production, making it an excellent fermentation agent.

[0029] Animal studies have shown that the animal Bifidobacterium lactis subsp. B7 and its postbiotic of the present invention have good safety, good tolerance to gastrointestinal fluids and bile salts, and can be taken orally and used in vivo.

[0030] Bifidobacterium animalis subsp. lactis B7 can efficiently metabolize sulforaphane and synthesize sulforaphane on MRS-GRP medium with a conversion rate as high as 43.42%, and can significantly promote the synthesis of short-chain fatty acids.

[0031] Bifidobacterium animalis subsp. lactis B7 exhibits significant antibacterial activity against common pathogens such as Escherichia coli 8099, Helicobacter pylori ATCC 26695, Streptococcus mutans CGMCC 1.2499, Staphylococcus aureus CMCC 26003, Clostridium perfringens ATCC 13124, and Candida albicans ATCC 10231. In particular, Bifidobacterium animalis subsp. lactis B7 cultured on MRS-GRP medium shows even higher antibacterial activity, indicating a significant synergistic effect between Bifidobacterium animalis subsp. lactis B7 and its metabolite sulforaphane in antibacterial activity.

[0032] Animal experiments have verified that the present invention, either Bifidobacterium lactis subsp. B7 or a mixture of Bifidobacterium lactis subsp. B7 and glucosinolates, can significantly improve constipation in zebrafish; Bifidobacterium lactis subsp. B7 can effectively improve and treat constipation, ulcerative colitis or inflammatory bowel disease.

[0033] Preservation of biological materials Bifidobacterium animalis subspecies B7 ( Bifidobacterium animalis subsp.lactis B7) was deposited at the China Center for Type Culture Collection on April 19, 2024, with accession number CCTCC NO:M2024734, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Attached Figure Description

[0034] Figure 1 —This is a colony diagram of Bifidobacterium lactis subsp. B7, the present invention. Figure 2 —A microscopic morphological image of Bifidobacterium lactis subsp. B7 of the present invention; Figure 3 —This is the phylogenetic tree of Bifidobacterium lactis subspecies B7 of the present invention; Figure 4 —This is a graph showing the growth of Bifidobacterium lactis subsp. B7 and the pH changes in the fermentation broth of the present invention. Figure 5 —This is a diagram showing the acid production of Bifidobacterium lactis subspecies B7, which is the present invention. Figure 6 —This is a graph showing the accelerated stability analysis of viable counts of Bifidobacterium lactis subsp. B7 in animals according to the present invention; Figure 7 —This is a comparative analysis of the body weight of different groups of mice with ulcerative colitis or inflammatory bowel disease according to the present invention; Figure 8 —This is a graph showing the DAI (Disease Activity Index) of inflammatory bowel disease in mice of different ulcerative colitis or inflammatory bowel disease mouse groups within 7 days of modeling. Figure 9 —This is a staining and pathological analysis diagram of mouse colon tissue sections from different groups of mice with ulcerative colitis or inflammatory bowel disease according to the present invention; Figure 10 —This is a comparative analysis of periodic acid Schiff staining and goblet cell counting in colon tissue sections from different groups of mice with ulcerative colitis or inflammatory bowel disease according to the present invention. Among them: (a) periodic acid-Schiff staining image; (b) goblet cell count comparison image; Figure 11 —This is a graph showing the changes in inflammatory and anti-inflammatory factors in mice in different groups of mice with ulcerative colitis or inflammatory bowel disease according to the present invention; Among them: (a) pro-inflammatory factor IL-6; (b) pro-inflammatory factor IL-17; (c) pro-inflammatory factor IL-22; (d) pro-inflammatory factor TNF-α; (e) anti-inflammatory factor IL-10; Figure 12 —This is a graph showing the analysis of serum LPS concentration in mice of different ulcerative colitis or inflammatory bowel disease groups according to the present invention; Figure 13 —This is a graph showing the effect of Bifidobacterium lactis subsp. B7 on D-LA in mouse serum according to the present invention; Figure 14 —This is a graph showing the effect of Bifidobacterium lactis subspecies B7 on IPA in mouse serum.

[0035] in: Figures 4-13 In the middle, ns represents no statistical significance; * represents P <0.05; ** represents P <0.01; *** represents P <0.001; **** represents P <0.0001. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0037] The following are the culture media involved in the embodiments of the present invention: MRS medium (g / L): peptone 10.0, beef meal 5.0, yeast powder 4.0, glucose 20.0, Tween 80 1.0, K2HPO4·7H2O 2.0, anhydrous sodium acetate 5, diammonium citrate 2.0, MgSO4·7H2O 0.2, MnSO4·H2O 0.038 (add 15g / L agar powder for solid medium).

[0038] MRS-GRP medium (g / L): peptone 10.0, beef meal 5.0, yeast powder 4.0, broccoli seed water extract (sulforaphane 13g / 100g) 10.0, Tween 80 1.0, K2HPO4·7H2O 2.0, anhydrous sodium acetate 5, diammonium citrate 2.0, MgSO4·7H2O 0.2, MnSO4·H2O 0.038 (with 15 g / L agar powder for solid medium).

[0039] Example 1 Screening and identification of Bifidobacterium animalis subsp. lactis B7 Bifidobacterium animalis subspecies B7 ( Bifidobacterium animalis subsp.lactis B7 was deposited at the China Center for Type Culture Collection on April 19, 2024, with accession number CCTCC NO:M2024734.

[0040] 1) Screening of Bifidobacterium animalis subsp. lactis B7 strain Samples were collected from the intestines of healthy infants in Chengdu, Sichuan Province. 1g of the collected sample was weighed and placed in 9mL of sterile physiological saline. After thorough mixing by shaking, the sample was serially diluted 10-fold and plated onto MRS solid medium. The culture was then strictly anaerobic at 37℃ for 48 hours. Single colonies of different shapes and sizes were visually observed and streaked for purification at least four times. Microscopic observation and the calcium dissolution method preliminarily identified the bacteria as lactic acid bacteria. The purified strain was stored in 45% glycerol at -80℃ for future use.

[0041] 2) Strain morphology and microscopic observation See the colony morphology of Bifidobacterium animalis subsp. lactis B7 strain. Figure 1 The colonies are smooth with intact edges, milky white in color, shiny, and have a soft texture. See also: [microscopic morphology description]. Figure 2 Bifidobacterium animalis subspecies B7 is rod-shaped.

[0042] 3) Molecular biological identification of strains The purified *Bifidobacterium animalis* subsp. *lactamase* B7 strain was identified using 16S rRNA. The 16S rRNA sequence of this strain is shown in SEQ ID NO: 1. The obtained 16S rRNA sequence was compared with *Bifidobacterium animalis* subsp. *lactamase* in Genebank using NCBIBLAST. Bifidobacterium animalis subsp.lactis With a similarity greater than 99%, this strain can be identified as *Bifidobacterium animalis* subsp. *lactam*. Bifidobacterium animalis subsp.lactis Based on the 16S rRNA gene sequence alignment results Scardovia inopinata The Neighbor-Joining phylogenetic tree constructed using DSM 10107 (AB029087) as an outbranch is shown below. Figure 3 As shown, it is named Bifidobacterium animalis subspecies B7 (… Bifidobacterium animalis subsp.lactis B7).

[0043] 4) Fermentation substrate utilization capacity Freshly cultured Bifidobacterium lactis subsp. B7 from the test animal was punctured and inoculated into the culture medium, or freshly cultured bacterial solution was added dropwise to a melted soft agar column (temperature 47±1℃). After mixing, a layer of 2% agar about 7 mm thick was added on top, and the culture was placed at 37℃ for anaerobic incubation for 48 h. The change in the culture medium from purple to yellow indicated that the strain was using a carbon source to produce acid.

[0044] The experimental results are shown in Table 1. The animal Bifidobacterium lactis subspecies B7 strain can ferment and produce acid using carbon source substrates such as glucose, aesculin, maltose, melibiose, sucrose, raffinose, 2-fucosylated lactose, and lactose-N-neotetraose. Among them, 2-fucosylated lactose and lactose-N-neotetraose are human milk oligosaccharides.

[0045] Table 1. Physiological and biochemical characteristics of Bifidobacterium animalis subspecies B7 – Acid production using carbon sources

[0046] 5) Growth performance testing Bifidobacterium animalis subsp. lactis B7 strain was inoculated into MRS liquid medium at a 5% inoculum and activated at 37°C for 24 hours, repeated twice. The activated B7 culture was then inoculated into MRS liquid medium at a 5% inoculum, mixed thoroughly, and dispensed into sterile test tubes (18mm × 180mm) at 8ml / tube. The dispensed cultures were placed in a 37°C incubator for static incubation, and the absorbance (OD) of three tubes was measured. 600 Calculate the average value; at regular intervals, take 3 test tubes to measure the total acidity and absorbance (OD) of the bacterial culture. 600 And plot the growth curve.

[0047] A line graph was plotted based on the growth performance data of strain Bifidobacterium animalis subsp. lactis B7, as shown below. Figure 4 As shown, within 4 hours of incubation, OD 600 No significant increase was observed in either OD value, and pH showed no significant change; after 4-12 h of incubation, OD... 600 It exhibits logarithmic growth, with a rapid decrease in pH value, and OD values ​​increase after 12-24 hours of incubation. 600 The pH was maintained at approximately 1.85 ± 0.05 and 4.71 ± 0.05. Based on the growth curve, the optimal growth time for strain B7 was preliminarily determined to be 12 hours.

[0048] 6) Determination of acid production performance of the strain Bifidobacterium animalis subsp. lactis B7 strain was inoculated into MRS medium at a 5% inoculum and activated at 37°C for 24 hours, with activation repeated twice. The activated B7 culture was then inoculated into MRS and MRS-GRP liquid media at a 5% inoculum, mixed thoroughly, and dispensed into sterile test tubes (18mm × 180mm) at 8ml / tube. The dispensed B7 culture was placed in a 37°C incubator for static incubation, and three tubes were used to determine the total acid content at 8h, 10h, 12h, and 14h, and the short-chain fatty acid content was determined by HPLC over 24 hours.

[0049] The results of acid production by Bifidobacterium animalis subsp. lactis B7 are shown in Table 2 and Figure 5At culture times of 8, 10, 12, and 14 hours, the acid production on MRS medium was 0.37 g / 100 ml, 0.58 g / 100 ml, 0.82 g / 100 ml, and 0.84 g / 100 ml, respectively, while the acid production on MRS-GRP medium was 0.32 g / 100 ml, 0.54 g / 100 ml, 0.87 g / 100 ml, and 0.91 g / 100 ml, respectively. From 8 to 10 hours, B7 produced acid faster on MRS medium, while from 10 to 12 hours, B7 produced acid faster on MRS-GRP medium. The highest acid production was observed at 14 hours on both MRS medium and MRS-GRP medium.

[0050] Table 2. Analysis of acid-producing performance of Bifidobacterium animalis subsp. Lactobacillus B7

[0051] Short-chain fatty acids (SCFAs), also known as volatile fatty acids (VFAs), are important metabolic products of beneficial bacteria in the gut. Common examples include acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and hexanoic acid. They have been shown to have a good effect on intestinal immunity, constipation improvement, and inflammation regulation.

[0052] The results of the determination of short-chain fatty acid production by Bifidobacterium animalis subsp. lactis B7 are shown in Table 3. Among them, in MRS-GRP culture, acetic acid increased by 37.72%, propionic acid by 95.15%, isobutyric acid by 152.70%, butyric acid by 83.33%, isovaleric acid by 71.74%, valeric acid by 236.36%, and hexanoic acid by 50.77%, for a total increase of 38.03%. The results indicate that Bifidobacterium animalis subsp. lactis B7 can significantly increase the content of short-chain fatty acids in MRS-GRP culture.

[0053] Table 3. Detection and analysis of short-chain fatty acids produced by Bifidobacterium animalis subsp. Lactobacillus B7

[0054] Note: The quantitative unit for short-chain fatty acids is μg / ml.

[0055] 7) Determination of the oxygen tolerance of the strain Bifidobacterium animalis subsp. lactis B7 strain was inoculated into MRS liquid medium at a 5% inoculum and activated at 37°C for 24 hours, with the activation process repeated twice. The activated B7 culture was then inoculated into MRS liquid medium test tubes (18mm × 180mm) at a 5% inoculum. The inoculated cultures were then placed in a 37°C incubator (21% oxygen) and a 37°C anaerobic incubator (0% oxygen) for 12 hours, respectively, and the OD (exponential growth rate) of the cultures was measured. 600 .

[0056] The oxygen tolerance of Bifidobacterium animalis subsp. lactis B7 directly affects the production cost and manufacturing cost of the bacterial powder. Experimental results showed that the OD of B7 after 12 hours of incubation at 37℃ (oxygen content 21%) was significantly reduced. 600 OD values ​​were obtained by culturing at 1.982°C and 37°C in an anaerobic incubator (0% oxygen) for 12 hours. 600 The value is 1.839, therefore, the growth of strain Bifidobacterium animalis subsp. lactis B7 is less affected by oxygen content, and this strain has excellent processing characteristics.

[0057] 8) Determination of the temperature resistance of the strain Bifidobacterium animalis subsp. lactis B7 strain was inoculated into MRS liquid medium at a 5% inoculum and activated at 37°C for 24 hours, with the activation process repeated twice. The activated B7 bacterial solution was then inoculated into MRS liquid medium test tubes (18mm × 180mm) at a 5% inoculum. The inoculated bacterial solutions were then placed in anaerobic incubators at 37°C, 40°C, 43°C, and 46°C for 12 hours each, and the OD (octane rating) of the bacterial solutions was measured. 600 .

[0058] The temperature tolerance of Bifidobacterium animalis subsp. lactis B7 directly affects the selection of the bacterial powder manufacturing process and the production cost. The experimental results (Table 4) show that B7 can grow well at 37℃, 40℃, 43℃ and 46℃. Therefore, the growth temperature of strain B7 is wide and the strain has excellent temperature tolerance. The bacterial powder can be prepared by spray drying or freeze drying.

[0059] Table 4. Temperature resistance test of Bifidobacterium lactis subsp. B7

[0060] Example 2 Stability test of Bifidobacterium animalis subsp. lactis B7 powder a) Third-generation activation culture Bifidobacterium animalis subsp. lactis B7 strain was inoculated into MRS liquid medium at a 5% inoculum and activated at 37°C for 48 hours, with the activation process repeated twice. The activated B7 culture was then inoculated into 100 ml of MRS liquid medium at a 5% inoculum. The inoculated cultures were then placed in an anaerobic incubator at 37°C and incubated for 12 hours. The OD (octane rating) of the cultures was measured. 600 That is, to produce the second-generation B7 strain.

[0061] Under a sterile operating table, take about 100ml of second-generation B7 bacteria and inoculate it into 2000ml of sterilized culture medium at a temperature of 37±2℃, and shake to mix evenly.

[0062] Place the conical flask containing the second-generation B7 culture medium into a constant temperature incubator at 37±2℃ and incubate statically for 10±2 hours until OD is reached. 600 The incubation period is >1 hour. Place the cultured third-generation B7 strain in a refrigerator at 4°C or below for up to 3 days.

[0063] b) Fermentation and centrifugation Approximately 2000 ml of third-generation activated culture was transferred to a cleaned and sterilized fermenter. The fermenter temperature was controlled at 37±2℃. The mixture was stirred for 5 minutes and allowed to ferment statically for 10±2 hours. When the OD of the fermentation broth... 600 When the total acid content is >2 and the total acid content is >0.8%, the bacterial cells are obtained by centrifugation and then transferred for emulsification and encapsulation.

[0064] C) Emulsification and Encapsulation The centrifuged bacterial cells were dispersed and emulsified for 1 hour with a pre-prepared solution of maltodextrin, trehalose, and skim milk powder (1:1:1). The pH of the emulsion was then adjusted to 5.0-6.0 with sterile calcium hydroxide or sodium hydroxide before being subjected to low-temperature vacuum spray drying.

[0065] d) Powder preparation (1) Low-temperature vacuum spray drying During the spray drying process, the inlet air temperature is automatically controlled between 100-120℃, and the outlet air temperature is automatically controlled between 50-70℃ to carry out low-temperature spray drying.

[0066] (2) Screening, packaging and testing After being spray-dried, B7 is sieved through a 50-mesh rotary vibrating screen and stored in a stainless steel storage tank. Material that cannot pass through the vibrating screen is crushed by a pulverizer and then loaded into the stainless steel storage tank. The B7 in the stainless steel storage tank is then pumped into a bagging machine and fed into an automatic packaging machine through a pipeline. Each bag is automatically weighed at 1 kg, heat-sealed, and coded before being sealed and conveyed to the finished product temporary storage room for sampling and testing. The finished products must be white or off-white in appearance, odorless, have a bacterial count of not less than 150 billion CFU / g, not settle in the dispersibility and solubility test, and have a moisture content of less than 5%.

[0067] e) Stability testing The B7 bacterial powder was divided into 100g bags and placed in a temperature of 37±2℃, relative humidity of RH75±5%, and protected from direct sunlight to complete the stability test.

[0068] The experimental results are shown in Figure 6 The initial viable count of Bifidobacterium animalis subsp. lactis B7 was 1.5 × 10⁻⁶. 11The survival rate of *Bifidobacterium animalis* powder (cfu / g), stored at 37℃ and RH 75±5% for 5 weeks was 52.4%, while the survival rate of commercially available freeze-dried *Bifidobacterium animalis* powder stored at 37℃ and RH 75±5% for 1 week was only about 50%. Therefore, *Bifidobacterium animalis* subsp. lactis B7 powder (prepared by low-temperature spray drying process) has better stability and can meet the requirements of room temperature storage and various processing scenarios with high stability requirements, ensuring the required live bacteria count during the product's shelf life.

[0069] Example 3 Tests on human gastrointestinal tolerance and bile salt tolerance of Bifidobacterium animalis subsp. lactis B7. 1) Activation and culture of the strain In a sterile laminar flow hood, the preserved Bifidobacterium animalis subsp. lactis B7 was inoculated into a centrifuge tube containing 10 mL of MRS liquid medium and incubated at 37°C for 12 hours. The cultured bacterial solution was turbid with a white precipitate.

[0070] 2) Tolerance of Bifidobacterium animalis subsp. lactis B7 to gastric and intestinal juices. Preparation of electrolyte solution A: Weigh 0.064 g potassium chloride, 0.015 g potassium dihydrogen phosphate, 0.263 g sodium bicarbonate, 0.345 g sodium chloride, 0.003 g magnesium chloride hexahydrate, 0.006 g ammonium carbonate, 1.5 g tryptone, and 0.05 g L-cysteine ​​hydrochloride monohydrate. Add 95 mL of distilled water and dissolve thoroughly. Adjust the pH to 3.0 with concentrated hydrochloric acid or 1 mol / L sodium hydroxide solution. Make up to 100 mL and autoclave at 121 ℃ for 15 min.

[0071] Preparation of electrolyte solution B: Weigh 0.022 g of calcium chloride dihydrate, dissolve in water and bring the volume to 100 mL, then autoclave at 121 °C for 15 min.

[0072] Take 8.0 mL of electrolyte solution A and 1.0 mL of electrolyte solution B in a beaker, add the equivalent of 4000 U of pepsin, and conduct tests according to the principles of standard simulated gastric juice (pH 3.0, simulated gastric juice treatment time 2.0 h), fasting simulated gastric juice (pH 2.0, simulated gastric juice treatment time 0.5 h), and satiated simulated gastric juice (pH 4.0, simulated gastric juice treatment time 3 h). Adjust the pH value using 1 mol / L hydrochloric acid solution or 1 mol / L sodium hydroxide solution, and make up to 10 mL. After mixing, filter through a 0.22 μm sterile filter membrane to prepare simulated gastric juice. Prepare and use immediately.

[0073] Preparation of simulated gastric juice: NaCl 2.0 g / L, pH value adjusted to 2.0, 2.5, 3.0 and 4.0 respectively with HCl, autoclaved, pepsin 3.2 g / L, pepsin added fresh for use during the experiment.

[0074] Preparation of simulated intestinal fluid: 6.8 g / L potassium dihydrogen phosphate, pH adjusted to 7.5 with NaOH, autoclaved, 10.0 g / L trypsin, trypsin added fresh for use during the experiment.

[0075] Aseptically weigh 1.0 g of sample and place it in a sterile homogenizer bag containing 99 mL of dissolving solution (8.5 g of sodium chloride and 0.5 g of L-cysteine ​​hydrochloride monohydrate, dissolved in 1000 mL of distilled water, aliquoted, and autoclaved at 121 °C for 15 min). Mix thoroughly and then homogenize at 10 times / second for 2 min until the sample is evenly dispersed. Prepare the bacterial suspension immediately. Measure the total viable count of the bacterial suspension and record it as N1. Take 5.0 mL of simulated gastric fluid and 5.0 mL of the bacterial suspension and place them in a 50 mL centrifuge tube. Vortex to mix. Slowly add approximately 5 mL of mineral oil above the solution using a dropper to form an oil seal.

[0076] Under simulated gastric fluid conditions, centrifuge tubes were incubated in a 37°C water bath for the specified time. Mineral oil was removed using a dropper, and the total viable count was determined according to the viable bacteria assay method, and recorded as follows: Repeat the gastric juice tolerance test twice using the method described above, and record the result of the total viable bacterial count as follows: and The results of repeated determinations of the total number of viable bacteria in simulated gastric juice are recorded as follows: and .calculate , , and , and The relative average deviation of the total viable bacteria count results should not exceed 15% between the two sets of data; otherwise, the gastric juice tolerance test should be repeated.

[0077] Gastric juice tolerance is calculated according to formula (1): …………………(1) In the formula: A —Gastric acid tolerance, expressed as a percentage (%); —The initial total viable bacterial count for the first gastric juice tolerance test, in CFU / mL; —The initial total viable bacterial count of the second gastric juice tolerance test, in CFU / mL; —The initial total viable bacterial count of the third gastric juice tolerance test, in CFU / mL; —Total number of viable bacteria after the first gastric juice tolerance test, in CFU / mL; — Total viable bacteria count after the second gastric juice tolerance test, in CFU / mL; —Total viable bacteria count after the third gastric juice tolerance test, in CFU / mL; 2 —Dilution factor conversion factor; 1 / 3 —The average of three parallel data points; The calculation result is expressed as an integer.

[0078] The following simulated gastric juices were used: standard simulated gastric juice (pH 3.0, simulated gastric juice treatment time 2.0 h), fasting simulated gastric juice (pH 2.0, simulated gastric juice treatment time 0.5 h), and satiety simulated gastric juice (pH 4.0, simulated gastric juice treatment time 3 h). Table 5 shows the gastric juice tolerance data for *Bifidobacterium animalis* subsp. *lactospirum* B7. At pH 2.0, the survival rate of B7 was 75.00%; at pH 3.0, the survival rate was 97.73%; and at pH 4.0, the survival rate was 100.00%. Therefore, *Bifidobacterium animalis* subsp. *lactospirum* B7 exhibits excellent gastric juice tolerance.

[0079] Table 5. Gastric acid tolerance of Bifidobacterium lactis subsp. B7

[0080] 3) Tolerance of Bifidobacterium animalis subsp. lactis B7 to bile salts Bifidobacterium animalis subsp. lactis B7 strain was inoculated into MRS liquid medium at a 5% inoculum and activated at 37°C for 24 hours, with activation repeated twice. The activated B7 bacterial culture was then inoculated into MRS liquid medium at a 5% inoculum and incubated statically at 37°C for 12 hours in an anaerobic constant-temperature incubator. The cultured bacterial culture was collected by centrifugation at 5000 rpm for 10 minutes, and the cells were shaken thoroughly with sterile physiological saline.

[0081] Add 10% of the well-shaken bacterial suspension to MRS medium with bile salt concentrations of 1.0 g / L, 2.0 g / L, 3.0 g / L, and 0.0 g / L (initial bacterial suspension), with a bile salt concentration of 0.0 g / L serving as the control group. Incubate at 37°C for 3 hours. After incubation, remove the bacterial suspension and immediately dilute it 10-fold with sterile physiological saline. Mix thoroughly by tapping and then test for B7. Count the viable bacteria detected and calculate the survival rate using the following formula: Strain survival rate (%) = experimental group / control group × 100%.

[0082] Table 6 shows the bile salt tolerance data of Bifidobacterium animalis subsp. lactis B7: When the bile salt concentration was 1.0 g / L and 2.0 g / L, the survival rates of strain B7 were 98.25% and 90.13%, respectively. However, when the bile salt concentration reached 3.0 g / L, the survival rate of strain B7 still reached 82.45%. The fact that the bile salt concentration in the intestine did not exceed 3.0 g / L indicates that strain B7 can tolerate bile salts in the intestine.

[0083] Table 6. Bile salt tolerance of Bifidobacterium animalis subsp. lactis B7

[0084] In summary, the Bifidobacterium lactis subsp. B7 strain screened in this invention exhibits good tolerance to gastric acid and bile salts, and can exert its role in regulating the human gut microbiota and improving intestinal inflammation through the gastrointestinal tract.

[0085] Example 4 Determination of the metabolism of sulforaphane and indole-3-propionic acid by Bifidobacterium animalis subsp. lactis B7 1) Determination of sulforaphane produced from the metabolism of glucoraphane by Bifidobacterium animalis subsp. lactis B7 Bifidobacterium animalis subsp. lactis B7 strain was inoculated into MRS liquid medium at a 5% inoculum and activated at 37°C for 24 hours, with this activation process repeated twice. The activated B7 culture was then inoculated into MRS-GRP medium at a 5% inoculum and cultured for 12 hours. The ability of Bifidobacterium animalis subsp. lactis B7 to convert sulforaphane into sulforaphane was determined by analyzing the concentrations of glucoraphane sulforaphane (GRA) and sulforaphane sulfonamide (SFN) in the fermentation supernatant. The conversion efficiency of sulforaphane sulfonamide was calculated using uninoculated culture medium as a control.

[0086] Conversion rate (%) = Sulforaphane content at the end of fermentation / Initial sulforaphane content × 100%.

[0087] Sulforaphane is an active product of sulforaphane in vivo after hydrolysis by myrosinase or intestinal flora. Sulforaphane can increase intestinal peristalsis and regulate intestinal flora. The experimental results are shown in Table 7 below.

[0088] Table 7. Content of glucoraphane and sulforaphane in the supernatant of Bifidobacterium animalis subsp. lactis B7 fermentation broth

[0089] Experimental results showed that after culturing Bifidobacterium animalis subsp. lactis B7 in MRS-GRP medium for 12 hours, the conversion efficiency of sulforaphane to sulforaphane was 43.42%. Therefore, Bifidobacterium animalis subsp. lactis B7 can be used in combination with sulforaphane to develop probiotic preparations and other products, thereby improving the conversion efficiency of sulforaphane in vivo.

[0090] 2) Determination of high indolepropionic acid production by Bifidobacterium animalis subsp. lactis B7 After activating Bifidobacterium animalis subsp. lactis B7 twice on MRS medium, the activated B7 bacterial suspension was inoculated at a 5% inoculum into MRS and MRS-GRP media with different tryptophan concentrations (0.1 g / L, 0.2 g / L, and 0.3 g / L). After thorough mixing, the suspension was dispensed into sterile test tubes (18 × 180 mm) at 8 ml / tube. The dispensed candidate bacterial suspensions were then incubated at 37°C. The indolepropionic acid (IPA) content was determined using a microplate reader, as follows: The method for the microbial indolepropionic acid ELISA detection kit is as follows: First, remove the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes. Seal the remaining strips in a resealable bag and return them to 4°C. Set up standard wells and sample wells. Add 50 μL of different concentrations of standard to each standard well. Add 10 μL of the sample to be tested to each sample well, followed by 40 μL of sample diluent. Do not add any reagents to the blank wells. Except for the blank wells, add horseradish peroxidase (HRP)-labeled detection antibody to each standard and sample well. Add 100 μL of the solution to each well, seal the reaction wells with a sealing film, and incubate at 37°C in a water bath or incubator for 60 min. Discard the liquid, pat dry on absorbent paper, add washing buffer to each well, let stand for 1 min, discard the washing buffer, pat dry on absorbent paper, and repeat this washing process 5 times (or a plate washer can be used). Add 50 μL each of substrate A and B to each well, and incubate at 37°C in the dark for 15 min. Add 50 μL of stop solution to each well, and within 15 min, measure the OD value of each well at a wavelength of 450 nm.

[0091] Standard curve and sample determination Using the IPA standards (S0-S5) from the kit, at concentrations of 0, 3, 6, 12, 24, and 48 nmol / L (0.0000, 0.0006, 0.0011, 0.0023, 0.0045, and 0.0091 µg / g), the indolepropionic acid ELISA assay was performed according to the method described in the kit. In an Excel worksheet, a linear regression curve was plotted with OD values ​​on the x-axis and sample concentrations on the y-axis. The concentration values ​​for each sample were calculated using the curve equation. The results were used to plot the IPA standard curve, and the fitted formula was y = 0.0016 × 2 + 0.0019 × - 0.0001, R 2 = 0.9968.

[0092] For sample determination, the fermentation broth was centrifuged at 7000 r / min for 10 min, and the supernatant was collected. For MRS, 1 mL of the supernatant was diluted with water to 25 mL, and for MRS-GRP, 1 mL of the supernatant was diluted with water to 30 mL.

[0093] Table 8. IPA content of Bifidobacterium animalis subsp. lactis B7 in culture media with different tryptophan contents.

[0094] Note: IPA content is calculated by multiplying the result from the IPA standard curve by the dilution factor. Table 8 shows the results of IPA production by *Bifidobacterium animalis* subsp. *mammary* B7 in media with different tryptophan concentrations. When the tryptophan concentration was 0.2 g / L, the IPA concentrations in MRS and MRS-GRP media reached 0.2123 µg / g and 0.2683 µg / g, respectively. Further increasing the tryptophan concentration resulted in a gradual increase in IPA concentration. When the tryptophan concentrations were 0.1 g / L, 0.2 g / L, and 0.3 g / L, the IPA concentrations in MRS-GRP media were 21.69%, 26.38%, and 28.12% higher than those in MRS media, respectively. This indicates that *Bifidobacterium animalis* subsp. *mammary* B7 can metabolize tryptophan to produce high levels of indolepropionic acid. Furthermore, the higher IPA concentration in MRS-GRP media suggests that MRS-GRP media can synergistically promote the metabolism of tryptophan by B7 to produce indolepropionic acid.

[0095] 3) Antibacterial experiment of fermentation broth of Bifidobacterium animalis subsp. lactis B7 Pathogenic bacteria inhibition test: Pour 10 mL of water agar medium into a sterile plate. After cooling and solidification, place an Oxford cup on top. Add the indicator bacterial suspensions (Escherichia coli 8099, Helicobacter pylori ATCC 26695, Streptococcus mutans CGMCC 1.2499, Staphylococcus aureus CMCC 26003, Clostridium perfringens ATCC 13124, Candida albicans ATCC 10231) to the corresponding agar medium for the growth of the indicator bacteria, cooled to 50°C, to make the indicator bacterial concentration 10. 6 Mix CFU / mL, pour onto the bottom layer of water agar, and allow to solidify. Remove the Oxford cup with tweezers to create wells. Add 200 μL of the test sample (including *Bifidobacterium lactis* B7 fermentation broth on MRS medium and *Bifidobacterium lactis* B7 fermentation broth on MRS-GRP medium) to each well. Use uninoculated MRS and MRS-GRP medium as controls for *E. coli* 8099. Diffusion for 30 min, incubate at 37℃ for 15-24 h. Observe whether an inhibition zone appears around the wells and measure its diameter with calipers. Record the diameter of the inhibition zone. Finally, evaluate the antibacterial activity based on the presence and size of the inhibition zone.

[0096] Table 9. Analysis of the antibacterial activity of B7 fermentation broth obtained from different culture media.

[0097] The antibacterial activity of *Bifidobacterium animalis* subsp. *lactamase* B7 fermentation broth obtained from different culture media is shown in Table 9. Compared with the fermentation supernatant of *Bifidobacterium animalis* subsp. *lactamase* B7 obtained from MRS medium fermentation, the fermentation broth of *Bifidobacterium animalis* subsp. *lactamase* B7 obtained from MRS-GRP medium fermentation showed significantly improved antibacterial activity against pathogenic microorganisms, including *Escherichia coli* 8099, *Helicobacter pylori* ATCC 26695, *Streptococcus mutans* CGMCC 1.2499, *Staphylococcus aureus* CMCC 26003, *Clostridium perfringens* ATCC 13124, and *Candida albicans* ATCC. The inhibition zones of 10231 all reached over 21 mm, indicating a 49% improvement in antibacterial performance against Candida albicans. In contrast, no inhibition zones were observed in the control group on MRS or MRS-GRP media. However, after fermentation with Bifidobacterium lactis B7, the inhibition zones significantly increased. Combined with the aforementioned conclusion that Bifidobacterium lactis B7 can convert sulforaphane into sulforaphane, this suggests that the main reason for the significant increase in antibacterial activity of Bifidobacterium lactis B7 in MRS-GRP media is the production of sulforaphane in the fermentation broth. The synergistic effect of sulforaphane and Bifidobacterium lactis B7 enhances the inhibitory effect against pathogens.

[0098] Table 10. Analysis of the antibacterial activity of B7 fermentation broth obtained from different culture media with added tryptophan.

[0099] Table 10 shows that with the addition of 0.2 g / L tryptophan, the inhibition zones of Bifidobacterium animalis subsp. lactis B7 on both MRS and MRS-GRP media were larger than those without tryptophan. The fermentation broth on MRS-GRP medium with added tryptophan showed the best antibacterial activity. Combined with the increased indolepropionic acid content in MRS and MRS-GRP media with added tryptophan, and the fact that it can metabolize and synthesize sulforaphane in MRS-GRP medium, and that indolepropionic acid has good antibacterial properties, this indicates that the indolepropionic acid or sulforaphane produced by the fermentation metabolism of Bifidobacterium animalis subsp. lactis B7 has a synergistic effect on the antibacterial activity of the strain itself.

[0100] Example 5 Effects of Bifidobacterium animalis subsp. lactis B7 and sulforaphane on constipation Bifidobacterium animalis subsp. lactis B7 strain was inoculated into MRS medium at a 5% inoculum and activated anaerobically at 37°C for 24 hours. This activation was repeated twice before transferring the culture to MRS medium and incubating for another 24 hours. The fermentation broth was centrifuged at 8000 rpm for 5 minutes at 4°C. Cells were collected, divided into two aliquots, and one aliquot was washed twice with sterile PBS buffer and resuspended. The final bacterial concentration was 10-1. 8 CFU / ml, one sample was washed twice with sterile PBS buffer and resuspended in PBS buffer containing sulforaphane, with a final bacterial concentration of 10. 8 CFU / ml, glucoraphane sulfadiazine (GRA) final concentration 0.5 g / L, stored at 4℃.

[0101] Zebrafish processing: AB zebrafish embryos were purchased from the Zebrafish Platform of Zhejiang University School of Medicine. The culture conditions for the zebrafish were as follows: The electrical conductivity of water is 500-550 μS / cm. -1 The culture conditions were as follows: pH 7.20-7.60, temperature 28℃, with a culture density of 50 embryos per sterile culture dish. The culture medium was sterile E3 (1% NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM magnesium sulfate, and 0.00003% methylene blue, pH 7.00). 75% of the sterile E3 medium was changed daily to remove accumulated waste and increase dissolved oxygen. On day 5 post-fertilization, the juvenile fish were transferred to 6-well plates and cultured at a density of 20 fish per well.

[0102] Experimental design: Zebrafish with a 5 dpf were selected as experimental subjects and divided into 5 groups, with 15 fish in each group. The control group consisted of a normal control group on E3 culture medium. The other four groups used the loperamide hydrochloride method (loperamide hydrochloride concentration 10 μg / mL) to construct a zebrafish intestinal peristalsis model. These groups were then treated with a positive control or different bacterial solutions. The four groups were: the loperamide hydrochloride model group (LOP), the LOP + positive control group (AC group, domperidone AC, 50 μg / mL, H10910003, Xian Janssen Pharmaceutical Co., Ltd.), the LOP + B7 group (B7 group), and the LOP + B7 + glucosinolate group (B7 + GRA group). After soaking constipated zebrafish for 24 hours, they were anesthetized with an anesthetic. The time difference between two consecutive peristaltic peaks at the same location in the zebrafish intestine was observed under an integrated microscope. One-way ANOVA was performed using GraphPad Prism 9.5 software. Data are expressed as mean ± standard deviation. P<0.05 This indicates that it is statistically significant.

[0103] The experimental results are shown in Table 11. The time difference between the second peak of intestinal peristalsis in the loperamide hydrochloride model group (LOP) was higher than that in the control group, indicating successful constipation modeling. The time difference between the second peak of intestinal peristalsis in the positive control group (AC) and the probiotic B7 group was significantly lower than that in the model group (LOP), indicating a promoting effect on intestinal peristalsis. The positive control group showed the best effect, followed by Bifidobacterium animalis subsp. lactis B7+ GRA. P <0.01), Bifidobacterium animalis subsp. B7 ( P <0.01) showed the weakest effect in promoting intestinal peristalsis, with Bifidobacterium animalis subsp. lactis B7+GRA being superior to Bifidobacterium animalis subsp. lactis B7 alone. These results indicate that probiotic B7 and sulforaphane GRA can synergistically and significantly increase intestinal peristalsis, providing good relief for constipation.

[0104] Table 11 Effects of Bifidobacterium lactis subsp. B7 and glucosinolates on intestinal motility

[0105] Note: * indicates P <0.05, statistically significant difference, ** is P <0.01, extremely significant difference (compared to the model group) Example 6: Analysis of the effects of Bifidobacterium animalis subsp. lactis B7 on ulcerative colitis or inflammatory bowel disease. The Bifidobacterium lactis subsp. B7 bacterial powder used in this embodiment has a viable count of 1×10⁻⁶. 11CFU / g. The *Bifidobacterium animalis* subsp. lactis B7 bacterial powder was prepared by the following method: *Bifidobacterium animalis* subsp. lactis B7 strain was inoculated into MRS medium at an inoculum of 5%, activated anaerobically at 37°C for 24 hours, and this activation was repeated twice before transferring to MRS medium for another 24 hours. The fermentation broth was centrifuged at 8000 rpm for 5 minutes at 4°C, and the *Bifidobacterium animalis* subsp. lactis B7 bacterial sludge precipitate was collected. This precipitate was then spray-dried to obtain *Bifidobacterium animalis* subsp. lactis B7 bacterial powder (i.e., *Bifidobacterium animalis* subsp. lactis B7 strain spray-dried powder). 1) Construction of DSS-induced ulcerative colitis or inflammatory bowel disease model and its alleviating effect on weight loss caused by ulcerative colitis or inflammatory bowel disease. C57BL / 6 mice were used as the subjects of pharmacological studies. Mice were randomly divided into three groups (n=6): a blank control group (NC group), a model control group (VC group), and an *Bifidobacterium lactis* subsp. B7 group (B7). The VC and B7 groups were given 2.5% sodium dextran sulfate solution (DSS solution) as drinking water for 7 consecutive days to establish ulcerative colitis or inflammatory bowel disease models; the NC group received purified water as a normal physiological blank control. The NC and VC groups were administered 0.2 mL of physiological saline (0.9% NaCl) by gavage, while each mouse in the B7 group was administered 0.2 mL of *Bifidobacterium lactis* subsp. B7 bacterial suspension (diluted with the *Bifidobacterium lactis* subsp. B7 bacterial powder mentioned above in this embodiment, yielding a viable count of 5 × 10⁻⁶). 9 (CFU / mL bacterial suspension).

[0106] Changes in body weight of mice in each group after modeling are shown in the figure. Figure 7 Mice in the VC group showed a decreasing weight trend during the modeling period, with a rapid decline starting from day 5. Weight loss is one of the important pathological manifestations of ulcerative colitis or inflammatory bowel disease. Mice in the B7 group showed weight changes similar to the NC group, without a decreasing trend. This indicates that *Bifidobacterium lactis* subsp. B7 can effectively alleviate weight loss caused by ulcerative colitis or inflammatory bowel disease.

[0107] 2) The therapeutic effect of Bifidobacterium animalis subsp. lactis B7 on the disease activity index (DAI) of ulcerative colitis or inflammatory bowel disease. After modeling, the mice's body weight, diarrhea severity, and fecal occult blood levels were measured daily. Fecal occult blood was determined using the benzidine method. The Disease Activity Index (DAI) score for ulcerative colitis or inflammatory bowel disease was determined according to Table 12.

[0108] Table 12 Disease Activity Index Scoring Criteria

[0109] The disease activity index (DAI) of ulcerative colitis or inflammatory bowel disease in each group of mice within 7 days after the modeling period ended is shown in the figure. Figure 8 During the modeling period, the disease activity index of mice in the VC group showed a significant upward trend, while that in the NC group remained unchanged at 0. The disease activity index of mice in the B7 group showed a lower upward trend than that in the VC group. This indicates that Bifidobacterium animalis subsp. lactis B7 alleviated the disease activity index (DAI) of ulcerative colitis or inflammatory bowel disease, thus exerting a therapeutic effect.

[0110] 3) The repairing effect of Bifidobacterium animalis subsp. lactis B7 on colonic tissue damage caused by ulcerative colitis or inflammatory bowel disease. Results of hematoxylin-eosin staining and pathological analysis of mouse colon tissue sections are shown below. Figure 9 In the VC group mice, the tight junction and crypt structures of the colon were significantly damaged, and ulcer foci appeared. Submucosal edema was accompanied by a large number of inflammatory cell infiltrations. In the B7 group mice, the tight junction and crypt structures were less damaged, no ulcer foci were observed, and the submucosal inflammatory cell infiltration was less severe. Bifidobacterium animalis subsp. lactis B7 significantly reduced colonic damage in mice.

[0111] 4) Protection of the colonic mucosa by Bifidobacterium animalis subsp. lactis B7 in patients with ulcerative colitis or inflammatory bowel disease. Results of periodic acid-Schiff staining and goblet cell counting in mouse colon tissue sections are shown below. Figure 10 The mucosal layer of mice in the VC group was significantly damaged, the number of goblet cells decreased sharply, and the number of goblet cells disappeared in some sections. The number of goblet cells in mice in the B7 group recovered to some extent. Bifidobacterium animalis subsp. lactis B7 reduced the loss of goblet cells in colitis mice, maintained colonic mucus secretion to a certain extent, and protected the mucosal layer.

[0112] 5) Effects of Bifidobacterium animalis subsp. lactis B7 on serum inflammatory factor levels in patients with ulcerative colitis or inflammatory bowel disease. Changes in the levels of inflammatory factors are one of the indicators for assessing the severity of inflammation. This experiment used ELISA to measure the levels of pro-inflammatory factors IL-6, TNF-α, IL-17, and IL-22, as well as the anti-inflammatory factor IL-10, in the serum of mice in each group. The results are as follows: Figure 11 As shown.

[0113] After intervention with Bifidobacterium lactis subsp. B7, the levels of pro-inflammatory factors in the serum of colitis mice decreased, including IL-6 (compared to the model group). p <0.001), TNF-α (compared with the model group) p <0.05), IL-17 (compared with the model group) p <0.05), IL-22 (compared with the model group) p <0.001), increased levels of anti-inflammatory factors (compared to the model group). pThe value was <0.05, indicating that Bifidobacterium lactis subspecies B7 exerted an anti-inflammatory effect by regulating the expression levels of these specific inflammatory factors, thereby reducing the inflammation level in mice with colitis.

[0114] 6) Effect of Bifidobacterium animalis subsp. lactis B7 on serum LPS levels in patients with ulcerative colitis or inflammatory bowel disease. In ulcerative colitis or inflammatory bowel disease, the intestinal barrier is damaged and intestinal permeability is significantly increased, causing more LPS to pass through the intestinal barrier into the blood, resulting in a significant increase in LPS levels in mouse serum.

[0115] like Figure 12 As shown, compared with the NC group, the serum LPS level in the VC group mice was significantly higher ( P <0.01) increases, while after intervention with Bifidobacterium animalis subsp. lactis B7, LPS significantly decreases (compared to the model group). p The value <0.05 indicates that intervention with Bifidobacterium lactis subsp. B7 effectively improved intestinal barrier function and reduced intestinal permeability in mice with ulcerative colitis or inflammatory bowel disease.

[0116] 7) Effects of Bifidobacterium animalis subsp. lactis B7 on D-LA secretion in ulcerative colitis or inflammatory bowel disease. Increased intestinal permeability in ulcerative colitis or inflammatory bowel disease leads to the accumulation of more D-LA (D-lactic acid) in the serum. Figure 13 As shown, compared with the NC group, the serum D-LA level in the VC group mice was significantly increased ( P <0.001), intervention with Bifidobacterium animalis subsp. lactis B7 reduced serum D-LA levels (compared to the model group). p Intervention with Bifidobacterium lactis subsp. B7 (<0.01) can improve intestinal barrier damage in mice to some extent, reduce intestinal permeability, and decrease serum D-LA levels.

[0117] 8) Effects of Bifidobacterium animalis subsp. lactis B7 on IPA in ulcerative colitis or inflammatory bowel disease. In cases of constipation, ulcerative colitis, or inflammatory bowel disease, blood levels of indolepropionic acid (IPA) are decreased, leading to impaired intestinal homeostasis. For example... Figure 14 As shown, compared with the NC group, the serum IPA level in the VC group mice was significantly reduced ( P <0.05%, after intervention with Bifidobacterium animalis subsp. lactis B7, it promoted the conversion of tryptophan in food by intestinal microorganisms and increased the IPA content in the blood. P Intervention with Bifidobacterium animalis subsp. lactis B7 (<0.05) can restore IPA levels to some extent. IPA can repair the intestinal mucosa, activate the intestinal AhR pathway, and improve intestinal homeostasis.

[0118] In summary, *Bifidobacterium animalis* subsp. lactis B7 can alleviate weight loss caused by ulcerative colitis or inflammatory bowel disease, and also alleviate the disease activity index (DAI) of ulcerative colitis or inflammatory bowel disease. Hematoxylin-eosin staining and pathological analysis showed that *Bifidobacterium animalis* subsp. lactis B7 significantly reduced colonic damage in mice, reduced goblet cell loss in colitis mice, and to some extent maintained colonic mucus secretion and protected the mucosal layer. After B7 intervention, the serum levels of pro-inflammatory factors in colitis mice decreased, including IL-6 (compared to the model group). p <0.001), TNF-α (compared with the model group) p <0.05), IL-17 (compared with the model group) p <0.05), IL-22 (compared with the model group) p <0.001), increased levels of anti-inflammatory factors (compared to the model group). p <0.05), IPA increased (compared to the model group). p (<0.05), therefore, B7 exerts an anti-inflammatory effect by regulating these specific inflammatory factors and reducing inflammation levels in colitis mice. LPS was significantly reduced after B7 intervention (compared to the model group). p <0.05), D-LA content decreased significantly (compared with the model group). p (<0.01), IPA is significantly increased. Therefore, B7 intervention can improve intestinal barrier function, reduce intestinal permeability, and increase IPA content, thereby achieving the purpose of preventing and treating ulcerative colitis or inflammatory bowel disease.

[0119] Example 7: Application Experiment of Bifidobacterium animalis subsp. lactis B7 Powder in Improving Constipation-Predominant Ulcerative Colitis This solid beverage made from *Bifidobacterium animalis* subsp. lactis B7 is formulated using *Bifidobacterium animalis* subsp. lactis B7 bacterial powder. The *Bifidobacterium animalis* subsp. lactis B7 bacterial strain is prepared by the following method: *Bifidobacterium animalis* subsp. lactis B7 strain is inoculated into MRS medium at a 5% inoculum, activated anaerobically at 37°C for 24 hours, and then transferred to MRS-GRP medium for 24 hours. The fermentation broth is centrifuged at 8000 rpm for 5 minutes at 4°C, and the *Bifidobacterium animalis* subsp. lactis B7 bacterial sludge precipitate is collected. Then, it is spray-dried at low temperature to obtain *Bifidobacterium animalis* subsp. lactis B7 bacterial powder. The viable count of *Bifidobacterium animalis* subsp. lactis B7 in this bacterial powder is 2 × 10⁻⁶. 11CFU / g; simultaneously, the concentration of the supernatant from the centrifuged fermentation broth was determined, and the B7 metabolite powder was obtained after low-temperature spray drying. The *Bifidobacterium lactis* B7 bacterial powder, B7 metabolite spray-dried powder, and maltodextrin carrier prepared using the aforementioned method for improving constipation-type ulcerative colitis were compounded in a weight ratio of 1:3:4 to obtain a solid beverage containing *Bifidobacterium lactis* B7 for improving constipation-type ulcerative colitis, with a viable count of 2.5 × 10⁻⁶. 10 Solid beverages containing CFU / strip or 2g / strip.

[0120] Population-controlled experimental method: Ten volunteers (18-70 years old, informed consent) with constipation-predominant ulcerative colitis were selected. They exhibited bowel movements less than 3 times per week, difficulty defecating, anal pain during defecation, occasional abdominal distension, constipation with bloody pus, and were diagnosed with ulcerative proctitis. They had no serious complications, could understand and cooperate with the survey, and possessed normal cognitive abilities. Exclusion criteria: Patients with severe diseases of vital organs such as the liver, kidneys, lungs, and brain, or those with critical illness; cancer patients, patients with mental illness or comprehension disorders; and patients who voluntarily withdrew or were lost to follow-up for personal reasons. They were randomly divided into an observation group and a control group using a random number table, with 5 patients in each group. There were no statistically significant differences in baseline data between the two groups, including gender, age, body mass index, disease duration, and constipation grade. P >0.05). Control group (5 cases): took one 2g sachet of maltodextrin solid beverage twice a day, morning and evening. Intervention group (5 cases): used Bifidobacterium animalis subsp. lactis B7, taking one 2.5×10 sachet of Bifidobacterium animalis subsp. lactis solid beverage twice a day, morning and evening. 10 CFU / strip, 2g), for a total of 30 days.

[0121] Observation indicators: Symptom scores were assessed in both groups one month before and after the intervention. Scores were based on abdominal pain, frequency of bowel movements per week, time of each bowel movement, stool characteristics, and the Disease Activity Index (DAI) for ulcerative colitis or inflammatory bowel disease (IBD), with each item assigned 4 points, for a total of 20 points. Higher scores indicated more severe symptoms. One month after the intervention, the effectiveness and adverse reactions were assessed using the efficacy evaluation criteria for refractory constipation in the "Guidelines for Primary Care Treatment of Constipation (2019)," with symptom score improvement as the determining factor. Specifically, a total score decrease of at least 70% (total score ≤ 6, no significant adverse reactions) was considered significant; a total score decrease of 40-70% (6 < total score ≤ 14), no adverse reactions, was considered effective; a total score decrease of less than 40% or an increase (14 < total score), with serious adverse reactions, was considered ineffective.

[0122] Overall effectiveness = (Number of cases with significant effect + Number of cases with effect) / Total number of cases × 100%.

[0123] Statistical methods: SPSS 20.0 software was used for statistical analysis. Quantitative data are expressed as mean ± standard deviation (s, minutes). P <0.05 indicates a statistically significant difference.

[0124] Symptom score Before the intervention, there was no statistically significant difference in symptom scores between the two groups. P >0.05). One month after intervention, symptom scores in both groups decreased compared to before treatment. The intervention group had a lower symptom score than the control group, and the difference was statistically significant. Compared with the pre-intervention scores of the same group ( P <0.01), compared with the control group after intervention ( P <0.01), and the specific experimental results are shown in Table 13.

[0125] Table 13. Intervention results of B7 for constipation-predominant ulcerative colitis ( ±s, minutes)

[0126] Note: 1) Compared with the same group before intervention, P a <0.05; compared with the control group after intervention, 2) P b <0.05.

[0127] Human trial effects and adverse reactions One month after the intervention, the total effective rate in the control group was 20%, while the total effective rate in the intervention group was 80%, which was four times that of the control group (see Table 14). No serious adverse reactions occurred in either group.

[0128] Table 14 Effects of the two intervention groups

[0129] Based on the above experimental results, the applicant proposed the application of Bifidobacterium animalis subsp. lactis B7 in constipation-predominant ulcerative colitis, with a recommended dosage of 5 × 10⁻⁶ viable bacteria in the Bifidobacterium animalis subsp. lactis B7 powder. 10 CFU / day.

[0130] Example 8 The Bifidobacterium lactis subsp. B7 live bacterial powder of this embodiment is obtained by centrifuging the fermentation broth of Bifidobacterium lactis B7 on MRS medium and incubating it at 37°C for 24 hours, followed by centrifugation, precipitation, freeze-drying, or spray-drying to obtain the Bifidobacterium lactis B7 powder. The live count of Bifidobacterium lactis B7 for improving constipation-predominant ulcerative colitis is 1.5 × 10⁻⁶. 11 CFU / g.

[0131] The application of Bifidobacterium animalis subsp. Lactobacillus B7 bacterial powder for improving constipation-type ulcerative colitis in the preparation of drugs for the prevention / treatment of constipation-type ulcerative colitis.

[0132] A drug containing *Bifidobacterium lactis* subsp. B7, used to improve constipation-predominant ulcerative colitis, is obtained by mixing the *Bifidobacterium lactis* subsp. B7 bacterial powder (used in this embodiment) with a drug carrier at a weight ratio of 1:2. The viable count of *Bifidobacterium lactis* subsp. B7 in this drug is 5.0 × 10⁻⁶. 10 CFU / g, 0.5g / capsule. The recommended dosage of this drug is 5.0 × 10⁻⁶ CFU / g. 10 CFU / day.

[0133] In this embodiment, the live bacteria powder of Bifidobacterium animalis subsp. lactis B7 is used as a starter culture in fermented foods such as kimchi and sauerkraut, with a dosage of 2×10⁻⁶. 7 CFU / kg fermented food.

[0134] Example 9 The fermentation composition of *Bifidobacterium lactis* subsp. B7 in this embodiment is a metabiotic of *Bifidobacterium lactis* subsp. B7. Its preparation method is as follows: *Bifidobacterium lactis* subsp. B7 is cultured statically on MRS-GRP medium at 37°C for 24 hours to obtain the fermentation broth. The supernatant obtained by filtration is then subjected to high-temperature inactivation (121°C, 15 min), concentration, and spray drying. This metabolite contains sulforaphane active ingredient, and the dead bacterial count of *Bifidobacterium lactis* subsp. B7 is 1×10⁻⁶. 11 per g.

[0135] A solid beverage containing the present embodiment for improving constipation-type ulcerative colitis includes 40 parts by weight of Bifidobacterium lactis subsp. B7 postbiotic powder prepared in the present embodiment, 50 parts by weight of broccoli seed water extract (sulforaphane 13g / 100g), and 10 parts by weight of maltodextrin.

[0136] The raw materials of this composition are first passed through a 40-mesh sieve, then mixed evenly according to the formula, and bagged using a screw back-sealing packaging machine to prepare a 2g / bag solid beverage that improves constipation-type ulcerative colitis.

[0137] The application of the fermentation composition of Bifidobacterium lactis subsp. B7 for improving constipation-type ulcerative colitis in the preparation of drugs for the prevention or treatment of constipation-type ulcerative colitis, in this embodiment, is at a dosage of 100~1000mg / day.

[0138] Example 10 The fermentation composition of *Bifidobacterium lactis* subsp. B7 in this embodiment is a mixture of live *Bifidobacterium lactis* subsp. B7 powder and freeze-dried fermentation supernatant powder in a weight ratio of 1:1. Specifically, *Bifidobacterium lactis* subsp. B7 is cultured statically in an MRS medium supplemented with tryptophan at 37°C for 24 hours. The resulting fermentation broth is centrifuged, and the precipitate is then freeze-dried or spray-dried to obtain *Bifidobacterium lactis* subsp. B7 powder. The live *Bifidobacterium lactis* subsp. B7 count for improving constipation-predominant ulcerative colitis is 1.5 × 10⁻⁶. 11 CFU / g, its supernatant was freeze-dried to obtain the supernatant freeze-dried powder of Bifidobacterium animalis subsp. lactis B7, which contains active ingredients such as indolepropionic acid and short-chain fatty acids.

[0139] The fermentation composition of Bifidobacterium animalis subsp. lactis B7 in this embodiment is used in the preparation of products for regulating intestinal flora, promoting intestinal motility, enhancing immunity, anti-inflammatory, antibacterial, or improving constipation.

[0140] Example 11 The fermentation composition of Bifidobacterium lactis B7 in this embodiment is a solid powder of Bifidobacterium lactis B7 metabolites. Specifically, Bifidobacterium lactis B7 is cultured statically in an MRS-GRP medium supplemented with 0.2 g / L tryptophan at 37°C for 24 h. The supernatant of the fermentation broth obtained by centrifugation (sulforaphane content ≥ 0.5 g / L) is then freeze-dried or spray-dried to obtain a solid powder of Bifidobacterium lactis B7 metabolites. The indolepropionic acid content in this fermentation composition is ≥ 2 μg / g.

[0141] A solid beverage containing a fermented composition of Bifidobacterium lactis B7 of this embodiment comprises 70 parts by weight of solid powder of Bifidobacterium lactis B7 metabolite, 20 parts by weight of broccoli seed water extract (sulforaphane 13g / 100g), and 10 parts by weight of maltodextrin.

[0142] The raw materials of this composition are first passed through a 40-mesh sieve, then mixed evenly according to the formula, and bagged using a screw back-sealing packaging machine to prepare a 2g / bag solid beverage for regulating constipation.

[0143] The fermentation composition of Bifidobacterium animalis subsp. lactis B7 in this embodiment is used in the preparation of products for regulating constipation, with a dosage of 100~1000 mg / day.

[0144] Example 12 The fermentation composition of Bifidobacterium animalis subsp. lactis B7 in this embodiment is a composition of live Bifidobacterium animalis subsp. lactis B7 powder and Bifidobacterium animalis subsp. lactis B7 postbiotic in a weight ratio of 2:1. The live Bifidobacterium animalis subsp. lactis B7 powder is prepared by the method described in Example 6, and the live bacteria content is 1×10⁻⁶. 11 CFU / g, Bifidobacterium animalis subsp. lactis B7 postbiotic was prepared as described in Example 9, wherein the cell number was 5 × 10⁻⁶. 10 per g.

[0145] The fermentation composition of Bifidobacterium lactis subsp. B7 in this embodiment is used in the preparation of a medicament for the prevention or treatment of ulcerative colitis or inflammatory bowel disease, with a dosage of 0.2-3 g / day.

[0146] Example 13 The fermentation composition of Bifidobacterium animalis subsp. lactis B7 in this embodiment includes live Bifidobacterium animalis subsp. lactis B7 powder, specifically, the freeze-dried or spray-dried powder of the bacterial cells obtained by centrifuging and drying the fermentation broth of Bifidobacterium animalis subsp. lactis B7 in sterilized fresh milk with added 10 g / L glutamic acid at 37°C for 24 h, containing 2 × 10⁻⁶ cells. 10 CFU / g live bacteria.

[0147] This embodiment describes the application of Bifidobacterium animalis subsp. lactis B7 in the preparation of biological agents or functional foods for regulating intestinal flora or promoting intestinal peristalsis. The biological agent is a powder, and the preparation method of the powder is as follows: by weight, it comprises 2 × 10⁻⁶ live Bifidobacterium animalis subsp. lactis B7 powder. 10 40 parts by weight of CFU / g), 30 parts by weight of broccoli seed water extract (sulforaphane 13g / 100g), 20 parts by weight of indolepropionic acid, and 10 parts by weight of maltodextrin.

[0148] The raw materials of this composition are first passed through a 40-mesh sieve, then mixed evenly according to the formula, and bagged using a screw back-sealing packaging machine to prepare a 2g / bag solid powder that has the effect of improving constipation, ulcerative colitis, and inflammatory bowel disease.

[0149] This embodiment describes the use of a fermentation composition of Bifidobacterium lactis subsp. B7 in the preparation of a medicament for treating ulcerative colitis, inflammatory bowel disease, constipation, or constipation-predominant ulcerative colitis.

[0150] In this embodiment, the fermentation composition of Bifidobacterium animalis subsp. lactis B7 is used as a starter in fermented foods such as kimchi and sauerkraut, with a dosage of 2×10⁻⁶. 7 CFU / Kg fermented food.

[0151] Example 14 This embodiment provides a drug for the prevention and / or treatment of ulcerative colitis, comprising live Bifidobacterium animalis subsp. lactis B7 powder and Bifidobacterium animalis subsp. lactis B7 metabolites in a weight ratio of 2:1. The live Bifidobacterium animalis subsp. lactis B7 powder is prepared using the method described in Example 6, and the live bacteria content is 3 × 10⁻⁶. 10 CFU / g, the metabolite of Bifidobacterium lactis B7 is prepared by fermenting Bifidobacterium lactis B7 in MRS-GRP medium at 37°C for 24 hours, and then centrifuging the supernatant obtained by centrifugation of the fermentation broth, followed by high-temperature inactivation (121°C, 15 min), concentration, and spray drying.

[0152] The recommended adult dosage for the medication described in this embodiment for the prevention and / or treatment of ulcerative colitis or inflammatory bowel disease is 0.2-3 g / day.

[0153] Example 15 This embodiment describes a drug for the prevention or treatment of ulcerative colitis, namely, Bifidobacterium animalis subsp. lactis B7 postbiotic. The Bifidobacterium animalis subsp. lactis B7 postbiotic is prepared by fermenting Bifidobacterium animalis subsp. lactis B7 in MRS medium at 37°C for 24 hours, followed by cell lysis, high-temperature inactivation (121°C, 15 min), concentration, and spray drying.

[0154] The drug described in this embodiment is used for the prevention or treatment of ulcerative colitis. The recommended dosage is 0.5~3g / day, and it has good efficacy.

[0155] Example 16 The drug used in this embodiment for the prevention or treatment of mixed ulcerative colitis includes live Bifidobacterium animalis subsp. lactis B7 powder. This live Bifidobacterium animalis subsp. lactis B7 powder is a lyophilized or spray-dried powder obtained by centrifuging and precipitating the fermentation broth of Bifidobacterium animalis subsp. lactis B7 in MRS medium at 37°C for 20 hours, followed by centrifugation and drying. It contains 8 × 10⁻⁶ cells. 10 CFU / g live bacteria.

[0156] The drug is in capsule form, and the preparation method of the capsule is as follows: by weight, it includes *Bifidobacterium animalis* subsp. *lactamase* B7 (4.0 × 10⁻⁶). 10 40 parts by weight of CFU / g, 40 parts by weight of starch, 5 parts by weight of glucose, 5 parts by weight of hydroxypropyl methylcellulose, 5 parts by weight of lactitol, and 5 parts by weight of magnesium stearate.

[0157] The raw materials for this composition are first passed through a 40-mesh sieve, then mixed evenly according to the formula, and then encapsulated or packaged into powder.

[0158] The components and dosage of the *Bifidobacterium lactis* subsp. B7 fermentation composition of the present invention can be selected according to different applications. For example, in Example 6, the dosage of *Bifidobacterium lactis* subsp. B7 bacterial powder in the preparation of a microbial preparation for improving ulcerative colitis or inflammatory bowel disease can also be 5 × 10⁻⁶. 8 CFU / day, the Bifidobacterium lactis B7 metabolite in Example 11 can also be used as a composition in the production of sulforaphane and / or tryptophan metabolites. The Bifidobacterium lactis B7 postbiotic in Example 9 can also be used as a composition in the preparation of a drug for the prevention or treatment of constipation, and its dosage can be 2×10. 9 The changes to the above technical features can be understood and implemented by those skilled in the art through textual description, so no further drawings are required.

Claims

1. A strain of Bifidobacterium animalis subsp. Lactobacillus B7, characterized in that, The animal Bifidobacterium lactis subspecies B7 ( Bifidobacterium animalis subsp.lactis B7 was deposited at the China Center for Type Culture Collection on April 19, 2024, with accession number CCTCC NO:M2024734.

2. A fermentation composition, characterized in that, The fermentation composition is prepared by fermenting Bifidobacterium lactis subsp. B7 as described in claim 1, comprising live Bifidobacterium lactis subsp. B7, inactivated bacterial cells, fermentation broth supernatant, fermentation precipitate, and Bifidobacterium lactis subsp. B7 post-biotic or spray-dried / lyophilized powder; the fermentation composition is prepared by fermenting Bifidobacterium lactis subsp. B7 in MRS medium, or MRS-GRP medium supplemented with broccoli seed water extract or glucosinolates, or MRS medium supplemented with tryptophan.

3. A probiotic preparation, characterized in that, The probiotic preparation comprises Bifidobacterium lactis subsp. B7 as described in claim 1 and / or the fermentation composition as described in claim 2; the content of Bifidobacterium lactis subsp. B7 in the probiotic preparation is ≥2×10⁻⁶. 7 CFU / g or 2×10 7 CFU / ml.

4. The probiotic preparation as described in claim 3, characterized in that, The probiotic preparation also includes glucosinolates or broccoli seed water extract or IPA.

5. The use of Bifidobacterium animalis subsp. Lactobacillus B7 as described in claim 1 in the production of sulforaphane and / or tryptophan metabolites.

6. The use of Bifidobacterium animalis subsp. lactis B7 as described in claim 1 or the fermentation composition as described in claim 2 in the preparation of products for regulating intestinal flora, promoting intestinal motility, enhancing immunity, anti-inflammatory, antibacterial, or improving constipation.

7. The use of Bifidobacterium lactis subsp. B7 as described in claim 1 and / or the fermentation composition as described in claim 2 in the preparation of medicaments for the prevention, relief or treatment of ulcerative colitis, inflammatory bowel disease, constipation and constipation-predominant ulcerative colitis.

8. The application of Bifidobacterium animalis subsp. Lactobacillus B7 as described in claim 1 as a starter culture in the preparation of fermented foods.

9. A drug for the prevention and treatment of ulcerative colitis, inflammatory bowel disease, constipation, and constipation-predominant ulcerative colitis, characterized in that, The drug comprises Bifidobacterium lactis subsp. B7 as described in claim 1, the fermentation composition as described in claim 2, or the probiotic preparation as described in claim 3 or 4.

10. The medicament as claimed in claim 9, characterized in that, The drug has at least one of the following effects: a) High production of short-chain fatty acids acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and hexanoic acid, improving the level of short-chain fatty acids in metabolites; b) Increase intestinal peristalsis; c) It can alleviate weight loss caused by ulcerative colitis or inflammatory bowel disease, and at the same time alleviate the disease activity index (DAI) of ulcerative colitis or inflammatory bowel disease; d) Significantly reduces colonic damage and protects the mucosal layer; reduces the levels of pro-inflammatory factors IL-6, TNF-α, IL-17, and IL-22 in the serum of patients with colitis, increases the level of anti-inflammatory factor IL-10, and alleviates intestinal inflammation; e) It can significantly reduce LPS and D-LA levels, improve intestinal barrier function, and reduce intestinal permeability; f) It can significantly increase IPA content and increase intestinal homeostasis.

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