Animal bifidobacterium capable of relieving ulcerative colitis related colorectal cancer and application thereof

Abstract

The application discloses an animal bifidobacterium capable of relieving ulcerative colitis related colorectal cancer and an application thereof, and belongs to the technical field of microorganisms and the technical field of medicines. The application provides an animal bifidobacterium lactis (Bifidobacterium animalis subsp. lactis) CCFM1383 with a preservation number of GDMCC No: 64443. The animal bifidobacterium lactis CCFM1383 can effectively improve the symptoms of ulcerative colitis related colorectal cancer, specifically in relieving the weight loss of mice, improving the survival rate, prolonging the colon length, reducing the number of tumor nodules, reducing the spleen index, reducing the tumor tissue proliferation marker, promoting the apoptosis of tumor cells, regulating the content of inflammatory factors, improving the content of tight junction proteins, inhibiting the expression of cancer signal pathway genes, and improving the content of colon indole propionic acid.

Description

Technical Field

[0001] This invention relates to an animal Bifidobacterium that can alleviate ulcerative colitis-associated colorectal cancer and its application, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] Colorectal cancer is a malignant digestive tract tumor that seriously threatens human health. Its incidence rate ranks third among cancers worldwide, and its mortality rate is the second highest among malignant tumors globally. The risk of colorectal cancer in my country is also increasing year by year. Colorectal cancer is mainly divided into sporadic colorectal cancer and ulcerative colitis-related colorectal cancer. The former is mainly caused by changes in genetic factors, while the latter's risk comes from external environmental factors such as inflammation, diet, and lifestyle. Currently, the main clinical treatment for colorectal cancer is various types of radiotherapy and chemotherapy, which can produce certain side effects on patients. Probiotics, as a novel intervention method, have had their allergic and therapeutic effects on various diseases gradually verified, such as enhancing immunity, inhibiting intestinal inflammation, alleviating allergic reactions, and preventing tumors.

[0003] The gut is the most direct lesion area for colorectal cancer, and it also contains millions of gut microbiota. Tryptophan is an essential amino acid that can be metabolized by gut microbes into indole-3-carboxaldehyde (IAld), indoleacetic acid, indolelactic acid, and other indole compounds. Previous studies have shown that total indole products of tryptophan are significantly reduced in colorectal cancer patients. Studies have reported that tryptophan-indole metabolites have good anti-inflammatory capabilities. For example, restoring gut microbiota dysbiosis and increasing the production of indoleacetic acid by gut microbes can alleviate colitis. Supplementing with indolelactic acid-producing *Lactobacillus reuteri* or *Lactobacillus plantarum* can alleviate colorectal cancer. Whether other indole compounds have anti-inflammatory and anti-cancer effects has not been fully investigated. Although there is evidence that IAld can repair intestinal barrier damage in colitis mice and enhance the efficacy of cytotoxic T-lymphocyte-associated protein 4 against colorectal cancer, *Bifidobacterium lactis* subsp. *animal*, which can produce high levels of IAld and alleviate the development of ulcerative colitis-associated colorectal cancer, has not yet been found to produce high levels of IAld. Summary of the Invention

[0004] To address the existing problems, this invention provides a *Bifidobacterium animalis* subsp. *lactis* CCFM1383, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 64443.

[0005] In one embodiment of the present invention, the Bifidobacterium animalis subsp. lactis CCFM1383 is isolated from human feces.

[0006] In one embodiment of the present invention, the growth characteristics of the Bifidobacterium animalis subsp. lactis CCFM1383 are as follows: This strain is a facultative anaerobic bacterium, and it is optimal to inoculate it into mMRS liquid medium and culture it anaerobically at 37°C for 24 hours.

[0007] In one embodiment of the present invention, the colony characteristics of Bifidobacterium animalis subsp. lactis CCFM1383 are as follows: on mMRS solid medium, it is milky white, opaque, round and raised, with neat and smooth edges.

[0008] The present invention also provides a microbial preparation containing the aforementioned Bifidobacterium lactis subsp. CCFM1383.

[0009] In one embodiment of the present invention, the microbial preparation is a liquid or solid preparation containing Bifidobacterium lactis subsp. CCFM1383.

[0010] In one embodiment of the present invention, the number of viable Bifidobacterium lactis subsp. CCFM1383 in the microbial preparation is ≥1×10⁻⁶. 8 CFU / mL or ≥1×10 8 CFU / g.

[0011] In one embodiment of the present invention, the microbial preparation further contains glycerol and water.

[0012] The present invention also provides a method for preparing the microbial preparation, wherein the animal Bifidobacterium lactis subsp. CCFM1383 is fermented in a culture medium.

[0013] The present invention also provides the use of the aforementioned Bifidobacterium lactis subsp. CCFM1383, or the aforementioned microbial preparation, in the preparation of drugs for alleviating colon cancer.

[0014] In one embodiment of the present invention, the viable count of Bifidobacterium animalis subsp. lactis CCFM1383 in the drug is not less than 1 × 10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0015] In one embodiment of the present invention, the Bifidobacterium animalis subsp. lactis CCFM1383 can be used to prevent and alleviate ulcerative colitis-related colorectal cancer.

[0016] In one embodiment of the present invention, the Bifidobacterium lactis subsp. CCFM1383, or the microbial preparation, has at least one of the following effects: (a) to (g)

[0017] (a) It alleviates weight loss, improves survival rate, and reduces rectal bleeding in colon cancer model mice;

[0018] (b) Reduce the number of tumors in colon cancer model mice;

[0019] (c) Significantly reduced the levels of pro-inflammatory factors IL-17A, IL-1β, and IFN-γ in colon cancer model mice;

[0020] (d) Increase the level of the anti-inflammatory factor IL-10 in colon cancer model mice;

[0021] (e) Inhibits the proliferation of cancer cells and promotes apoptosis in a colon cancer model mouse;

[0022] (f) Inhibit the PI3K-Akt-mTOR signaling pathway;

[0023] (g) Increase the colonic IAld level in mice with colon cancer.

[0024] Beneficial effects:

[0025] The *Bifidobacterium animalis* subsp. *lactis* CCFM1383 described in this invention can effectively improve symptoms of ulcerative colitis-associated colorectal cancer, specifically in the following ways:

[0026] (1) The body weight of the colon cancer model mice was significantly reduced, from 24.33±0.51g to 26.35±0.48g;

[0027] (2) The survival rate of colon cancer model mice increased from 50% to 75%;

[0028] (3) The colon length of the colon cancer model mice increased from 4.55±0.21cm to 5.9±0.26cm, which was significantly different from that of the model group mice;

[0029] (4) The number of tumor nodules in the colon cancer model mice decreased from 6 to 3;

[0030] (5) The spleen index of the colon cancer model mice decreased from 0.0096±0.003 to 0.005±0.001;

[0031] (6) The positive rate of Ki67, a marker of colon tumor proliferation, was reduced in colon cancer model mice from 13.97% to 10.26%.

[0032] (7) The percentage of Tunel-positive cells in the colon tumor tissue of colon cancer model mice increased from 0.33% to 1.02±0.30%;

[0033] (8) The concentration of the inflammatory factor IL-1β in the colon of a colon cancer model mouse decreased from 71.33±5.27 pg / mg protein to 36.88±5.61 pg / mg protein;

[0034] (9) The concentration of the inflammatory factor IFN-γ in the colon of a colon cancer model mouse decreased from 4.63±0.26 pg / mg protein to 2.63±0.28 pg / mg protein;

[0035] (10) The concentration of the inflammatory factor IL-17A in the colon of a colon cancer model mouse decreased from 20.95±2.49 pg / mg protein to 12.60±2.50 pg / mg protein;

[0036] (11) The concentration of the anti-inflammatory factor IL-10 in the colon of a colon cancer model mouse increased from 2.61±0.90 pg / mg protein to 25.00±6.64 pg / mg protein;

[0037] (12) The relative expression level of ZO-1 mRNA in the colon of colon cancer model mice increased from 0.17±0.10 to 1.03±0.17, which was significantly different from that in the model group; the relative expression level of Claudin-1 mRNA in the colon of colitis mice increased from 0.30±0.19 to 1.30±0.32; and the relative expression level of Muc-2 mRNA in the colon of colon cancer model mice increased from 0.28±0.14 to 1.07±0.03.

[0038] (13) The relative expression level of PI3K mRNA in the colon of colon cancer model mice decreased from 3.04±0.09 to 0.86±0.40; the relative expression level of Akt mRNA in the colon of colon cancer model mice decreased from 2.56±0.22 to 0.63±0.52; the relative expression level of mTOR mRNA in the colon of colon cancer model mice decreased from 1.79±0.17 to 1.16±0.17;

[0039] (14) The relative expression level of Bax mRNA in the colon of colon cancer model mice increased from 0.23±0.07 to 1.12±0.17; the relative expression level of Bcl-2 mRNA in the colon of colon cancer model mice decreased from 3.72±0.19 to 2.60±0.26; the relative expression level of Bcl-XL mRNA in the colon of colon cancer model mice decreased from 2.43±0.12 to 0.36±0.07, and these values ​​were significantly different from those in the model group.

[0040] (15) The concentration of indole-3-carboxaldehyde in the colon contents of colon cancer model mice increased from 18.572 ng / mL to 157.346 ng / mL, which was significantly different from the model group and not significantly different from the control group.

[0041] Therefore, Bifidobacterium animalis subsp. lactis CCFM1383 has great potential for application in the prevention and / or relief of ulcerative colitis-associated colorectal cancer.

[0042] Preservation of biological materials

[0043] A strain of *Bifidobacterium animalis* subsp. *lactis*, CCFM1383, taxonomically named *Bifidobacterium animalis* subsp. *lactis*, was deposited on March 25, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 64443. Attached Figure Description

[0044] Figure 1 The effect of this strain on the body weight of mice with colorectal cancer;

[0045] Figure 2 The effect of this strain on the survival rate of a mouse model of colorectal cancer;

[0046] Figure 3 The effect of this strain on colon length in a mouse model of colorectal cancer;

[0047] Figure 4 The effect of this strain on the number of tumor nodules in a mouse model of colorectal cancer;

[0048] Figure 5 The effect of this strain on the spleen index in a mouse model of colorectal cancer;

[0049] Figure 6This is a representative immunohistochemical image of this strain against the proliferation marker Ki67 in a mouse model of colorectal cancer.

[0050] Figure 7 The effect of this strain on the staining results of Ki67, a proliferation marker, in a mouse model of colorectal cancer;

[0051] Figure 8 This is a representative image of Tunel staining in a mouse model of colorectal cancer using this strain;

[0052] Figure 9 The effect of this strain on the Tunel staining results of a mouse model of colorectal cancer;

[0053] Figure 10 The effect of this strain on the inflammatory factor IL-1β in the colonic tissue of a mouse model of colorectal cancer;

[0054] Figure 11 The effect of this strain on the inflammatory factor IFN-γ in the colonic tissue of a mouse model of colorectal cancer;

[0055] Figure 12 The effect of this strain on the inflammatory factor IL-17A in the colon tissue of a mouse model of colorectal cancer;

[0056] Figure 13 The effect of this strain on the anti-inflammatory factor IL-10 in the colon tissue of a mouse model of colorectal cancer;

[0057] Figure 14 The effect of this strain on the relative expression levels of intestinal barrier-related genes ZO-1, Claudin-1, and Muc2 mRNA in colon tissue of a mouse model of colorectal cancer;

[0058] Figure 15 The effect of this strain on the relative expression levels of PI3K, Akt, and mTOR mRNAs in colon tissue of a mouse model of colorectal cancer.

[0059] Figure 16 The effect of this strain on the relative expression levels of apoptosis-related genes Bax, Bcl-XL, and Bcl-2 mRNA in colon tissue of a mouse model of colorectal cancer;

[0060] In the above images: *, **, and *** indicate p-values ​​less than 0.05, 0.01, and 0.001, respectively; unlabeled values ​​indicate p-values ​​greater than 0.05. Detailed Implementation

[0061] The C57BL / 6 male mice used in the following examples were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. IL-1β, IFN-γ, IL-17A, and IL-10 ELISA kits were purchased from R&D Company, USA.

[0062] The culture media involved in the following examples are as follows:

[0063] mMRS liquid medium (L): peptone 10g, beef extract 10g, yeast powder 5g, anhydrous glucose 20g, anhydrous sodium acetate 2g, magnesium sulfate (MgSO4·7H2O) 0.5g, manganese sulfate (MnSO4·H2O) 0.25g, diammonium hydrogen citrate 2g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 2.6g, Tween 80 1mL, cysteine ​​0.5g, pH 6.2-6.4.

[0064] mMRS solid medium (L): Add 15g of agar to the liquid medium formula, pH 6.2-6.4.

[0065] Example 1: Screening of Bifidobacterium animalis subsp. lactis using CCFM1383:

[0066] Using fecal samples from healthy individuals in Wuxi, Jiangsu Province, one spoonful of the sample was added to 5 mL of PBS (with 0.05% cysteine ​​added), mixed well, and serially diluted. A 10⁻⁶ dilution was selected. -5 ~10 -7 The serially diluted solutions were spread onto the above-mentioned mMRS solid medium and incubated at 37°C for 48 h. Typical colonies were picked and streaked onto mMRS solid medium for purification, and incubated upside down in a 37°C incubator for 48 h. Single colonies were picked and inoculated into 5 mL of liquid mMRS medium, and incubated at 37°C for 16-18 h. 1.5 mL of bacterial culture was centrifuged at 6000 r / min for 3 min to remove the supernatant, and 1 mL of 30% sterile glycerol was added for preservation. At the same time, 1.5 mL of bacterial culture was centrifuged, the supernatant was removed, and the culture was resuspended in sterile water for bacterial identification.

[0067] The results showed that the obtained strain was Bifidobacterium animalis subsp. lactis CCFM1383, named Bifidobacterium animalis subsp. lactis CCFM1383, and deposited at Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 64443.

[0068] Example 2: High in vitro production of IAld by Bifidobacterium animalis subsp. lactis CCFM1383

[0069] The specific steps are as follows:

[0070] The strain was activated for two generations using 5 mL of mMRS liquid medium at a 4% inoculum. The entire process was conducted in an anaerobic workstation (37°C, 10% hydrogen, 10% carbon dioxide, 80% nitrogen). The strain was removed when it reached the logarithmic growth phase in the third generation, and the viable count of *Bifidobacterium animalis* subsp. *lactamella* CCFM1383 in the culture medium reached 1 × 10⁻⁶. 8 CFU / mL, 2 mL of culture medium was centrifuged at 6000 rpm for 3 min to obtain bacterial sludge and supernatant. The supernatant was used for targeted tryptophan metabolomics detection. The obtained bacterial sludge was washed three times with 0.9% sterile physiological saline, resuspended in pre-cooled 30% sucrose solution for counting, and stored at -80℃ for later use. 100 μL of fermentation supernatant was thoroughly mixed with 400 μL of pre-cooled methanol and then allowed to stand at -20℃ for 30 min. To remove proteins and other grade 5 impurities, the mixture was centrifuged at 20000 g for 15 min. Then, 300 μL of supernatant was vacuum dried at 45℃. The obtained dry matter was resuspended in 100 μL of methanol dilution (water:methanol = 4:1) and filtered through a 0.22 μm microporous membrane. Targeted tryptophan metabolomics was used for determination. Vanquish UHPLC Q-Exactive Plus MS was used to quantify tryptophan metabolites in fecal samples. An ACQUIRE UPLC BEH C18 column was used for chromatographic separation. The column temperature was maintained at 35°C, the autosampler temperature at 4°C, the sample volume was 2 μL, the total time was 20 min, and the flow rate was 0.3 mL / min. The gradient elution system consisted of mobile phases A (0.1% formic acid and acetonitrile) and B (0.1% formic acid and water). The yield of Bifidobacterium animalis subsp. lactis CCFM1383 in vitro was determined to be 421.765 ng / mL.

[0071] Example 3: Effects of Bifidobacterium animalis subsp. lactis on body weight and survival rate in CCFM1383 colorectal cancer mice.

[0072] Obtaining the bacterial suspension of Bifidobacterium animalis subsp. lactis CCFM1383: Take the bacterial sludge stored at -80℃ in Example 2, centrifuge at 6000 r / min for 3 min to remove the supernatant, and prepare the bacterial suspension (viable count 1×10⁻⁶) using sterile physiological saline. 8 (CFU / mL)

[0073] The specific steps are as follows:

[0074] This study investigated the effects of *Bifidobacterium animalis* subsp. *lactis* CCFM1383 on colorectal cancer in mice. Five-week-old SPF-grade male C57BL / 6J mice were randomly divided into three groups: a control group, a model group, an experimental group (A4), and a drug group (aspirin). The experimental group received *Bifidobacterium animalis* subsp. *lactis* CCFM1383 via gavage, while the drug group received aspirin via gavage. Eight mice were housed in each group at the Experimental Animal Center of Jiangnan University under a constant temperature of 21-26℃, humidity of 40-70%, noise level ≤60dB, and illumination of 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University). An AOM / DSS-induced mouse model of orthotopic ulcerative colitis-associated colorectal cancer was established.

[0075] The experiment lasted a total of 13 weeks.

[0076] The first week is the adaptation period. During this period, each cage of mice has free access to water and is given a normal growth and reproduction diet.

[0077] After the adaptation period ends, weeks 2 through 13:

[0078] On day 8, mice in the model group, experimental group, and drug group were given a single intraperitoneal injection of AOM (10 mg / kg). -1 In the second week, mice had free access to plain water. In the third week, mice were given drinking water containing 2.5% DSS for one week, followed by two weeks of plain water. This three-week cycle was repeated twice. After the third cycle, mice were given sterile water for two weeks. Starting from the third week, the model group was given 0.2 mL of physiological saline by gavage every two days; the experimental group was given 0.2 mL of 1×10⁻⁶ DSS by gavage every two days. 8 The treatment group received a CFU / mL suspension of *Bifidobacterium animalis* subsp. *lactis* CCFM1383; the drug group received 0.2 mL of 2 mg / mL aspirin by gavage every two days. The control group received the same volume of physiological saline by gavage at the same time points.

[0079] The mice's weight was monitored weekly during the experiment, and the final weight changes were as follows: Figure 1 As shown, the final survival rate is as follows Figure 2 As shown.

[0080] like Figure 1As shown, on day 91 of the experiment, the average weight of mice in the control group was 29.37±0.57g, and the average weight of mice in the model group was 24.33±0.51g. After gavage administration of CCFM1383 bacterial solution, the average weight of mice increased to 26.35±0.48g, similar to the effect in the drug group (27.30±0.89g). Figure 2 As shown, on day 91 of the experiment, the survival rate of mice in the model group was 50%, while the survival rate increased to 75% after intervention with CCFM1383 strain. Therefore, Bifidobacterium animalis subsp. lactis CCFM1383 can effectively reduce weight loss and improve the survival rate of mice.

[0081] Example 4: Effects of Bifidobacterium animalis subsp. lactis CCFM1383 on colon length and tumor nodule number in mice with colorectal cancer.

[0082] The experimental setup was the same as in Example 3. After the experiment, the mice were dissected in accordance with ethical requirements, and the length of the colon and rectum was measured and the number of tumor nodules was recorded.

[0083] Colorectal length as Figure 3 As shown, compared to the control group (mean colon length 7.50±0.33 cm), the colon length in the model group was significantly reduced to 4.55±0.21 cm. Under the intervention of the probiotic CCFM1383, the colon length significantly increased to 5.9±0.26 cm, approaching that of the drug group (6.79±0.54 cm). The number of colorectal tumor nodules is as follows... Figure 4 As shown, the average number of tumor nodules in the model group was 6, which decreased to 3 after intervention with the probiotic CCFM1383. Therefore, Bifidobacterium animalis subsp. lactis CCFM1383 can effectively slow down the shortening of colon length and reduce the number of tumor nodules in mice.

[0084] Example 5: Effect of Bifidobacterium animalis subsp. lactis CCFM1383 on spleen index in mice with colorectal cancer.

[0085] The experimental setup was the same as in Example 3. After the experiment, the mice were dissected according to ethical requirements, and their spleens were weighed. The spleen index was calculated using the body weight. The spleen index results are as follows: Figure 5As shown, the spleen index of the model group mice was 0.0096±0.003, and the index decreased to 0.005±0.001 after intervention with the probiotic SHXXA4M. Therefore, Bifidobacterium animalis subsp. lactis CCFM1383 can reduce the spleen index in mice.

[0086] Example 6: Effect of Bifidobacterium animalis subsp. lactis CCFM1383 on Ki67, a proliferation marker in colorectal cancer mice.

[0087] The experimental setup was the same as in Example 3. After the experiment, the mice were dissected in accordance with ethical requirements. The distal cecal end of the colon (0.5 cm) was fixed in 4% paraformaldehyde solution for 36 h. After being dehydrated by ethanol gradient, cleared with xylene, embedded in paraffin, stained with Ki67, and then the colon section was observed by scanning.

[0088] A representative image of Ki67 immunohistochemical staining is shown below. Figure 6 As shown, the results of Ki67 positive cells are as follows: Figure 7 As shown, the Ki67 positive cell rate in the model group was 13.97%, which decreased to 10.26% after intervention with the probiotic CCFM1383, close to that in the drug group (8.99%). Therefore, Bifidobacterium animalis subsp. lactis CCFM1383 can inhibit tumor cell proliferation.

[0089] Example 7: Effect of Bifidobacterium animalis subsp. lactis CCFM1383 on Tunel staining in mice with colorectal cancer.

[0090] The experimental setup was the same as in Example 3. After the experiment, the mice were dissected in accordance with ethical requirements. The distal cecal end of the colon (0.5 cm) was fixed in 4% paraformaldehyde solution for 36 h. After dehydration by gradient ethanol, clearing with xylene, embedding in paraffin, and TUNEL staining, the colon section was observed by scanning.

[0091] A representative image of Tunel staining is shown below. Figure 8 As shown, the results of TUNEL positive cells are as follows: Figure 9As shown, the proportion of Tunel-positive cells in the model group was 0.33%, which increased to 1.02% after intervention with the probiotic CCFM1383, close to that in the drug group (1.27%). Therefore, Bifidobacterium animalis subsp. lactis CCFM1383 can promote tumor cell apoptosis.

[0092] Example 8: Effects of Bifidobacterium animalis subsp. lactis CCFM1383 on inflammatory factors in mice with colorectal cancer.

[0093] The experimental setup was the same as in Example 3. After the experiment, mice were dissected according to ethical requirements. Colonic tissue was weighed and mixed with an appropriate volume of RIPA lysis buffer and a mixture of protease phosphatase inhibitors. The tissue was ground and the supernatant was extracted. The concentrations of IL-1β, IFN-γ, IL-17A, and IL-10 in the colonic tissue were determined using ELISA. The final concentrations were calculated based on the total protein concentration obtained from BCA quantification.

[0094] Colon tissue IL-1β concentrations are shown in the figure. Figure 10 The IL-1β concentration in the control group was 3.29 ± 1.00 pg / mg protein, while in the model group it increased to 71.33 ± 5.27 pg / mg protein. After CCFM1383 intervention, it decreased to 36.88 ± 5.61 pg / mg protein. Therefore, *Bifidobacterium animalis* subsp. *lactis* CCFM1383 can effectively inhibit IL-1β levels in mouse colon tissue.

[0095] The concentration of IFN-γ in colon tissue is shown in the figure. Figure 11 The IFN-γ concentration in the control group was 0.68±0.18 pg / mg protein, while in the model group it increased to 4.63±0.26 pg / mg protein. After CCFM1383 intervention, it decreased to 2.63±0.28 pg / mg protein. Therefore, *Bifidobacterium animalis* subsp. *lactis* CCFM1383 can effectively inhibit IFN-γ levels in mouse colon tissue.

[0096] Colon tissue IL-17A concentrations are shown in the figure. Figure 12The IL-17A concentration in the control group was 1.91 ± 0.48 pg / mg protein, while in the model group it increased to 20.95 ± 2.49 pg / mg protein. After CCFM1383 intervention, it decreased to 12.60 ± 2.50 pg / mg protein. Therefore, *Bifidobacterium animalis* subsp. *lactis* CCFM1383 can effectively inhibit IL-17A levels in mouse colon tissue.

[0097] Colon tissue IL-10 concentrations are shown in the figure. Figure 13 The IL-10 concentration in the control group was 6.83±1.65 pg / mg protein, while in the model group it decreased to 2.61±0.90 pg / mg protein. After CCFM1383 intervention, it increased to 25.00±6.64 pg / mg protein. Therefore, Bifidobacterium animalis subsp. lactis CCFM1383 can effectively increase the IL-10 level in mouse colon tissue.

[0098] Example 9: Effects of Bifidobacterium animalis subsp. lactis CCFM1383 on intestinal barrier-related protein mRNA in mice with colorectal cancer.

[0099] The experimental setup was the same as in Example 3. After the experiment, mice were dissected according to ethical requirements, and colon tissue was weighed and extracted. Total RNA was extracted from the colon tissue using RNA-easy reagent for reverse transcription. The relative cDNA content was analyzed using a ChamQ SYBRqPCR Master Mix quantitative real-time PCR thermal cycler. The mRNA expression levels of GAPDH-normalized ZO-1, Claudin-1, and Muc2 were calculated using 2-ΔΔCT.

[0100] Depend on Figure 14 Quantitative results from A showed that the mRNA expression level of Claudin-1 in the model group was 0.30, while its mRNA expression level increased to 1.30 after intervention with the probiotic CCFM1383. Figure 14 Quantitative results from B showed that the mRNA expression level of ZO-1 in the model group was 0.17, while the mRNA expression level of ZO-1 significantly increased to 1.03 under the intervention of probiotic CCFM1383. Figure 14The quantitative results of C showed that the mRNA expression level of Muc2 in the model group was 0.28, while the mRNA expression level increased to 1.07 under the intervention of probiotic CCFM1383. Therefore, Bifidobacterium animalis subsp. lactis CCFM1383 can effectively promote the mRNA expression levels of tight junction proteins ZO-1, Claudin-1, and mucin Muc2 in colon tissue.

[0101] Example 10: Effects of Bifidobacterium animalis subsp. lactis CCFM1383 on cancer signaling pathway mRNA in colorectal cancer mice.

[0102] The experimental setup was the same as in Example 3. After the experiment, mice were dissected according to ethical requirements, and colon tissue was weighed and extracted. Total RNA was extracted from the colon tissue using RNA-easy reagent for reverse transcription. The relative cDNA content was analyzed using a ChamQ SYBRqPCR Master Mix quantitative real-time PCR thermal cycler. The mRNA expression levels of GAPDH-normalized PI3K, Akt, and mTOR were calculated using 2-ΔΔCT.

[0103] Depend on Figure 15 Quantitative results from A showed that the mRNA expression level of PI3K in the model group was 3.04, while the mRNA expression level decreased to 0.86 under the intervention of probiotic CCFM1383. Figure 15 Quantitative results from B showed that the Akt mRNA expression level in the model group was 2.56, while the Akt mRNA expression level decreased to 0.63 under the intervention of probiotic CCFM1383. Figure 15 The quantitative results of C showed that the mTOR mRNA expression level in the model group was 1.79, which decreased to 1.16 under the intervention of probiotic CCFM1383. Therefore, Bifidobacterium animalis subsp. lactis CCFM1383 can effectively inhibit the mRNA expression of genes related to the PI3K-Akt-mTOR signaling pathway in mice.

[0104] Example 11 Effects of Bifidobacterium animalis subsp. lactis CCFM1383 on apoptosis-related gene mRNA in colorectal cancer mouse cells

[0105] The experimental setup was the same as in Example 3. After the experiment, mice were dissected according to ethical requirements, and colon tissue was weighed and extracted. Total RNA was extracted from the colon tissue using RNA-easy reagent for reverse transcription. The relative cDNA content was analyzed using a ChamQ SYBRqPCR Master Mix quantitative real-time PCR thermal cycler. The mRNA expression levels of GAPDH-normalized Bax, Bcl-XL, and Bcl-2 were calculated using 2-ΔΔCT.

[0106] Depend on Figure 16 Quantitative results from A showed that the mRNA expression level of the pro-apoptotic gene Bax in the model group was 0.23, while its mRNA expression level increased to 1.12 under the intervention of probiotic CCFM1383. Figure 16 Quantitative results of B showed that the mRNA expression level of the anti-apoptotic gene Bcl-XL in the model group was 2.43, while the mRNA expression level of Bcl-XL was significantly reduced to 0.40 under the intervention of probiotic CCFM1383. Figure 16 The quantitative results of C showed that the mRNA expression level of Bcl-2 in the model group was 3.72, which decreased to 2.60 under the intervention of probiotic CCFM1383. Therefore, Bifidobacterium animalis subsp. lactis CCFM1383 can effectively inhibit the mRNA expression levels of apoptosis-related genes Bax, Bcl-XL, and Bcl-2 in mice.

[0107] Example 12 Effect of Bifidobacterium animalis subsp. lactis CCFM1383 on indole-3-carboxaldehyde in the colon of mice with colorectal cancer

[0108] The experimental setup was the same as in Example 3. After the experiment, mice were dissected according to ethical requirements, and the contents of the mouse colon were collected. After freeze-drying, the contents were mixed with methanol and homogenized at 65 Hz for 3 minutes. After being placed at -20°C for 10 minutes, the mixture was centrifuged at 15000g for 10 minutes at 4°C. 400 μL of the supernatant was vacuum-dried at 45°C and resuspended in 100 μL of 20% methanol-water mixture. The supernatant obtained under the above centrifugation conditions was filtered through a 0.22 μm microporous membrane to remove impurities and then used for UHPLC-MS analysis of tryptophan indole metabolites.

[0109] The results of the detection of indole derivatives in mouse colonic contents are shown in Table 1. By comparing the levels of each indole derivative after probiotic CCFM1383 intervention with the control and model groups, it was found that the level of IAld in mouse colonic contents increased after probiotic CCFM1383 intervention, with the most significant statistical difference compared to the model group, and the level was closest to that of the control group. Therefore, Bifidobacterium animalis subsp. lactis CCFM1383 can effectively promote the increase of IAld content in mouse colon.

[0110] Table 1. Levels of indole metabolites in mice from different groups

[0111]

[0112]

[0113] () represents (P) 25 P 75 The value before the parentheses represents the median.

[0114] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Bifidobacterium animalis subsp. lactis, CCFM1383, has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 64443, and deposit date of March 25, 2024.

2. A microbial preparation containing the Bifidobacterium lactis subsp. CCFM1383 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The microbial preparation is a liquid or solid preparation containing Bifidobacterium animalis subsp. lactis CCFM1383.

4. The microbial preparation according to claim 3, characterized in that, The microbial preparation contains ≥1×10⁻⁶ viable bacteria of Bifidobacterium lactis subsp. lactis CCFM1383. 8 CFU / mL or ≥1×10 8 CFU / g.

5. The microbial preparation according to claim 4, characterized in that, The microbial preparation also contains glycerol.

6. A method for preparing the microbial preparation according to any one of claims 2 to 5, characterized in that, The animal Bifidobacterium lactis subsp. CCFM1383 described in claim 1 was fermented in a culture medium.

7. The use of Bifidobacterium lactis subsp. CCFM1383 as described in claim 1, or the microbial preparation as described in any one of claims 2 to 5, in the preparation of a drug for alleviating colon cancer.

8. The application as described in claim 7, characterized in that, In the drug, the viable count of Bifidobacterium lactis subsp. CCFM1383 is not less than 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

9. The application as described in claim 8, characterized in that, The relief of colon cancer includes one or more of the following: (a) It alleviates weight loss, improves survival rate, and reduces rectal bleeding in colon cancer model mice; (b) Reduce the number of tumors in colon cancer model mice; (c) Significantly reduced the levels of pro-inflammatory factors IL-17A, IL-1β, and IFN-γ in colon cancer model mice; (d) Increase the level of the anti-inflammatory factor IL-10 in colon cancer model mice; (e) Inhibits the proliferation of cancer cells and promotes apoptosis in a colon cancer model mouse; (f) Inhibit the PI3K-Akt-mTOR signaling pathway; (g) Increase the colonic IAld level in mice with colon cancer.

10. The application as described in claim 9, characterized in that, The drug also includes a drug carrier and drug excipients.

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