Bifidobacterium animalis lactis strain b7, composition and application thereof

Abstract

This paper discusses a strain of Bifidobacterium animalis subsp. lactis B7 and its composition and applications, belonging to the field of microbial technology. The specific strain is Bifidobacterium animalis subsp. lactis B7. Bifidobacterium animalis subsp. lactis Bifidobacterium lactis subspecies B7 was deposited at the China Center for Type Culture Collection on April 19, 2024, with accession number CCTCC NO: M2024734. This invention also discloses the application of the B7 fermentation composition in the preparation of drugs for the prevention and treatment of ulcerative colitis, constipation, and constipation-predominant ulcerative colitis. This animal bifidobacterium lactis subspecies B7 produces high levels of short-chain fatty acids and indolepropionic acid, utilizes sulforaphane to generate sulforaphane, increases intestinal peristalsis, effectively alleviates the disease activity index (DAI) and weight loss caused by ulcerative colitis or inflammatory bowel disease, significantly reduces colonic damage in mice, protects the mucosal layer, has anti-inflammatory effects, inhibits pathogens, and improves intestinal barrier function.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to an animal Bifidobacterium lactis B7, a composition and application thereof. BACKGROUND

[0002] In recent years, with the change of life rhythm and eating habits, the prevalence of inflammatory bowel disease and constipation has increased year by year. Inflammatory bowel disease is a chronic and recurrent inflammatory gastrointestinal disease, which can be divided into ulcerative colitis (UC) and Crohn's disease (CD). The main clinical manifestations of the diagnostic criteria for ulcerative colitis at home and abroad are diarrhea and mucous blood stool. A large number of epidemiological and clinical data show that although constipation type UC is rare, the number of patients with constipation symptoms related to UC has increased year by year. In a study on the intestinal directed pelvic floor behavior therapy of patients with inflammatory bowel disease, 55% of the 40 patients were constipated, and 45% were incontinent, which was much higher than that of ordinary people (SPERBER AD, 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 type ulcerative colitis involves genetic susceptibility, intestinal flora imbalance, intestinal barrier defect, anorectal dysfunction, individual immunity, environmental factors, and living habits. In the prior art, drug therapy is generally used for constipation type UC patients, such as traditional Chinese medicine treatment. CN118717861A discloses a traditional Chinese medicine enema soup for treating constipation type ulcerative colitis. The enema soup is made of raw medicinal materials such as Raphanus sativus, Fructus Aurantii, Camellia japonica, Coptis chinensis, Scutellaria baicalensis, Rehmannia glutinosa and Angelica sinensis. The enema soup can effectively solve the problem of treating constipation type ulcerative colitis. In addition, guanylyl cyclase C agonists, such as plenapanth and docanatide, are also used to treat constipation type ulcerative colitis. However, the biological agent is expensive, and long-term use can cause many adverse reactions to patients.

[0004] In recent years, more and more studies have found that the severe imbalance of intestinal flora in constipation type ulcerative colitis patients can aggravate the disease, and the use of related drugs for treatment can destroy the balance of intestinal flora, while the use of probiotics for treatment can directly or indirectly regulate intestinal flora or play a therapeutic role through metabolic products. For example, CN119331785A discloses animal Bifidobacterium lactis for relieving ulcerative colitis and constipation and application thereof. The animal Bifidobacterium lactis in the mother milk source can regulate the intestinal flora of the patient, and the metabolites of the animal Bifidobacterium lactis can directly or indirectly regulate the intestinal flora of the patient, thereby relieving the symptoms of ulcerative colitis and constipation. Bifidobacterium animalis subsp. lactisLIHUO 01, which was preserved in the Guangdong Microbial Culture Collection Center on November 20, 2024, has a preservation number of GDMCC No: 65512. The animal Bifidobacterium lactis LIHUO01 has good in-vitro probiotic properties, has the effects of relieving ulcerative colitis, improving constipation, and regulating intestinal flora, and has a good application prospect in the preparation of drugs for relieving ulcerative colitis or improving constipation or intestinal flora regulation. However, in the existing disclosed technology, the efficacy target is not clear, and only constipation or ulcerative colitis is studied alone, so it is of great significance to develop specific strains or postbiotics of specific strains that can improve constipation type ulcerative colitis for the prevention and control of constipation type ulcerative colitis intestinal diseases. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the shortcomings of the prior art and provide an animal Bifidobacterium lactis B7 that can produce high yields of sulforaphane and indole propionic acid, as well as a new application of a fermentation composition of the animal Bifidobacterium lactis B7, specifically an application in the preparation of sulforaphane, or tryptophan metabolites, or probiotic preparations or drugs for regulating intestinal peristalsis, relieving ulcerative colitis or inflammatory bowel disease, especially using the animal Bifidobacterium lactis B7 strain and its metabolites, including but not limited to sulforaphane and tryptophan metabolites, to further improve intestinal barrier function, increase intestinal peristalsis, and prevent and treat ulcerative colitis, especially constipation type ulcerative colitis.

[0006] One of the technical solutions adopted by the present application to solve its technical problems is:

[0007] An animal Bifidobacterium lactis B7, wherein the animal Bifidobacterium lactis B7 (B7) Bifidobacterium animalis subsp. lactis B7) was preserved in the China Center for Type Culture Collection on April 19, 2024, and has a preservation number of CCTCC NO: M2024734.

[0008] The animal Bifidobacterium lactis B7 is isolated from the intestinal tract of a healthy infant in Chengdu, Sichuan. The isolated and purified strain is identified by 16S rRNA, and the 16S rRNA identification sequence of the strain is shown as SEQ ID NO: 1. The measured 16S rRNA sequence is subjected to NCBI BLAST comparison, and the similarity with the animal Bifidobacterium lactis in Genebank is greater than 99%, so the strain can be identified as the animal Bifidobacterium lactis. Bifidobacterium animalis subsp. Lactis Bifidobacterium animalis subsp. lactis Based on the 16S rRNA gene sequence comparison result, the animal Bifidobacterium lactis B7 is identified as the animal Bifidobacterium lactis. Scardovia inopinata ​DSM10107 (AB029087) is the Neighbor-Joining phylogenetic tree constructed by outgroup, named Bifidobacterium animalis lactis B7 (AB029087) Bifidobacterium animalis subsp. lactis B7).

[0009] The Bifidobacterium animalis lactis B7 of the present application forms colonies on MRS medium, which are smooth, complete in edge, milky white, shiny and soft in texture, and rod-shaped under microscope. The suitable growth temperature thereof is 37-46℃, and the growth pH is 3.0-7.0.

[0010] Preferably, the Bifidobacterium animalis lactis B7 produces short-chain fatty acids by fermentation, and synthesizes sulforaphane from glucoraphanin and indole-3-propionic acid (also known as indole propionic acid, IPA) from tryptophan, and other tryptophan metabolites, thereby increasing intestinal peristalsis and relieving or treating constipation-predominant ulcerative colitis or inflammatory bowel disease.

[0011] One of the technical solutions adopted by the present application to solve the technical problems is:

[0012] A fermentation composition is produced by fermenting the Bifidobacterium animalis lactis B7 described above.

[0013] The fermentation composition includes live Bifidobacterium animalis lactis B7, inactivated Bifidobacterium animalis lactis B7, fermentation broth supernatant, fermentation precipitate, Bifidobacterium animalis lactis B7 postbiotic, or spray-dried powder / freeze-dried powder; the fermentation composition is produced by fermenting Bifidobacterium animalis lactis B7 in MRS medium, or MRS-GRP medium added with water extract of broccoli seeds or glucoraphanin, or MRS medium added with tryptophan.

[0014] Preferably, the fermentation supernatant of the Bifidobacterium animalis lactis B7 is prepared as follows: the Bifidobacterium animalis lactis B7 is fermented in MRS medium at 37℃ for 24 hours, then activated twice in succession with 5% inoculation amount into fresh MRS medium, and cultured under the same conditions for 12 hours, and then centrifuged to collect the bacterial cells, which are then inoculated into MRS medium, MRS-GRP medium added with water extract of broccoli seeds or glucoraphanin, or MRS medium added with tryptophan, and cultured at 37℃ for 24 hours, and then centrifuged at low temperature (10000 r / min, 10 min, 4℃) to collect the fermentation supernatant, which is then inactivated by high-temperature sterilization (121℃, 15 min), concentrated, and spray-dried to obtain the fermentation supernatant. The fermentation supernatant contains active ingredients such as glucoraphanin and tryptophan metabolites. The MRS medium added with water extract of broccoli seeds or glucoraphanin can promote the synthesis of glucoraphanin during the fermentation of the Bifidobacterium animalis lactis B7, and the MRS medium added with tryptophan can produce a large amount of indole-3-propionic acid during the fermentation of the Bifidobacterium animalis lactis B7, so as to further improve the intestinal peristalsis performance, increase the level of anti-inflammatory factors in the intestine, and increase the anti-inflammatory effect.

[0015] Preferably, the Bifidobacterium animalis lactis B7 probiotic is prepared as follows: after the fermentation of the Bifidobacterium animalis lactis B7 in MRS medium, MRS-GRP medium added with water extract of broccoli seeds or glucoraphanin, or MRS medium added with tryptophan is completed, the cells are lysed, inactivated, concentrated, and spray-dried to obtain a solid powder containing inactivated bacterial cells of the Bifidobacterium animalis lactis B7 and metabolites of the Bifidobacterium animalis lactis B7, including dead cells, cell lysates, and / or fermentation broth.

[0016] Another technical solution adopted by the present application to solve the technical problems is as follows:

[0017] The probiotic preparation contains the Bifidobacterium animalis lactis B7 or the fermentation composition described above, and the content of the Bifidobacterium animalis lactis B7 in the probiotic preparation is ≥2×10 7 CFU / g or 2×10 7 CFU / ml.

[0018] Preferably, the probiotic preparation further contains glucoraphanin, water extract of broccoli seeds, or indole-3-propionic acid.

[0019] Another technical solution adopted by the present application to solve the technical problems is as follows:

[0020] The application of the Bifidobacterium animalis lactis B7 in the production of glucoraphanin and / or tryptophan metabolites. In a certain exemplary embodiment, the tryptophan metabolite is indole-3-propionic acid.

[0021] Use of animal Bifidobacterium lactis B7 or a fermentation composition of animal Bifidobacterium lactis B7 in the preparation of a product for regulating intestinal flora or promoting intestinal peristalsis or enhancing immunity or anti-inflammation or inhibiting bacteria or improving constipation.

[0022] Use of animal Bifidobacterium lactis B7 and / or the fermentation composition in the preparation of a medicine for preventing, alleviating or treating ulcerative colitis, inflammatory bowel disease, constipation and constipation-type ulcerative colitis.

[0023] Use of animal Bifidobacterium lactis B7 as a fermenting agent in the preparation of fermented foods such as pickles and sauerkraut.

[0024] In this application, the amount of live animal Bifidobacterium lactis B7 in the composition is 5×10 9 ~2×10 11 CFU / day, the amount of inactivated animal Bifidobacterium lactis B7 is 1×10 10 ~2×10 11 CFU / day, the amount of animal Bifidobacterium lactis B7 metabolites is 100-1000 mg / day, and the amount of animal Bifidobacterium lactis B7 postbiotics is 5.0×10 9 ~1×10 11 CFU / day.

[0025] The present application provides a medicine for preventing and / or treating ulcerative colitis, inflammatory bowel disease, constipation and constipation-type ulcerative colitis, which comprises the above-mentioned animal Bifidobacterium lactis B7 and / or the above-mentioned fermentation composition.

[0026] The medicine achieves the prevention and treatment effect on ulcerative colitis, especially constipation-type ulcerative colitis, through at least one of the following functions:

[0027] a) high production of short-chain fatty acids acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and caproic acid, and improvement of the level of short-chain fatty acids in metabolites;

[0028] b) increase of intestinal peristalsis;

[0029] c) alleviation of weight loss caused by ulcerative colitis or inflammatory bowel disease, and alleviation of disease activity index DAI of ulcerative colitis or inflammatory bowel disease;

[0030] d) significant reduction of colon damage and protection of the mucosa layer; reduction of IL-6, TNF-α, IL-17 and IL-22 in serum pro-inflammatory factors of colitis, and increase of IL-10 level in anti-inflammatory factors to alleviate intestinal inflammation;

[0031] e) significant reduction of LPS and D-LA (D-lactic acid) content, improvement of intestinal barrier function and reduction of intestinal permeability;

[0032] f) can significantly increase the content of IPA, increase the intestinal homeostasis.

[0033] The medicine also includes a pharmaceutically acceptable carrier and / or a pharmaceutical adjuvant.

[0034] In an embodiment, the pharmaceutically acceptable carrier includes, but is not limited to, one or more of a filler, a binder, a wetting agent, a disintegrant, or a lubricant.

[0035] Preferably, the medicine is an oral medicine.

[0036] In an 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 sugar syrup; the wetting agent is one or more of glycerol or ethanol; the disintegrant is one or more of cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, or cross-linked sodium carboxymethyl starch; and the lubricant is one or more of silicon dioxide, magnesium stearate, or sodium stearyl fumarate.

[0037] Advantages of the animal Bifidobacterium lactis B7 of the present application:

[0038] The animal Bifidobacterium lactis B7 of the present application has a wide growth temperature and can grow well at 37-46℃, has good oxygen tolerance; has good stability in long-term storage at room temperature and 5-week survival rate of 52.4% at accelerated temperature of 37℃; the spray-dried bacterial powder prepared from the fermentation broth has good stability; the animal Bifidobacterium lactis B7 can produce short-chain fatty acids, convert glucoraphanin to sulforaphane, increase intestinal peristalsis, and at the same time, the animal Bifidobacterium lactis B7 can metabolize tryptophan or broccoli seed water extract to produce high levels of indole-3-propionic acid (IPA), can alleviate weight loss caused by ulcerative colitis or inflammatory bowel disease, at the same time, alleviate the disease activity index DAI of ulcerative colitis or inflammatory bowel disease, significantly reduce the colon injury of mice, and protect the mucosal layer; reduce the levels of pro-inflammatory factors IL-6, TNF-α, IL-17, and IL-22 in the serum of colitis, and increase the level of anti-inflammatory factor IL-10, thereby playing an anti-inflammatory role. At the same time, the B7 intervention can significantly reduce the contents of LPS and D-LA, improve the intestinal barrier function, and reduce the intestinal permeability, and therefore has great application prospects in the preparation of medicines for preventing and / or relieving constipation-type ulcerative colitis or inflammatory bowel disease.

[0039] The acid content after 12h fermentation is as high as 0.82g / 100ml, has the advantages of fast and persistent acid production, and is a good ferment.

[0040] Animal experiments show that the animal Bifidobacterium lactis B7 and its postbiotic of the present application have good safety, good gastrointestinal fluid tolerance, and good bile salt tolerance, and can be orally taken and used in vivo.

[0041] The animal Bifidobacterium lactis B7 can efficiently metabolize glucoraphanin and synthesize sulforaphane on the MRS-GRP culture medium, and the conversion rate is as high as 43.42%, and can significantly promote the synthesis of short-chain fatty acids.

[0042] The animal Bifidobacterium lactis B7 has significant bacteriostatic effect on common pathogenic bacteria 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, especially the animal Bifidobacterium lactis B7 cultivated on the MRS-GRP culture medium, which has higher bacteriostatic performance, indicating that the animal Bifidobacterium lactis B7 and its metabolite sulforaphane have significant synergistic effect in bacteriostasis.

[0043] Through animal experiment verification, the animal Bifidobacterium lactis B7 or the mixture of the animal Bifidobacterium lactis B7 and glucoraphanin can significantly improve the constipation of zebrafish; the animal Bifidobacterium lactis B7 can effectively improve and treat constipation, ulcerative colitis or inflammatory bowel disease.

[0044] Biological material preservation

[0045] The animal Bifidobacterium lactis B7 ( Bifidobacterium animalis subsp. lactis B7) was preserved in the China Center for Type Culture Collection on April 19, 2024, and the preservation number is CCTCC NO: M2024734, and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 It is a colony map of the animal Bifidobacterium lactis B7 of the present application;

[0047] Figure 2 It is a microscope morphology map of the animal Bifidobacterium lactis B7 of the present application;

[0048] Figure 3 It is a phylogenetic tree of the animal Bifidobacterium lactis B7 of the present application;

[0049] Figure 4 It is a growth and fermentation liquid pH change map of the animal Bifidobacterium lactis B7 of the present application;

[0050] Figure 5 It is an acid production map of the animal Bifidobacterium lactis B7 of the present application;

[0051] Figure 6 It is a live bacteria number accelerated stability analysis map of the animal Bifidobacterium lactis B7 of the present application;

[0052] Figure 7 Figure 6 is a graph showing the body weight comparison of different groups of mice with ulcerative colitis or inflammatory bowel disease according to the present application;

[0053] Figure 8 Figure 7 is a graph showing the disease activity index (DAI) of different groups of mice with ulcerative colitis or inflammatory bowel disease within 7 days of modeling according to the present application;

[0054] Figure 9 Figure 8 is a graph showing the staining and pathological analysis of colon tissue sections of different groups of mice with ulcerative colitis or inflammatory bowel disease according to the present application;

[0055] Figure 10 Figure 9 is a graph showing the staining and pathological analysis of colon tissue sections of different groups of mice with ulcerative colitis or inflammatory bowel disease according to the present application;

[0056] Figure 9 is a graph showing the staining and pathological analysis of colon tissue sections of different groups of mice with ulcerative colitis or inflammatory bowel disease according to the present application;

[0057] Figure 11 Figure 10 is a graph showing the changes of inflammatory factors and anti-inflammatory factors in different groups of mice with ulcerative colitis or inflammatory bowel disease according to the present application;

[0058] Figure 10 is a graph showing the changes of inflammatory factors and anti-inflammatory factors in different groups of mice with ulcerative colitis or inflammatory bowel disease according to the present application;

[0059] Figure 12 Figure 11 is a graph showing the serum LPS concentration in different groups of mice with ulcerative colitis or inflammatory bowel disease according to the present application;

[0060] Figure 13 Figure 12 is a graph showing the effect of Bifidobacterium animalis lactis B7 on D-LA in the serum of mice according to the present application;

[0061] Figure 14 Figure 13 is a graph showing the effect of Bifidobacterium animalis lactis B7 on IPA in the serum of mice according to the present application.

[0062] Wherein: Figures 4-13 ns represents no statistical significance; * represents P <0.05; ** represents P <0.01; *** represents P <0.001; **** represents P <0.0001. DETAILED DESCRIPTION

[0063] The present application will be further described below in conjunction with the accompanying drawings and examples. The present application will be further described below in conjunction with the accompanying drawings and examples.

[0064] The following is the culture medium involved in the embodiments of the present application:

[0065] MRS medium (g / L): Proteose peptone 10.0, beef powder 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 agar powder 15 g / L for solid medium).

[0066] MRS-GRP medium (g / L): Proteose peptone 10.0, beef powder 5.0, yeast powder 4.0, broccoli seed water extract (sulforaphane 13 g / 100 g) 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, (add agar powder 15 g / L for solid medium).

[0067] Example 1

[0068] Screening and identification of animal bifidobacterium lactis B7

[0069] Animal bifidobacterium lactis B7 Bifidobacterium animalis subsp. lactis B7) was preserved in China Center for Type Culture Collection on April 19, 2024, and the preservation number is CCTCC NO: M2024734.

[0070] 1) Screening of animal bifidobacterium lactis B7 strain

[0071] The sample was collected from the intestinal tract of healthy infants in Chengdu, Sichuan. 1 g of the collected sample was placed in 9 mL of sterile normal saline. After thorough shaking and mixing, 10-fold gradient dilution was performed and plated on MRS solid medium, and incubated at 37°C under strict anaerobic conditions for 48 h. Under visual observation, single colonies of different sizes and shapes in the culture medium were picked and streaked for purification for more than 4 times. Microscopic observation and calcium dissolution method were used to preliminarily determine the strain as a lactic acid bacteria, and the purified strain was stored in 45% glycerol at -80°C refrigerator for standby.

[0072] 2) Strain morphology and microscopic observation

[0073] The colony morphology of animal bifidobacterium lactis B7 strain is shown in Figure 1 , which is smooth, complete in edge, milky white, shiny and soft in texture. The microscopic morphology is shown in Figure 2 , and animal bifidobacterium lactis B7 is rod-shaped.

[0074] 3) Molecular biology identification of the strain

[0075] The purified animal Bifidobacterium lactis B7 strain was identified by 16S rRNA, and the 16S rRNA identification sequence of the strain is shown as SEQ ID NO: 1. The measured 16S rRNA sequence was subjected to NCBI BLAST alignment, and the similarity with the animal Bifidobacterium lactis (B7) in Genebank was greater than 99%, which can identify the strain as animal Bifidobacterium lactis (B7). Bifidobacterium animalis subsp. lactis ) similarity greater than 99%, which can identify the strain as animal Bifidobacterium lactis (B7). Bifidobacterium animalis subsp. lactis ) similarity greater than 99%, which can identify the strain as animal Bifidobacterium lactis (B7). Scardovia inopinata The Neighbor-Joining phylogenetic tree constructed based on the 16S rRNA gene sequence alignment result with DSM 10107 (AB029087) as the outgroup is shown in Figure 3 . Bifidobacterium animalis subsp. lactis B7) was named.

[0076] 4) Fermentation substrate utilization ability

[0077] Fresh slant culture of the animal Bifidobacterium lactis B7 to be tested was inoculated into the culture medium, or fresh culture liquid was added dropwise into the melted soft agar column (temperature 47±1℃), and after mixing, a layer of about 7mm thick 2% agar was added on top, and placed in 37℃ anaerobic culture for 48h, and the culture medium was determined to produce acid by the strain utilizing carbon source.

[0078] The experimental results are shown in Table 1, and the animal Bifidobacterium lactis B7 strain can utilize glucose, escin, maltose, melibiose, sucrose, raffinose, 2-fucosyllactose, lacto-N-neotetraose, etc. carbon source substrate for fermentation to produce acid, among which 2-fucosyllactose, lacto-N-neotetraose are human milk oligosaccharides.

[0079] Table 1 Physiological and biochemical characteristics of animal Bifidobacterium lactis B7 – utilization of carbon source to produce acid

[0080]

[0081] 5) Growth performance determination

[0082] The animal Bifidobacterium lactis B7 strain was inoculated into the MRS liquid medium at a inoculation amount of 5%, and cultured at 37℃ for 24 hours, and continuously activated twice; the activated B7 liquid was inoculated into the MRS liquid medium at a inoculation amount of 5%, and mixed uniformly, and then divided into sterile test tubes (18mm×180mm) at 8ml / branch; the divided liquid was placed in a 37℃ constant temperature incubator for static culture, and 3 test tubes were taken to measure the absorbance value OD 600 of the liquid, and the average value was calculated; every certain time, 3 test tubes were taken to measure the total acid and absorbance value OD 600 of the liquid, and the growth curve was drawn.

[0083] The growth performance data of the strain animal Bifidobacterium lactis B7 were plotted as a line graph as shown in Figure 4 There was no significant increase in OD 600 and no significant change in pH; from 4 to 12 h, OD 600 presented logarithmic growth, and the pH value decreased rapidly; from 12 to 24 h, OD 600 was basically maintained at 1.85±0.05, and the pH was maintained at 4.71±0.05. According to the growth curve, it can be preliminarily determined that the optimal growth time of the strain B7 is 12 h.

[0084] 6) Strain acid production performance determination

[0085] The animal Bifidobacterium lactis B7 strain was inoculated into MRS medium at a 5% inoculation amount, activated and cultured at 37°C for 24 hours, and continuously activated twice. The activated B7 bacterial liquid was inoculated into MRS and MRS-GRP liquid culture media at a 5% inoculation amount, mixed uniformly, and then divided into sterile test tubes (18 mm x 180 mm) at 8 ml / tube. The divided B7 bacterial liquid was placed in a 37°C constant temperature incubator for static culture, and 3 test tubes were taken to determine the total acid at 8 h, 10 h, 12 h, and 14 h, and the 24-hour short-chain fatty acid content was determined by HPLC.

[0086] The acid production results of the animal Bifidobacterium lactis B7 are shown in Table 2 and Figure 5 , the acid production in the MRS medium was 0.37 g / 100 ml, 0.58 g / 100 ml, 0.82 g / 100 ml, and 0.84 g / 100 ml at 8 h, 10 h, 12 h, and 14 h, respectively, and the acid production in the MRS-GRP culture 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 h to 10 h, B7 produced acid faster in the MRS medium, while from 10 h to 12 h, B7 produced acid faster in the MRS-GRP culture, and the highest acid production was at 14 h in the MRS medium and the MRS-GRP culture.

[0087] Table 2 Analysis of acid production performance of animal Bifidobacterium lactis B7

[0088]

[0089] Short-chain fatty acid (SCFA), also known as volatile fatty acid (VFA), is one of the important metabolic products of beneficial bacteria in the intestinal tract, and common ones are acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and caproic acid, which have been confirmed to have good effects on intestinal immunity, constipation improvement and inflammation regulation.

[0090] The results of the determination of the production of short-chain fatty acids by Bifidobacterium animalis lactis B7 are shown in Table 3. Among them, on the MRS-GRP culture, acetic acid increased by 37.72%, propionic acid increased by 95.15%, isobutyric acid increased by 152.70%, butyric acid increased by 83.33%, iso-valeric acid increased by 71.74%, valeric acid increased by 236.36%, caproic acid increased by 50.77%, and the total increased by 38.03%. The results show that Bifidobacterium animalis lactis B7 can significantly increase the content of short-chain fatty acids on the MRS-GRP culture.

[0091] Table 3 Detection and analysis of the production of short-chain fatty acids by Bifidobacterium animalis lactis B7

[0092]

[0093] Note: The quantitative unit of short-chain fatty acid is μg / ml.

[0094] 7) Determination of the oxygen tolerance of the strain

[0095] The Bifidobacterium animalis lactis B7 strain was inoculated into MRS liquid medium at a 5% inoculation amount, and cultured at 37°C for 24 hours for continuous activation twice. The activated B7 bacterial liquid was inoculated into MRS liquid medium test tubes (18mmx180mm) at a 5% inoculation amount. The inoculated bacterial liquid was placed in a 37°C constant temperature incubator (oxygen content 21%) and a 37°C anaerobic incubator (oxygen content 0%) for 12h of static culture, and the OD 600 of the bacterial liquid was determined.

[0096] The oxygen tolerance of Bifidobacterium animalis lactis B7 directly affects the production cost of the bacterial powder. The experimental results show that the OD 600 of B7 cultured in a 37°C constant temperature incubator (oxygen content 21%) for 12h is 1.982, and the OD 600 of B7 cultured in a 37°C anaerobic incubator (oxygen content 0%) for 12h is 1.839. Therefore, the growth amount of the Bifidobacterium animalis lactis B7 strain is less affected by the oxygen content, and the strain has excellent processing characteristics.

[0097] 8) Determination of the temperature tolerance of the strain

[0098] The animal Bifidobacterium lactis B7 strain was inoculated into MRS liquid medium at an inoculation amount of 5%, activated and cultured at 37°C for 24 hours, and continuously activated twice; the activated B7 bacterial liquid was inoculated into MRS liquid medium test tubes (18mmx180mm) at an inoculation amount of 5%; and the inoculated bacterial liquid was placed in an anaerobic incubator at 37°C, 40°C, 43°C and 46°C respectively and statically cultured for 12 hours, and the OD 600 .

[0099] The temperature resistance of the animal Bifidobacterium lactis B7 directly affects the selection of the bacterial powder manufacturing process and the production cost of the strain. The experimental results show (Table 4) that B7 can grow well at 37°C, 40°C, 43°C and 46°C, so the strain B7 has a wide growth temperature range, and the strain has excellent temperature resistance, and the bacterial powder can be prepared by spray drying or freeze drying.

[0100] Table 4 Determination of temperature resistance of animal Bifidobacterium lactis B7

[0101]

[0102] Example 2

[0103] Stability test of animal Bifidobacterium lactis B7 bacterial powder

[0104] a) Three generations of activated culture

[0105] The animal Bifidobacterium lactis B7 strain was inoculated into MRS liquid medium at an inoculation amount of 5%, activated and cultured at 37°C for 48 hours, and continuously activated twice; the activated B7 bacterial liquid was inoculated into 100ml MRS liquid medium at an inoculation amount of 5%; and the inoculated bacterial liquid was placed in an anaerobic incubator at 37°C and statically cultured for 12 hours, and the OD 600 of the bacterial liquid was determined.

[0106] Under a sterile operating table, about 100ml of the second generation B7 strain was inoculated into 2000ml of sterilized medium with a temperature of 37±2°C, and shaken to mix uniformly.

[0107] The conical flask containing the inoculated second generation B7 strain was placed in a constant temperature culture room at 37±2°C, and statically cultured for 10±2 hours. When the OD 600 of the culture liquid was greater than 1, the culture was ended. The cultured third generation B7 strain culture liquid was placed in a refrigerator below 4°C for cold storage, and the storage period was 3 days.

[0108] b) Fermentation and centrifugation

[0109] About 2000 ml of the third generation activated culture seed was transferred to a clean and sterilized fermentation tank, the temperature of which was controlled at 37±2℃, and stirred for 5 min and then left to ferment for 10±2 h. When the fermentation broth OD 600 >2 and total acid >0.8%, the bacterial cells were obtained by centrifugation and transferred for emulsification and embedding.

[0110] C) Emulsification and embedding

[0111] The centrifuged bacterial cells were dispersed and emulsified with a solution of malt dextrin, trehalose and skimmed milk powder (1:1:1) prepared in advance for 1 h, and then the emulsion was adjusted to pH 5.0-6.0 with sterile calcium hydroxide or sodium hydroxide, and then subjected to low-temperature vacuum spray drying.

[0112] d) Powder preparation

[0113] (1) Low-temperature vacuum spray drying

[0114] During the spray drying process, the inlet air temperature was automatically controlled at 100-120℃, and the outlet air temperature was automatically controlled at 50-70℃, for low-temperature spray drying.

[0115] (2) Screening, packaging and testing

[0116] After spray drying, B7 was screened through a 50-mesh rotary vibrating screen and stored in a stainless steel storage tank. The material that could not pass through the vibrating screen was crushed by a crusher and then loaded into the stainless steel storage tank. B7 in the stainless steel storage tank was pumped into a bagging machine and then input into an automatic packaging machine through a pipeline. The B7 was automatically weighed at 1 kg per bag, heat sealed, coded and then sealed and conveyed to a finished product temporary storage room for sampling and testing. The appearance was white or white-like, there was no odor, the bacterial count was not less than 150 billion CFU / g, the dispersion and dissolution test did not settle, and the moisture content was less than 5%.

[0117] e) Stability test

[0118] B7 bacterial powder was divided into 100 g per bag and placed in a condition of temperature 37±2℃, relative humidity RH 75±5% and avoiding direct light to complete the stability test experiment.

[0119] The experimental results are shown in Figure 6 The initial viable bacterial count of B7 bacterial powder of animal Bifidobacterium lactis was 1.5×10 11 cfu / g, the survival rate was 52.4% at 37℃ and relative humidity RH 75±5% after 5 weeks of storage, while the survival rate of animal Bifidobacterium lactis bacterial powder prepared by freeze-drying on the market was only about 50% after 1 week of storage at 37℃ and relative humidity RH 75±5%. Therefore, the stability of B7 bacterial powder of animal Bifidobacterium lactis (prepared by low-temperature spray drying process) is good, which can meet the requirements of normal temperature storage and various processing scenarios with high stability requirements, and ensure the product shelf life requirement of viable bacterial count.

[0120] Example 3

[0121] Animal Bifidus lactis B7 gastrointestinal tolerance and bile salt tolerance test

[0122] 1) Activation and culture of the strain

[0123] In a sterile clean bench, the preserved animal Bifidus lactis B7 was inoculated into a centrifuge tube containing 10 mL of MRS liquid medium, and incubated at 37°C in a constant temperature biochemical incubator for 12 h. The cultured bacterial solution was turbid with white precipitate.

[0124] 2) Animal Bifidus lactis B7 tolerance to gastric juice and intestinal juice

[0125] Electrolyte solution A: weigh potassium chloride 0.064 g, potassium dihydrogen phosphate 0.015 g, sodium bicarbonate 0.263 g, sodium chloride 0.345 g, magnesium chloride hexahydrate 0.003 g, ammonium carbonate 0.006 g, 1.5 g of tryptone, 0.05 g of L-cysteine hydrochloride monohydrate, add 95 mL of distilled water to dissolve thoroughly, adjust the pH to 3.0 with concentrated hydrochloric acid or 1 mol / L sodium hydroxide solution, and dilute to 100 mL. High pressure sterilization at 121°C for 15 min.

[0126] Electrolyte solution B: weigh calcium chloride dihydrate 0.022 g, dissolve in water and dilute to 100 mL. High pressure sterilization at 121°C for 15 min.

[0127] Take 8.0 mL of electrolyte solution A and 1.0 mL of electrolyte solution B in a beaker, add an equivalent of 4,000 U of pepsin, and test according to the principles of standard simulated gastric juice (pH 3.0, simulated gastric juice treatment time 2.0 h), empty stomach simulated gastric juice (pH 2.0, simulated gastric juice treatment time 0.5 h), and full stomach simulated gastric juice (pH 4.0, simulated gastric juice treatment time 3 h). Adjust the pH with 1 mol / L hydrochloric acid solution or 1 mol / L sodium hydroxide solution, dilute to 10 mL, mix well, and filter through a 0.22 μm sterile filter to prepare the simulated gastric juice, which is prepared and used immediately.

[0128] Prepare simulated gastric juice: NaCl 2.0 g / L, adjust the pH to 2.0, 2.5, 3.0 and 4.0 with HCl, high pressure sterilization, pepsin 3.2 g / L, pepsin is added fresh during the experiment.

[0129] Prepare simulated intestinal fluid: potassium dihydrogen phosphate 6.8 g / L, adjust pH to 7.5 with NaOH, autoclave, trypsin 10.0 g / L, trypsin is added fresh during the experiment.

[0130] Weigh 1.0 g of sample with sterile operation, place it in a sterile homogenization bag containing 99 mL of dissolution solution (weigh 8.5 g of sodium chloride, 0.5 g of L-cysteine hydrochloride monohydrate, add 1000 mL of distilled water to dissolve thoroughly, and autoclave at 121 ℃ for 15 min after dispensing), mix thoroughly, then pass through the homogenizer at 10 times / sec for 2 min until the sample is evenly dispersed, to prepare the sample bacterial solution, which is prepared fresh. Determine the total number of viable bacteria in the sample bacterial solution, denoted as N1. Take 5.0 mL of simulated gastric juice and 5.0 mL of sample bacterial solution in a 50 mL centrifuge tube, vortex mix, and slowly add about 5 mL of mineral oil on top of the solution with a rubber bulb dropper to form an oil seal layer.

[0131] Select the simulated gastric juice conditions, place the centrifuge tube in a 37 ℃ constant temperature water bath for the specified treatment time, remove the mineral oil with a rubber bulb dropper, and determine the total number of viable bacteria according to the viable bacteria determination method, denoted as . Repeat the determination of gastric juice tolerance test twice according to the above method, where the total number of viable bacteria is denoted as and , the total number of viable bacteria in the simulated gastric juice is denoted as and . Calculate the relative average deviation of , , and , and total number of viable bacteria determination results, the relative average deviation of the two groups of data should not be more than 15%, otherwise the gastric juice tolerance test should be re-done.

[0132] Gastric juice tolerance is calculated according to formula (1):

[0133] …………………(1)

[0134] In the formula:

[0135] A gastric acid tolerance, expressed in percentage (%);

[0136] initial total number of viable bacteria in the first gastric juice tolerance test, unit: CFU / mL;

[0137] initial total number of viable bacteria in the second gastric juice tolerance test, unit: CFU / mL;

[0138] - the initial total number of viable bacteria, in CFU / mL, for the third gastric juice resistance test;

[0139] - the total number of viable bacteria, in CFU / mL, after treatment for the first gastric juice resistance test;

[0140] - the total number of viable bacteria, in CFU / mL, after treatment for the second gastric juice resistance test;

[0141] - the total number of viable bacteria, in CFU / mL, after treatment for the third gastric juice resistance test;

[0142] 2 - the dilution factor conversion coefficient;

[0143] 1 / 3 - the average of the three parallel data;

[0144] The calculation result is expressed to the integer.

[0145] According to the standard simulated gastric juice (pH 3.0, simulated gastric juice treatment time 2.0 h), empty stomach simulated gastric juice (pH 2.0, simulated gastric juice treatment time 0.5 h), and full stomach simulated gastric juice (pH 4.0, simulated gastric juice treatment time 3 h). The animal Bifidobacterium lactis B7 gastric juice resistance data are shown in Table 5. At pH 2.0, the survival rate of B7 was 75.00%; at pH 3.0, the survival rate of B7 was 97.73%; at pH 4.0, the survival rate of B7 was 100.00%; therefore, the animal Bifidobacterium lactis B7 has very excellent gastric juice resistance.

[0146] Table 5 Gastric acid resistance of animal Bifidobacterium lactis B7

[0147]

[0148] 3) Animal Bifidobacterium lactis B7 tolerance to bile salts

[0149] The animal Bifidobacterium lactis B7 strain was inoculated into the MRS liquid medium at an inoculation amount of 5%, activated and cultured at 37°C for 24 hours, and continuously activated twice. The activated B7 bacterial liquid was inoculated into the MRS liquid medium at an inoculation amount of 5%, and statically cultured at 37°C in an anaerobic constant temperature incubator for 12 h. The cultured bacterial liquid was centrifuged at 5000 rpm for 10 min to collect the bacterial bodies, and the bacterial bodies were uniformly shaken with sterile normal saline.

[0150] The shock uniform bacterial liquid was added into MRS medium with bile salt concentration of 1.0 g / L, 2.0 g / L, 3.0 g / L and 0.0 g / L (initial bacterial liquid) at an addition amount of 10%, and the bile salt concentration of 0.0 g / L was used as a control group. Then, the bacterial liquid was incubated in a constant temperature incubator at 37℃ for 3h. The incubated bacterial liquid was taken out, immediately diluted with sterile normal saline at 10 times, mixed uniformly by beating, and then B7 was detected; the viable bacteria for detection were counted, and the survival rate was calculated, and the calculation formula was as follows:

[0151] Strain survival rate (%) = test group / control group x 100%.

[0152] The bile salt tolerance data of animal Bifidobacterium lactis B7 are shown in Table 6: when the bile salt concentration is 1.0 g / L and 2.0 g / L, the survival rates of B7 strain are 98.25% and 90.13% respectively, but when the bile salt concentration reaches 3.0 g / L, the survival rate of B7 strain still reaches 82.45%. The bile salt concentration in the intestinal tract is not more than 3.0 g / L, which indicates that the B7 strain can tolerate the bile salt in the intestinal tract.

[0153] Table 6 Bile salt tolerance of animal Bifidobacterium lactis B7

[0154]

[0155] In summary, the animal Bifidobacterium lactis B7 screened by the present application has good gastric acid and bile salt tolerance, and can play a role in regulating human intestinal flora and improving intestinal inflammation through the gastrointestinal tract.

[0156] Example 4

[0157] Determination of metabolism of animal Bifidobacterium lactis B7 to generate sulforaphane and indole-3-propionic acid from glucoraphanin and tryptophan

[0158] 1) Determination of metabolism of animal Bifidobacterium lactis B7 to generate sulforaphane from glucoraphanin

[0159] The animal Bifidobacterium lactis B7 strain was inoculated into MRS liquid medium at an inoculation amount of 5%, and incubated at 37℃ for 24 hours, and continuously incubated twice. The activated B7 bacterial liquid was inoculated into MRS-GRP medium at an inoculation amount of 5% and cultured for 12h, and the concentration of glucoraphanin (GRA) and sulforaphane (SFN) in the fermentation supernatant was analyzed to determine the ability of animal Bifidobacterium lactis B7 to convert glucoraphanin to sulforaphane, and the culture liquid without inoculation of microorganisms was used as a control, and the conversion efficiency of sulforaphane was calculated as follows.

[0160] Conversion rate (%) = sulforaphane content at the end of fermentation / initial glucoraphanin content x 100%.

[0161] Sulforaphane is the active product of glucoraphanin in vivo after hydrolysis by myrosinase or intestinal flora, and sulforaphane can increase intestinal peristalsis and regulate the role of intestinal flora. The experimental results are shown in Table 7.

[0162] Table 7 Content of glucoraphanin and sulforaphane in animal Bifidobacterium lactis B7 fermentation supernatant

[0163]

[0164] The experimental results show that the conversion efficiency of animal Bifidobacterium lactis B7 in MRS-GRP medium for 12h to metabolize glucoraphanin to generate sulforaphane is 43.42%, therefore, animal Bifidobacterium lactis B7 can be used to develop probiotic preparations and other products in combination with glucoraphanin to improve the in vivo conversion efficiency of glucoraphanin.

[0165] 2) Determination of high indole propionic acid production of animal Bifidobacterium lactis B7

[0166] After animal Bifidobacterium lactis B7 was activated twice in MRS medium, the activated B7 bacterial liquid was inoculated into MRS and MRS-GRP media with different tryptophan concentrations (tryptophan addition amount was 0.1g / L, 0.2g / L, 0.3g / L) at an inoculation amount of 5%, mixed uniformly, and then divided into sterile test tubes (18x180mm) at 8ml / branch; the divided candidate strain bacterial liquid was placed in a 37℃ constant temperature incubator for static culture, and the indole propionic acid (IPA) content was determined by an enzyme label instrument, and the method was as follows:

[0167] Microbial indole propionic acid ELISA detection kit method, first take out the required board from the aluminum foil bag after 20min of room temperature balance, and the remaining board is sealed with a self-sealing bag and put back at 4℃; set the standard sample hole and the sample hole, add different concentrations of standard sample 50μL to each standard sample hole; add 10μL of sample to be tested to the sample hole, and then add 40μL of sample diluent; blank hole does not add; except for the blank hole, add 100μL of horseradish peroxidase (HRP) labeled detection antibody to each well of the standard sample hole and the sample hole, cover the reaction hole with a sealing film, and incubate in a 37℃ water bath or constant temperature box for 60min; discard the liquid, dry on the blotting paper, fill each well with washing solution, stand for 1min, shake off the washing solution, dry on the blotting paper, repeat the plate washing 5 times (also can use plate washing machine to wash the plate); add 50μL of substrate A and B to each well, and incubate at 37℃ in the dark for 15min; add 50μL of stop solution to each well, and measure the OD value of each well at 450nm wavelength within 15min.

[0168] Standard curve and sample determination

[0169] Using the IPA standard in the kit (S0-S5), the concentration is: 0, 3, 6, 12, 24, 48 nmol / L (0.0000, 0.0006, 0.0011, 0.0023, 0.0045, 0.0091 μg / g) in turn, according to the method of indolepropionic acid ELISA detection kit, in the Excel worksheet, with OD value as abscissa, sample concentration as ordinate, draw the standard linear regression curve, calculate the sample concentration value according to the curve equation. The results of IPA standard curve fitting are y = 0.0016x 2 + 0.0019x - 0.0001, R 2 = 0.9968.

[0170] Sample determination, centrifuge the fermentation broth at 7000 r / min for 10 min, collect the supernatant, dilute 1 mL of supernatant to 25 mL with water for MRS, and dilute 1 mL of supernatant to 30 mL with water for MRS-GRP.

[0171] Table 8 IPA content produced by Bifidobacterium animalis lactis B7 in different tryptophan content culture medium

[0172]

[0173] Note: IPA content is calculated according to the results of IPA standard curve multiplied by the dilution factor

[0174] The results of Bifidobacterium animalis lactis B7 in different tryptophan content culture medium are shown in Table 8. When the tryptophan content is 0.2 g / L, the IPA content in MRS and MRS-GRP culture medium reaches 0.2123 μg / g and 0.2683 μg / g, respectively, and the increasing trend of IPA content tends to be flat with the increase of tryptophan addition. When the tryptophan content is 0.1 g / L, 0.2 g / L and 0.3 g / L, the IPA content in MRS-GRP medium is increased by 21.69%, 26.38% and 28.12% respectively compared with that in MRS medium, which shows that Bifidobacterium animalis lactis B7 can metabolize tryptophan to produce indolepropionic acid, and the IPA content is higher in MRS-GRP medium, which shows that MRS-GRP medium can synergistically promote B7 to metabolize tryptophan to produce indolepropionic acid.

[0175] 3) Bifidobacterium animalis lactis B7 fermentation broth antibacterial experiment

[0176] Pathogenic bacteria inhibition experiment: pour 10 mL of water agar medium in a sterile plate, after cooling and solidification, put the Oxford cup, add the indicator bacteria suspension (Escherichia coli 8099, Helicobacter pylori ATCC 26695, Streptococcus mutans CGMCC 1.2499, Staphylococcus aureus CMCC 26003, Clostridium perfringens ATCC 13124, Candida albicans ATCC 10231) into the agar medium corresponding to the growth of the indicator bacteria, which is cooled to 50℃, so that the concentration of the indicator bacteria is 10 6 CFU / mL, mix well, pour it on the bottom layer of water agar, after it solidifies, remove the Oxford cup with tweezers, which forms a hole, add 200 μL of the sample to be tested (the sample to be tested includes MRS medium animal Bifidobacterium lactis B7 fermentation broth, MRS-GRP medium animal Bifidobacterium lactis B7 fermentation broth) to each hole, use un-inoculated MRS medium and MRS-GRP medium as controls for Escherichia coli 8099, diffuse for 30 min, and culture at 37℃ for 15-24 h. Observe whether there is an inhibition zone around the culture hole and measure its diameter with a vernier caliper, record the diameter of the inhibition zone, and finally evaluate the inhibition activity according to the presence or absence and size of the inhibition zone.

[0177] Table 9 Analysis of the inhibition ability of B7 fermentation broth obtained by different media

[0178]

[0179] The inhibition ability of animal Bifidobacterium lactis B7 fermentation broth obtained by different media is shown in Table 9. Compared with the animal Bifidobacterium lactis B7 fermentation supernatant obtained by MRS medium fermentation, the animal Bifidobacterium lactis B7 fermentation broth obtained by MRS-GRP medium fermentation has significantly improved inhibition ability on pathogenic microorganisms. The inhibition zone of 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 all reached more than 21 mm, and the inhibition performance on Candida albicans increased by 49%. Combined with the control group MRS medium and MRS-GRP medium, no inhibition zone was observed, but after adding animal Bifidobacterium lactis B7 fermentation, the inhibition zone increased significantly. Combined with the previous conclusion that animal Bifidobacterium lactis B7 can convert sinigrin to sulforaphane, it is concluded that the main reason for the significant improvement of the inhibition of animal Bifidobacterium lactis B7 in MRS-GRP medium is the production of sulforaphane in the fermentation broth, which synergistically improves the inhibition of pathogenic bacteria with animal Bifidobacterium lactis B7.

[0180] Table 10 Analysis of the inhibition ability of B7 fermentation broth obtained by different media with added tryptophan

[0181]

[0182] As can be seen from Table 10: adding tryptophan 0.2 g / L, Bifidobacterium animalis lactis B7 in MRS medium and MRS-GRP medium, the inhibition zone of all pathogenic bacteria is increased compared with the inhibition zone without adding tryptophan, among which the inhibition of the fermentation broth of MRS-GRP medium with added tryptophan is the best. Combined with the increase of indolepropionic acid content of Bifidobacterium animalis lactis B7 in MRS and MRS-GRP medium with added tryptophan, and the metabolism and synthesis of sulforaphane in MRS-GRP medium, indolepropionic acid has good antibacterial property, which shows that indolepropionic acid or sulforaphane produced by Bifidobacterium animalis lactis B7 has a synergistic effect on the antibacterial effect of the strain itself.

[0183] Example 5

[0184] Effect of Bifidobacterium animalis lactis B7 and sulforaphane glucosinolate on constipation

[0185] Bifidobacterium animalis lactis B7 strain was inoculated into MRS medium at an inoculation amount of 5%, activated at 37°C for 24 hours under anaerobic culture, and then transferred to MRS medium for culture for 24 hours after continuous activation for two times. The fermentation broth was centrifuged at 8000 rpm for 5 minutes at 4°C. The cells were collected, and one part was washed twice with sterile PBS buffer and resuspended, with a final concentration of 10 8 CFU / ml, and the other part was washed twice with sterile PBS buffer and resuspended with PBS buffer containing sulforaphane glucosinolate, with a final concentration of 10 8 CFU / ml and a final concentration of 0.5 g / L of sulforaphane glucosinolate (GRA), and stored at 4°C.

[0186] Zebrafish treatment: AB zebrafish embryos were purchased from the Zebrafish Platform of the Medical College of Zhejiang University. The culture conditions of zebrafish are as follows:

[0187] The conductivity of water is 500-550 μS cm -1 , the pH value is 7.20-7.60, the temperature is 28°C, the culture density of each sterile culture dish is 50 embryos per culture dish, and the culture temperature is 28°C. The culture medium is sterile E3 (1% NaCl, 0.17 mM KCI, 0.33 mM CaCl2, 0.33 mM magnesium sulfate and 0.00003% methylene blue, pH 7.00). 75% sterile E3 medium is replaced every day to remove accumulated waste and increase dissolved oxygen. On the 5th day after fertilization, the larvae were transferred to a 6-well plate for breeding at a density of 20 per well.

[0188] Experimental design: 5 dpf zebrafish were selected as experimental objects, and divided into 5 groups, 15 fish in each group. They are E3 medium normal control group (Control), the other 4 groups are all constructed by using loperamide hydrochloride method to construct intestinal peristalsis zebrafish model (the concentration of loperamide hydrochloride is 10 μg / mL), and then treated with positive drug or different bacterial liquid, the four groups are loperamide hydrochloride modeling group (LOP), LOP+positive drug control group (referred to as AC group, domperidone AC, 50 μg / mL, H10910003, Xi'an Yangsen Pharmaceutical Co., Ltd.), LOP+B7 group (abbreviated as B7 group) and LOP+B7+glucoraphanin (abbreviated as B7+GRA) group. After the constipated zebrafish were soaked for 24 hours, the zebrafish were anesthetized with anesthetic, and the time difference of two consecutive peristaltic wave peaks at the same position of the intestinal tract of the zebrafish was observed under an integrated microscope. The data were expressed as "mean ± standard deviation" by one-way ANOVA program for single factor analysis of variance using GraphPad Prism 9.5 statistical software, P<0.05 indicates that it has statistical significance.

[0189] The test results are shown in Table 11. The time difference of two peristaltic wave peaks of the zebrafish intestinal tract in the loperamide hydrochloride modeling group (LOP) is higher than that in the control group, indicating that the constipation modeling is successful. The time difference of two peristaltic wave peaks of the zebrafish intestinal tract in the positive drug AC group and the probiotic B7 group is significantly lower than that in the modeling group (LOP), indicating that they have a promoting effect on intestinal peristalsis, among which the positive drug is the best, followed by animal Bifidobacterium lactis B7+GRA, P <0.01), animal Bifidobacterium lactis B7 ( P <0.01) has the weakest promoting effect on intestinal peristalsis, and animal Bifidobacterium lactis B7+GRA is better than animal Bifidobacterium lactis B7 alone. The above results show that probiotic B7 and glucoraphanin GRA can significantly increase intestinal peristalsis and have a good effect on relieving constipation.

[0190] Table 11 Effect of animal Bifidobacterium lactis B7 and glucoraphanin on intestinal peristalsis

[0191]

[0192] Note: * is P <0.05, significant difference, ** is P <0.01, extremely significant difference (compared with the modeling group)

[0193] Example 6 Analysis of animal Bifidobacterium lactis B7 on ulcerative colitis or inflammatory bowel disease

[0194] The animal Bifidobacterium lactis B7 bacterial powder used in this example has a viable bacterial count of 1×10 11CFU / g. The Bifidobacterium animalis lactis B7 bacterial powder was prepared by the following method: the Bifidobacterium animalis lactis B7 strain was inoculated into MRS medium at an inoculation amount of 5%, activated anaerobically at 37°C for 24 hours, and subcultured into MRS medium for 24 hours after two times of continuous activation. The fermentation broth was centrifuged at 8000 rpm for 5 minutes at 4°C, and the Bifidobacterium animalis lactis B7 bacterial slurry was collected. Then, the bacterial slurry was spray-dried to obtain the Bifidobacterium animalis lactis B7 bacterial powder (i.e., the Bifidobacterium animalis lactis B7 strain spray-dried powder).

[0195] 1) Construction of a DSS-induced ulcerative colitis or inflammatory bowel disease model and alleviating effect of Bifidobacterium animalis lactis B7 on weight loss caused by ulcerative colitis or inflammatory bowel disease

[0196] C57BL / 6 mice were used as the pharmacological research object, and the mice were randomly divided into 3 groups (n = 6), namely, a blank control group (NC group), a model control group (VC group), and a Bifidobacterium animalis lactis B7 group (B7). The VC group and the B7 group were given 2.5% dextran sodium sulfate solution (DSS solution) as drinking water for 7 consecutive days to construct an ulcerative colitis or inflammatory bowel disease model. The NC group used pure water as drinking water as a normal physiological blank control. The NC group and the VC group were given 0.2 mL of normal saline (0.9% NaCl) by gavage, and the B7 group was given 0.2 mL of Bifidobacterium animalis lactis B7 bacterial suspension (diluted with the Bifidobacterium animalis lactis B7 bacterial powder of the preceding embodiment to obtain a bacterial suspension with a viable bacterial count of 5×10 9 CFU / mL) by gavage.

[0197] The body weight changes of the mice in each group after modeling are shown in Figure 7 The body weight of the mice in the VC group showed a decreasing trend during the modeling period, and the body weight decreased rapidly from the fifth day. Weight loss is one of the important pathological manifestations of ulcerative colitis or inflammatory bowel disease. The body weight change of the mice in the B7 group was close to that of the NC group, and no decreasing trend was observed. This indicates that Bifidobacterium animalis lactis B7 can effectively alleviate the weight loss caused by ulcerative colitis or inflammatory bowel disease.

[0198] 2) Therapeutic effect of Bifidobacterium animalis lactis B7 on the disease activity index DAI of ulcerative colitis or inflammatory bowel disease

[0199] After modeling, the body weight of the mice was measured every day, and the degree of diarrhea and fecal occult blood were observed. The fecal occult blood was determined by the benzidine method. The disease activity index DAI of ulcerative colitis or inflammatory bowel disease was scored according to Table 12.

[0200] Table 12: Scoring criteria for the disease activity index

[0201]

[0202] 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.

[0203] 3) The repairing effect of Bifidobacterium animalis subsp. lactis B7 on colonic tissue damage caused by ulcerative colitis or inflammatory bowel disease.

[0204] 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.

[0205] 4) Protection of the colonic mucosa by Bifidobacterium animalis subsp. lactis B7 in patients with ulcerative colitis or inflammatory bowel disease.

[0206] 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.

[0207] 5) Effects of Bifidobacterium animalis subsp. lactis B7 on serum inflammatory factor levels in patients with ulcerative colitis or inflammatory bowel disease.

[0208] 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.

[0209] 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). p The 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.

[0210] 6) Effect of Bifidobacterium animalis subsp. lactis B7 on serum LPS levels in patients with ulcerative colitis or inflammatory bowel disease.

[0211] 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.

[0212] 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.

[0213] 7) Effects of Bifidobacterium animalis subsp. lactis B7 on D-LA secretion in ulcerative colitis or inflammatory bowel disease.

[0214] 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.

[0215] 8) Effects of Bifidobacterium animalis subsp. lactis B7 on IPA in ulcerative colitis or inflammatory bowel disease.

[0216] 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 the intervention of Bifidobacterium animalis lactis B7, the intestinal microorganisms promoted the conversion of tryptophan in food, and increased the content of IPA in blood P <0.05), the intervention of Bifidobacterium animalis lactis B7 could restore the level of IPA to a certain extent, IPA could repair the intestinal mucosa, activate the AhR pathway of the intestine, and improve the intestinal homeostasis.

[0217] In summary, Bifidobacterium animalis lactis B7 can alleviate the 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; hematoxylin-eosin staining and pathological analysis results show that Bifidobacterium animalis lactis B7 can significantly reduce the colon damage of mice, reduce the loss of the number of goblet cells in colitis mice, maintain the mucus secretion of the colon to a certain extent, and protect the mucosa layer; after the intervention of B7, the levels of pro-inflammatory factors in the serum of colitis mice decreased, among which IL-6 (compared with 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), and the levels of anti-inflammatory factors increased (compared with the model group p <0.05), and IPA increased (compared with the model group p <0.05), so B7 reduced the inflammation level of colitis mice by regulating these specific inflammatory factors, and played an anti-inflammatory role. After the intervention of B7, LPS was significantly reduced (compared with the model group p <0.05), the content of D-LA was extremely significantly reduced (compared with the model group p <0.01), and IPA was significantly increased, so B7 intervention can improve the intestinal barrier function, reduce the intestinal permeability, and increase the content of IPA, thereby achieving the purpose of preventing and treating ulcerative colitis or inflammatory bowel disease.

[0218] Example 7 Application experiment of Bifidobacterium animalis lactis B7 bacterial powder in improving constipation type ulcerative colitis

[0219] The solid preparation of *Bifidobacterium animalis* subsp. lactis B7 is formulated from *Bifidobacterium animalis* subsp. lactis B7 bacterial powder. The *Bifidobacterium animalis* subsp. lactis B7 bacterial powder 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. 11 CFU / 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-predominant ulcerative colitis were compounded at a weight ratio of 1:3:4 to obtain a solid preparation of *Bifidobacterium lactis* B7 with a viable count of 2.5 × 10⁻⁶ CFU / g; the concentration of the supernatant was determined, and the B7 metabolite powder was obtained after low-temperature spray drying. 10 Solid dosage form, CFU / strip, 2g / strip.

[0220] 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). The control group (5 cases) took one 2g sachet of maltodextrin solid beverage twice daily, morning and evening. The intervention group (5 cases) used Bifidobacterium animalis subsp. lactis B7, taking one 2.5×10g sachet of Bifidobacterium animalis subsp. lactis solid preparation twice daily, morning and evening. 10 CFU / strip, 2g), for a total of 30 days.

[0221] Observation indicators:

[0222] Symptom scores were assessed in both groups before and 1 month after intervention. The scores were based on abdominal pain, weekly stool frequency, stool time, stool character, ulcerative colitis or inflammatory bowel disease disease activity index DAI (degree of fecal occult blood), each scored 4 points, and the total score was 20 points. The higher the score, the more severe the symptoms. The effectiveness and adverse reactions of the population were evaluated 1 month after intervention. The efficacy evaluation criteria for refractory constipation in the Constipation Primary Diagnosis and Treatment Guidelines (2019) were used to determine the improvement of symptom scores. That is, a total score decrease of at least 70% (total score ≤ 6, no significant adverse reactions, judged to be effective; total score decreased by 40-70% (6 < total score ≤ 14), no adverse reactions, judged to be effective; total score decreased by less than 40% or increased (14 < total score), severe adverse reactions occurred, judged to be ineffective.

[0223] Total effective rate = (number of cases with significant effect + number of cases with effective effect) / total number of cases × 100%.

[0224] Statistical methods: SPSS 20.0 software was used for statistical analysis. Measurement data was expressed as (±s, points). P < 0.05 was considered statistically significant. P <0.05 for a statistically significant difference.

[0225] Symptom scores

[0226] Before intervention, there was no statistically significant difference in symptom scores between the two groups (P > 0.05). One month after intervention, the symptom scores of both groups were lower than before treatment, and the symptom scores of the intervention group were lower than those of the control group, with statistically significant differences compared with the same group before intervention (P < 0.01) and compared with the control group after intervention (P < 0.01). The specific experimental results are shown in Table 13. P P P

[0227] Table 13 B7 intervention results for constipation-type ulcerative colitis ±s, points

[0228]

[0229] Note: 1) Compared with the same group before intervention, P a P < 0.05; compared with the control group after intervention, 2) P b P < 0.05.

[0230] Population experimental effectiveness and adverse reactions

[0231] One month after intervention, the total effective rate of the control group was 20%, and the total effective rate of the intervention group was 80%, which was 4 times that of the control group, as shown in Table 14. No serious adverse reactions occurred in either group.

[0232] ​​​Table 14 Effect after intervention of two groups

[0233]

[0234] Based on the above experimental results, the applicant proposes the application of the animal Bifidobacterium lactis B7 in the treatment of constipation-type ulcerative colitis, and recommends a dosage of 5x10 10 CFU / day.

[0235] Example 8

[0236] The live Bifidobacterium lactis B7 powder of this example, i.e., the Bifidobacterium lactis B7 powder obtained by centrifuging the fermentation broth obtained after 24h static culture of Bifidobacterium lactis B7 in MRS medium in a 37℃ constant temperature incubator, and then freeze-drying or spray-drying the centrifugal precipitate, wherein the live Bifidobacterium lactis B7 for improving constipation-type ulcerative colitis is 1.5x10 11 CFU / g.

[0237] The Bifidobacterium lactis B7 powder for improving constipation-type ulcerative colitis of this example is used for preparing a medicine for preventing / treating constipation-type ulcerative colitis.

[0238] A medicine containing Bifidobacterium lactis B7 for improving constipation-type ulcerative colitis is prepared by mixing the Bifidobacterium lactis B7 powder for improving constipation-type ulcerative colitis of this example with a pharmaceutical carrier at a weight ratio of 1:2, to obtain a medicine containing Bifidobacterium lactis B7 for improving constipation-type ulcerative colitis, wherein the live Bifidobacterium lactis B7 is 5.0x10 10 CFU / g, and the capsule is 0.5g. The recommended dosage of the medicine is 5.0x10 10 CFU / day.

[0239] The live Bifidobacterium lactis B7 powder of this example is used as a ferment in the fermentation of pickles, sauerkraut and other fermented foods, and the dosage is 2x10 7 CFU / kg of fermented food.

[0240] Example 9

[0241] The animal Bifidobacterium lactis B7 fermentation composition of the present embodiment is a metabolite of animal Bifidobacterium lactis B7, which is prepared by filtering the fermentation broth obtained by culturing animal Bifidobacterium lactis B7 in a MRS-GRP medium at 37°C in an incubator for 24 hours, and then high-temperature inactivating (121°C, 15 minutes), concentrating, and spray drying the obtained fermentation broth supernatant. The metabolite contains sulforaphane active ingredients, and the dead cell count of the animal Bifidobacterium lactis B7 is 1×10 11 CFU / g.

[0242] The animal Bifidobacterium lactis B7 fermentation composition for improving constipation-type ulcerative colitis of the present embodiment is used for preparing a drug for preventing or treating constipation-type ulcerative colitis, and the dosage is 100-1000 mg / day.

[0243] Example 10

[0244] The animal Bifidobacterium lactis B7 fermentation composition of the present embodiment is a mixture of animal Bifidobacterium lactis B7 live bacteria powder and fermentation supernatant freeze-dried powder in a weight ratio of 1:1, i.e., the fermentation broth obtained by culturing animal Bifidobacterium lactis B7 in a MRS medium added with 0.2 g / L tryptophan at 37°C in an incubator for 24 hours is centrifuged, the centrifugal precipitate is freeze-dried or spray-dried to obtain animal Bifidobacterium lactis B7 powder. The live bacteria count of the animal Bifidobacterium lactis B7 for improving constipation-type ulcerative colitis is 1.5×10 11 CFU / g, and the supernatant is freeze-dried to obtain animal Bifidobacterium lactis B7 supernatant freeze-dried powder containing indolepropionic acid, short-chain fatty acids, and other active ingredients.

[0245] The animal Bifidobacterium lactis B7 fermentation composition of the present embodiment is used for preparing a product for regulating intestinal flora, promoting intestinal peristalsis, enhancing immunity, resisting inflammation, inhibiting bacteria, or improving constipation.

[0246] Example 11

[0247] The animal Bifidobacterium lactis B7 fermentation composition of the present embodiment is a solid powder of animal Bifidobacterium lactis B7 metabolites, i.e., the fermentation broth supernatant (sulforaphane content ≥0.5 g / L) obtained by culturing animal Bifidobacterium lactis B7 in a MRS-GRP medium added with 0.2 g / L tryptophan at 37°C in an incubator for 24 hours is centrifuged, and then freeze-dried or spray-dried to obtain a solid powder of animal Bifidobacterium lactis B7 metabolites. The indolepropionic acid content in the fermentation composition is ≥2 μg / g.

[0248] The animal Bifidobacterium lactis B7 fermentation composition of the present embodiment is used for preparing a product for regulating constipation, and the dosage is 100-1000 mg / day.

[0249] Example 12

[0250] The animal Bifidobacterium lactis B7 fermentation composition of the present embodiment is a composition of animal Bifidobacterium lactis B7 viable powder and animal Bifidobacterium lactis B7 postbiotic at a weight ratio of 2:1, wherein the animal Bifidobacterium lactis B7 viable powder is prepared by the method described in Example 6, and the viable bacterial content is 1×10 11 CFU / g, and the animal Bifidobacterium lactis B7 postbiotic is prepared by the method described in Example 9, wherein the cell number is 5×10 10 CFU / g.

[0251] The animal Bifidobacterium lactis B7 fermentation composition of the present embodiment is used for preparing a medicine for preventing or treating ulcerative colitis or inflammatory bowel disease, and the dosage is 0.2-3 g / day.

[0252] Example 13

[0253] The animal Bifidobacterium lactis B7 fermentation composition of the present embodiment includes animal Bifidobacterium lactis B7 viable powder, i.e., the freeze-dried powder or spray-dried powder of the bacterial bodies obtained by drying the fermentation broth after centrifugation, wherein the fermentation broth is obtained by culturing animal Bifidobacterium lactis B7 in sterilized fresh cow milk added with 0.10 g / L tryptophan at 37°C for 24 h, and the viable bacterial content is 2×10 10 CFU / g.

[0254] The animal Bifidobacterium lactis B7 of the present embodiment is used for preparing a biological preparation for regulating intestinal flora or promoting intestinal peristalsis, wherein the biological preparation is a powder, and the preparation method is as follows: 40 parts by weight of animal Bifidobacterium lactis B7 viable powder (2×10 10 CFU / g), 30 parts by weight of water extract of broccoli seeds (13 g / 100 g of glucoraphanin), 20 parts by weight of indolepropionic acid, and 10 parts by weight of malt dextrin.

[0255] The raw materials of the composition are first sieved through a 40-mesh screen, then uniformly mixed according to the ratio, and packaged by a screw back-seal packaging machine to prepare a solid powder with a dosage of 2 g / bag, which has the effects of improving constipation, ulcerative colitis, and inflammatory bowel disease.

[0256] The animal Bifidobacterium lactis B7 fermentation composition of the present embodiment is used for preparing a medicine for treating ulcerative colitis, inflammatory bowel disease, constipation, or constipation-type ulcerative colitis.

[0257] The fermented composition of animal Bifidobacterium lactis B7 in this embodiment is used as a fermenting agent in fermented food such as pickled vegetable and sauerkraut, and the amount is 2×10 7 CFU / Kg of fermented food.

[0258] Example 14

[0259] The drug for preventing and / or treating ulcerative colitis in this embodiment is a mixture of animal Bifidobacterium lactis B7 viable powder and animal Bifidobacterium lactis B7 metabolite in a weight ratio of 2:1, wherein the animal Bifidobacterium lactis B7 viable powder is prepared by the method described in Example 6, and the viable bacteria content is 3×10 10 CFU / g, and the animal Bifidobacterium lactis B7 metabolite is prepared by high-temperature inactivation (121℃, 15min), concentration, and spray drying of the centrifugal supernatant obtained by centrifugation of the fermentation broth after 24h fermentation of animal Bifidobacterium lactis B7 in MRS-GRP medium at 37℃.

[0260] The drug for preventing and / or treating ulcerative colitis or inflammatory bowel disease in this embodiment is recommended for adults in an amount of 0.2-3g / day.

[0261] Example 15

[0262] The drug for preventing or treating ulcerative colitis in this embodiment is animal Bifidobacterium lactis B7 postbiotic, wherein the animal Bifidobacterium lactis B7 postbiotic is prepared by cell lysis, high-temperature inactivation (121℃, 15min), concentration, and spray drying of the fermentation broth after 24h fermentation of animal Bifidobacterium lactis B7 in MRS medium at 37℃.

[0263] The drug for preventing or treating ulcerative colitis in this embodiment is used for preventing or treating ulcerative colitis, and the recommended amount is 0.5-3g / day, which has good efficacy.

[0264] Example 16

[0265] The drug for preventing or treating mixed ulcerative colitis in this embodiment comprises animal Bifidobacterium lactis B7 viable powder, which is the freeze-dried powder or spray-dried powder of the centrifugal precipitate obtained by centrifugation of the fermentation broth after 20h fermentation of animal Bifidobacterium lactis B7 in MRS medium at 37℃, and contains 8×10 10 CFU / g of viable bacteria.

[0266] The drug is in the form of capsules, and the preparation method of the capsules is as follows: 4.0×10 1040 parts by weight, starch 40 parts by weight, glucose 5 parts by weight, hydroxypropyl methylcellulose 5 parts by weight, lactitol 5 parts by weight, magnesium stearate 5 parts by weight.

[0267] The raw materials of the composition are mixed uniformly according to the ratio after being passed through a 40-mesh screen, and then are filled into capsules or packaged into powders to obtain the package.

[0268] The animal Bifidobacterium lactis B7 of the present application can select the ingredients and the dosage of the animal Bifidobacterium lactis B7 fermentation composition according to different applications, for example, the application of the animal Bifidobacterium lactis B7 powder in the preparation of a microbial preparation for improving ulcerative colitis or inflammatory bowel disease in Example 6, the dosage can also be 5*10 8 The application of the animal Bifidobacterium lactis B7 metabolite as a composition in the production of sulforaphane and / or tryptophan metabolites in Example 11, the application of the animal Bifidobacterium lactis B7 metabolite as a composition in the preparation of a drug for preventing or treating constipation in Example 9, the dosage can also be 2*10 9 The above technical features are changed, and those skilled in the art can understand and implement them through the description, so they will not be described again in the drawings.

Claims

1. A strain of Bifidobacterium animalis ssp. lactis B7, characterized in that, The animal bifidobacterium B7 (Bifidobacterium animalis subsp. lactis B7) Bifidobacterium animalis subsp. lactis B7) was deposited with the China Center for Type Culture Collection on April 19, 2024, and the deposit number is CCTCC NO: M2024734.

2. A fermentation composition, characterized in that, The fermentation composition is produced by fermenting the Bifidobacterium animalis subsp. lactis B7 according to claim 1, including live Bifidobacterium animalis subsp. lactis B7, inactivated Bifidobacterium animalis subsp. lactis B7, fermentation supernatant, fermentation precipitate, Bifidobacterium animalis subsp. lactis B7 postbiotic or spray-dried powder / lyophilized powder; the fermentation composition is produced by fermenting Bifidobacterium animalis subsp. lactis B7 in MRS medium, or MRS medium added with water extract of broccoli seeds or glucoraphanin, or MRS medium added with tryptophan.

3. A probiotic formulation characterized in that, including Bifidobacterium animalis lactis B7 as claimed in claim 1 or / and a fermentation composition as claimed in claim 2; the content of Bifidobacterium animalis lactis B7 in the probiotic preparation is > 2 x 10 7 CFU / g or 2 x 10 7 CFU / ml.

4. The probiotic formulation as claimed in claim 3, wherein, The probiotic preparation further includes glucoraphanin or water extract of broccoli seeds or indole-3-propionic acid.

5. Use of Bifidobacterium animalis subsp. lactis B7 according to claim 1 in the production of sulforaphane by metabolizing glucoraphanin and / or indole-3-propionic acid by metabolizing tryptophan.

6. Use of Bifidobacterium animalis lactis B7 according to claim 1 or of the fermentation composition according to claim 2 for the preparation of a product for modulating the intestinal flora, or for promoting intestinal peristalsis, or for strengthening the immune system, or for anti-inflammatory, or for bacteriostatic, or for improving constipation, characterized in that, The bacteriostasis refers to the bacteriostasis of Bifidobacterium animalis subsp. lactis B7 or the fermentation composition on Escherichia coli, Helicobacter pylori, Streptococcus mutans, Staphylococcus aureus, Clostridium perfringens and Candida albicans.

7. Use of Bifidobacterium animalis subsp. lactis B7 according to claim 1 and / or the fermentation composition according to claim 2 in the preparation of a medicament for relieving or treating ulcerative colitis, constipation.

8. The use according to claim 7, characterized in that, The ulcerative colitis is constipation-type ulcerative colitis.

9. Use of Bifidobacterium animalis subsp. lactis B7 according to claim 1 as a fermenting agent in the preparation of fermented food.

10. A medicament for preventing and treating ulcerative colitis and / or constipation, characterized by, The medicament contains Bifidobacterium animalis subsp. lactis B7 according to claim 1, the fermentation composition according to claim 2 or the probiotic preparation according to claim 3 or 4.

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