CN107629988B - Bifidobacterium bifidum capable of relieving colorectal cancer and use thereof - Google Patents

Abstract

The invention discloses Bifidobacterium bifidum capable of obviously inhibiting colorectal cancer and application thereof, belonging to the technical field of microorganisms. The bifidobacterium bifidum HNJ6 provided by the invention has the preservation number of GDMCC No.60255, can tolerate gastric acid and bile salt, can remarkably reduce the inflammatory reaction of colorectal cancer model mouse colorectal parts, and reduces the tumor number of the colorectal parts of the model mouse by regulating the expression of Notch1, Notch2 signal pathways and VEGFR2 molecules in colorectal tissues. In addition, bifidobacterium bifidum HNJ6 can also improve the intestinal flora structure and the short-chain fatty acid level in the intestinal tract. The bifidobacterium bifidum HNJ6 is used for preparing fermented food for inhibiting colorectal cancer, and has very wide application prospect.

Description

Bifidobacterium bifidum capable of relieving colorectal cancer and use thereof

technical field

The invention relates to the technical field of microorganisms, in particular to a bifidobacteria bifidum capable of significantly inhibiting the occurrence of colorectal cancer by regulating Notch1, Notch2 and VEGFR2 signaling pathways and uses thereof.

Background technique

Colorectal cancer is a malignant digestive tract tumor. As the incidence and mortality of colorectal cancer continue to rise in my country, its threat to the health of the Chinese people is gradually deepening. At present, the mainstream methods of clinical treatment of colorectal cancer have large side effects, which greatly affect the quality of life of patients. As a safe and edible food, probiotics have been gradually confirmed by research on their remission and therapeutic effects in various diseases, such as promoting digestion and absorption, enhancing immunity, preventing reproductive system infections, alleviating allergic reactions, preventing and suppressing Cancer, prevention and inhibition of cardiovascular and cerebrovascular diseases, prevention and inhibition of neurological diseases, etc.

The intestinal tract is not only the direct incidence area of colorectal cancer, but also the main place where probiotics exert their probiotic effects. Research on the effects of probiotics on colorectal cancer is increasing. Some studies have shown that probiotics, especially lactic acid bacteria, have the effect of antagonizing colon cancer cells or inhibiting colon tumorigenesis. They can inhibit colon cancer cell proliferation or induce colon cancer cell apoptosis to varying degrees. In addition, the antagonistic effect of probiotics on colorectal cancer is also manifested in the following aspects: combining or metabolizing carcinogens, inducing apoptosis of cancer cells, improving immunity, inhibiting the differentiation and proliferation of cancer cells, protecting the intestine, and maintaining intestinal bacteria The regulation of intestinal flora by probiotics will ultimately affect the concentration of short-chain fatty acids in the intestine, which further affects the occurrence of tumors.

Studies have shown that colon cancer can develop and grow despite higher concentrations of butyrate in the colon, and a butyrate-rich microenvironment can select tumor cells that can metabolize butyrate. Probiotics may also inhibit tumorigenesis by modulating tumor-related signaling pathways. There are many signaling pathways involved in colorectal cancer, including AKT signaling pathway, GPCR signaling pathway, MAPK signaling pathway, NF-κB signaling pathway, JAK signaling pathway, Wnt signaling pathway, TGF-β signaling pathway, ESC signaling pathway, Apoptosis signaling pathway , CCC signaling pathway and Angiogenesis signaling pathway. At present, there are many studies on the relationship between Wnt and NF-κB signaling pathways and colorectal cancer, but Notch signaling pathway, as a signaling pathway related to epithelial cell differentiation, is also closely related to colorectal cancer. For colorectal cancer, the Notch signaling pathway plays a role in inducing and promoting tumors to increase drug resistance. Many clinical studies have also shown that the Notch signaling pathway is over-activated in colon tumor tissue compared with paracancerous tissue or normal tissue.

In addition to the Notch signaling pathway, a large number of studies have also shown that vascular endothelial growth factor receptor (VEGFR) is highly expressed in most tumors. The main physiological functions of VEGF on endothelial cells are almost all achieved by activating VEGFR2, including stimulating endothelial cell proliferation, increasing vascular permeability, and chemotaxis of endothelial cells. Different researchers have observed that VEGF binds to VEGFR2 and activates the MAPK signal transduction pathway in a variety of endothelial cells. VEGFR2 not only promotes the division and proliferation of vascular endothelial cells, but also induces tumor angiogenesis and promotes the growth and metastasis of tumor cells. A large number of reports have also proved that VEGFR2 is also highly expressed in colorectal cancer tissues. Therefore, VEGFR2 can be targeted for therapy and diagnosis of tumorigenesis.

Probiotics may also regulate the Notch and VEGFR signaling pathways to alleviate the occurrence of colorectal cancer or inhibit the growth of colon cancer cells. Bifidobacterium bifidum is a kind of lactic acid bacteria, which widely exists in the intestinal tract of animals and humans. There are a large number of research reports on the physiological function of Bifidobacterium bifidum at home and abroad, and there are also a small number of reports on the inhibition of colorectal cancer by Bifidobacterium bifidum, but the mechanism is not clear. Research on the inhibition of colorectal carcinogenesis by Bifidobacterium bifidum is of great value to many disciplines such as food science, preventive medicine and microbiology. Screening excellent and safe Bifidobacterium bifidum with the function of inhibiting the occurrence of colorectal cancer is of great significance for the development of functional foods and drugs.

In the published literature, patents or patent applications, there are a few patents for probiotics or probiotics, fermentation products to inhibit tumors, but no specific probiotic strains with the function of inhibiting colon cancer are really involved. For example, CN105535650A discloses a probiotic composition with anti-tumor (subcutaneous transplantation of liver cancer cell lines) function. The composition involves a variety of probiotics and traditional Chinese medicine ingredients, but the tumor suppressing function of each component is not clarified, and all probiotics None of the strains have been identified. Since there are significant differences between different strains of the same probiotic in terms of probiotic functions, the formula is not universal; similarly, the probiotics in CN10468665 7A and CN101711775A have not been identified, and the fermented product has not been identified. The function of the ingredients is not clear.

CN104523761A discloses a genetically engineered Lactococcus lactis that can inducibly express interleukin 12, which can inhibit the growth of intestinal tumors, but the bacterium is not obtained from nature and can only be used for medicine but not for food. CN105441357A discloses a strain of Lactobacillus plantarum SKT109 which produces extracellular polysaccharide, which can inhibit the growth of human colon cancer cell HT-29 and the growth of tumor-bearing nude mice (inoculated colon cancer cells in the armpit) in vitro; CN103445068A and CN103468600A A strain of Lactobacillus plantarum Dy-1 with tumor-inhibiting effect is disclosed, and the bacteria and its fermentation products can inhibit the growth of human colon cancer cells HT-29 and gastric cancer cells SGC-7901 in vitro, and inhibit the growth of tumor-bearing nude mice (abdominal subcutaneous cells). inoculated colon cancer cells) tumor growth. The above three patented strains are clear and have clear functions, but the animal models involved use colon cancer cell lines to carry tumors under the skin, which are quite different from the tumor environment in the intestine, and their roles in the regulation of tumor-related signaling pathways are unclear. Therefore, it is necessary to screen edible probiotics from nature and have a clear tumor-inhibiting mechanism for inhibiting the growth of tumors in the colorectum.

SUMMARY OF THE INVENTION

In view of the above problems existing in the prior art, the present application provides a bifidobacteria bifidum and uses thereof. The single bacteria of this strain has a strong ability to regulate the Notch1, Notch2 signaling pathway and the expression of VEGFR2 molecules, which can significantly inhibit the occurrence of colorectal cancer, and also relieve colonic inflammation, improve the structure of intestinal flora and the level of short-chain fatty acids, etc. Multiple functions and uses.

The technical scheme of the present invention is as follows:

A kind of Bifidobacterium bifidum HNJ6 (Bifidobacterium bifidum), which was deposited in the Guangdong Provincial Microbial Culture Collection Center on October 25, 2017, and the preservation address is Guangdong Province Microbiological Research on the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City Institute, the deposit number is GDMCC No.60255.

A fermented food, the fermented food is dairy products, soybean products and fruit and vegetable products produced by fermentation with Bifidobacterium bifidum HNJ6.

The dairy products include milk, sour cream or cheese; the soy products include soy milk, tempeh or soybean paste; the fruit and vegetable products include cucumber, carrot, beet, celery or cabbage products.

The application of the Bifidobacterium bifidum HNJ6 or the fermented food is used to improve the intestinal flora, reduce the abnormally elevated short-chain fatty acid level in the intestinal tract, relieve colorectal inflammation, and inhibit the occurrence of colorectal cancer.

The Bifidobacterium bifidum HNJ6 has the following biological properties:

(1) Bacterial characteristics: Gram-positive, non-spore-forming, non-motile bacteria.

(2) Colony characteristics: anaerobic culture for 36 hours to form obvious colonies, the diameter is between 0.5-2mm, the front shape is round, the side shape is protruding, the edges are neat, milky white, opaque, the surface is moist and smooth, and no pigment is produced.

(3) Growth characteristics: cultured in mMRS medium for about 24 hours under constant temperature and anaerobic conditions at 37°C to reach the end logarithmic phase.

(4) It has good tolerance to artificial simulated gastrointestinal fluid;

(5) It can regulate the Notch1 and Notch2 signaling pathways and the expression level of VEGFR2 in HT-29 cells;

(6) It can significantly reduce the number of tumors in the colorectal cancer tissue of the colon cancer model mice, and improve the integrity of the intestinal tissue of the model mice;

(7) Can significantly alleviate the level of colorectal inflammation in colon cancer model mice;

(8) Regulate the expression levels of Notch1, Notch2 signaling pathway and VEGFR2 in colon tissue of colon cancer model mice;

(9) Improve intestinal microecology and short-chain fatty acid levels in colon cancer model mice.

The extraction method of this strain is:

(1) Isolation and screening of lactic acid bacteria

(1) Take 1 g of fresh feces from healthy male elderly from Changshou Village, Chaihu Town, Zhongxiang City, Hubei Province. After gradient dilution, it was spread on mMRS solid medium and cultured at 37°C for 72 hours in an anaerobic environment.

(2) Observe and record the colony morphology, pick colonies for streak purification.

(3) In mMRS liquid medium, cultured at 37°C for 48 hours, the obtained colonies were Gram-stained, and the colony morphology was recorded.

(4) discarding the gram-negative bacterial strains and gram-positive cocci in the colony, and selecting to obtain gram-positive bacilli.

(5) After the catalase analysis, the catalase-positive strains were discarded, and the catalase-negative strains were retained.

(2) Preliminary identification of Bifidobacterium bifidum: fructose-6-phosphate phosphatidase assay

(1) the lactic acid bacteria screened in step (1) are cultivated in liquid mMRS nutrient solution for 24h, then get 1mL culture 8000rpm centrifugal 2min;

(2) Wash twice with 0.05M KH ₂ PO ₄ solution of pH 6.5 containing 0.05% (mass percent) cysteine hydrochloride;

(3) Resuspend in 200 μL of the above-mentioned phosphate buffer added with 0.25% (mass percentage) Triton X-100;

(4) Add 50 μL of a mixture of 6 mg/mL sodium fluoride and 10 mg/mL sodium iodoacetate and 50 μL of 80 mg/mL fructose-6-phosphate, and incubate at 37°C for 1 h;

(5) Add 300 μL of hydroxylamine hydrochloride with a concentration of 0.139 g/mL and pH 6.5, and place at room temperature for 10 min;

(6) Add 200 μL of 15% (mass percent) trichloroacetic acid and 4M HCL respectively;

(7) 200 μL of 0.1M HCL containing 5% (mass percent) ferric chloride was added, and the system quickly turned red, which was F6PPK positive, and it was preliminarily determined to be bifidobacteria.

(3) Molecular biological identification of Bifidobacterium bifidum

(l) Single bacterial genome extraction

A. the lactic acid bacteria obtained by step (2) are cultivated overnight, get the bacterial suspension 1mL of the overnight culture in a 1.5mL centrifuge tube, centrifuge at 10000rpm for 2min, discard the supernatant to obtain thalline;

B. after rinsing the thalline with 1mL sterile water, centrifuge at 10000rpm for 2min, discard the supernatant to obtain the thalline;

C. Add 200 μL of SDS lysis solution, water bath at 80°C for 30min;

D. Add 200 μL of phenol-chloroform solution to the bacterial cell lysis solution, wherein the composition and volume ratio of the phenol-chloroform solution are Tris saturated phenol:chloroform:isoamyl alcohol=25:24:1, after inversion and mixing, centrifuge at 12000rpm 5-10min, take 200μL of supernatant;

E. Add 400μL ice ethanol or ice isopropanol to 200uL supernatant, let stand at -20℃ for 1h, centrifuge at 12000rpm for 5-10min, discard the supernatant;

F. Add 500 μL of 70% (volume percentage) ice ethanol to resuspend the pellet, centrifuge at 12000 rpm for 1-3 min, and discard the supernatant;

G.60℃ oven drying, or natural drying;

H. 50 μL ddH ₂ O to redissolve the pellet for PCR;

(2) 16S rDNA PCR

A. Bacterial 16S rDNA 50μL PCR reaction system:

10×Taq buffer, 5 μL; dNTP, 5 μL; 27F, 0.5 μL; 1492R, 0.5 μL; Taq enzyme, 0.5 μL; template, 0.5 μL; ddH ₂ O, 38 μL.

B. PCR conditions:

95℃5min; 95℃10s; 55℃30s; 72℃30s; step2-4 30×; 72℃5min; 12℃2min;

(3) Prepare a 1% agarose gel, then mix the PCR product with 10000× loading buffer, load 5 μL, run at 120V for 30 minutes, and then perform gel imaging;

(4) The PCR product of 16S rDNA is subjected to sequencing analysis, the obtained sequence results are searched and compared in GeneBank using BLAST, and the lactic acid bacteria identified as Bifidobacterium bifidum by the sequencing results are selected and stored at -80 °C for future use;

The beneficial technical effects of the present invention are:

The bifidobacteria bifidum HNJ6 of the invention has good resistance to gastric acid and bile salts, and can regulate the Notch1 and Notch2 signaling pathways and the expression of VEGFR2 molecules in the colorectal tissue to achieve the inhibitory effect on the occurrence of colorectal cancer, and significantly reduce the occurrence of colorectal cancer. Inflammation in the colon of rectal cancer model mice, and reduce the number of tumors in the colon and rectum of model mice. In addition, it can improve the intestinal flora and the level of short-chain fatty acids in the intestine.

The bacterium is superior to the commercial strain Lactobacillus rhamnosus GG strain (LGG) in the efficacy of inhibiting the occurrence of colorectal cancer, and the mechanism of inhibiting the occurrence of colorectal cancer is different from that of LGG. Therefore, the Bifidobacterium bifidum HNJ6 of the present invention can be used as an auxiliary means for clinical treatment of colorectal cancer, and does not have the toxic and side effects of drugs. Therefore, Bifidobacterium bifidum HNJ6 can be used to prepare pharmaceutical compositions and fermented foods for relieving and preventing colorectal cancer, and has a very wide application prospect.

Description of drawings

Figure 1 shows the effect of this strain on the expression levels of Notch1, Notch2 signaling pathway molecules and VEGFR2 in HT-29 cell line;

Figure 2 shows the effect of this strain on the expression levels of other cancer-related signaling pathway molecules in the HT-29 cell line;

Figure 3 shows the effect of this strain on the number of tumors in the colorectum of colorectal cancer model mice;

Figure 4 is the improvement of the colorectal cancer model mice colorectal tissue damage by this strain;

Figure 5 is the improvement of IL-17 and IFN-γ levels in the serum of colorectal cancer model mice by this strain;

Figure 6 is the effect of this strain on the expression levels of Notch1, Notch2 signaling pathway molecules and VEGFR2 in intestinal tissue of colorectal cancer model mice;

Fig. 7 is the effect of this strain on intestinal short-chain fatty acid levels in colorectal cancer model mice;

Figure 8 shows the improvement effect of this strain on the intestinal flora structure of colorectal cancer model mice.

Detailed ways

The present invention will be described in detail below with reference to the accompanying drawings, embodiments and test examples.

Example 1: Bifidobacterium bifidum HNJ6 has good tolerance to simulated gastrointestinal fluid

The cryopreserved Bifidobacterium bifidum HNJ6 was streaked in mMRS medium (MRS medium + 0.05% cysteine hydrochloride) solid medium, and cultured anaerobically at 37°C for 48 hours, and then cultured with mMRS. After subculture for 2 to 3 times, take Bifidobacterium bifidum HNJ6 culture medium, centrifuge at 8000 × g for 5 min to collect bacteria, and resuspend in (1:1) artificial simulated gastric juice (containing 1% pepsin, pH 2.5) at pH 2.5. = 2.5 mMRS medium), then cultured at 37 °C anaerobic, sampling at the beginning (0h), 0.5h, 1h and 2h respectively, poured with mMRS agar medium to count the plate colonies, and determined the number of viable bacteria. and calculate its survival rate. The survival rate is the ratio of the number of viable bacteria in the culture solution to the number of viable bacteria at 0 h, expressed in %.

Take the culture medium of Bifidobacterium bifidum HNJ6, centrifuge at 8000 × g for 5 min to collect the bacteria, and resuspend in (1:1) artificial simulated intestinal fluid (containing 0.3% bovine bile salts, 1% trypsin, pH=8.0 mMRS culture). Base), anaerobic culture at 37 °C, sampling at 0h, 0.5h, 1h, 2h, 3h and 4h respectively, and pouring culture with MRS agar medium to count the plate colonies, determine the number of viable bacteria and calculate their survival rate . The survival rate is the ratio of the number of viable bacteria at the time of sampling in the culture solution to the number of viable bacteria at 0 h, and is expressed in %. The experimental results are shown in Table 1 and Table 2. It can be seen that Bifidobacterium bifidum HNJ6 has good tolerance to artificial stomach and intestinal juice.

Table 1 Tolerance of Bifidobacterium bifidum HNJ6 in artificial simulated gastric juice

Table 2 Tolerance of Bifidobacterium bifidum HNJ6 in artificial simulated intestinal fluid

Example 2: Regulation of Bifidobacterium bifidum HNJ6 on Notch1, Notch2, VEGFR2 and colon cancer-related signaling pathways in HT-29 cells

Use antibiotic-free RPMI1640 cell culture medium to resuspend Bifidobacterium bifidum HNJ6 (or control Bifidobacterium bifidum BB37, LGG, E. coli E.coli) after washing twice with PBS, and adjust the cell density To about 2×10 ⁸ CFU/mL; add HNJ6 (or BB37, LGG, E.coli) suspension to the 6-well plate cultured with HT-29 according to the addition amount of 2 mL of bacterial suspension per well (the cell confluency reaches 95%), 2 mL of antibiotic-free cell culture medium was added to the blank group, and placed in a cell culture incubator containing 5% CO ₂ at 37°C for 2 h; after cells were washed 3 times with PBS, 1 mL was added to each well (6-well plate). TRIzol, after standing at room temperature for 5 minutes, repeated pipetting with an enzyme-free pipette tip, and then transferred to a non-enzyme 1.5mL EP tube, and the total cell RNA was extracted according to the TRIzol instructions; reverse transcription was performed according to the Takara RR047A instructions; fluorescence quantitative PCR Refer to the instructions of Bio-Rad iTaqUniversal SYBR Green Supermix, and the primers used are shown in Table 3.

Table 3 qPCR primer sequences

The results are shown in Figure 1 and Figure 2. Bifidobacterium bifidum HNJ6 down-regulated the transcription levels of Notch1, Notch2, Hes1, and VEGFR2 genes in HT-29 cells, while LGG only down-regulated the transcription levels of Notch1 and Hes1 genes, but not Notch2 and VEGFR2. Bifidobacterium bifidum BB37 and E. coli had no significant effect on the transcription levels of Notch1, Notch2, Hes1 and VEGFR2, but increased the transcription level of Notch1 to a certain extent. The detection results of other tumor-related signaling molecules showed that the transcriptional regulation effect of LGG on tumor-related signaling molecules except Notch1/2 and VEGFR2 was stronger than that of Bifidobacterium bifidum HNJ6. This indicates that not all Bifidobacterium bifidum can inhibit the occurrence of colorectal cancer. The development of colorectal tumors is achieved by regulating the Notch signaling pathway and the level of VEGFR2. E. coli in the control group only showed a certain down-regulation effect on the transcription level of CyclinD1, but had no obvious effect on the transcription levels of other molecules.

Example 3: Bifidobacterium bifidum HNJ6 has no acute toxicity to mice

Bifidobacterium bifidum HNJ6 was resuspended in 2% (w/v) sucrose solution at a cell density of 4.0×10 ⁹ CFU/mL. 10 healthy male BALB/c mice with a weight of about 25 g were taken, and the concentration of the suspension was administered by gavage once a day, observed for one week, and the death and body weight were recorded.

The results of these tests are listed in Table 4. These results show that the feeding concentration of Bifidobacterium bifidum HNJ6 at a concentration of 4.0×10 ⁹ CFU/mL has no obvious effect on the mice, and there is no significant change in body weight and no death phenomenon. The mice showed no obvious pathological symptoms.

Table 4 Changes in body weight and death of mice

time (days) 1 2 3 4 5 6 7 Weight (g) 24.7±1.8 25.1±1.2 25.4±1.3 25.5±1.5 25.7±1.3 25.8±1.1 26.0±1.7 death situation - - - - - - -

Note: -: Mice did not die

Example 4: Relief effect of Bifidobacterium bifidum HNJ6 on colorectal cancer in mice

Forty-eight healthy male BALB/c mice weighing 20-25 g were selected and randomly divided into 6 groups: blank control group, colorectal cancer model control group, Bifidobacterium bifidum HNJ6 intervention group, and Bifidobacterium bifidum BB37 control group , LGG control group, E.coli control group, each group contains 8 mice.

The AOM-DSS method was used to establish colorectal cancer models in the model control group, Bifidobacterium bifidum HNJ6 intervention group, Bifidobacterium bifidum BB37 control group, LGG control group and E.coli control group. That is, after intraperitoneal injection of AOM (7.5mg/kg), use drinking water containing 2% (m/v) DSS instead of normal drinking water for 4 days; return to normal drinking water for 7 days; change the normal drinking water to 2% (m/v) again ) DSS drinking water for 4 days; return to normal drinking water for 15 days.

During the modeling process, 0.25 mL of Bifidobacterium bifidum HNJ6 suspension prepared according to Example 3 of the present specification with a concentration of 4.0×10 ⁹ CFU/mL was given to the mice in the intervention group every day, and the same amount of BB37 was given to the BB37 control group. , the LGG control group was given the same amount of LGG, the E.coli control group was given the same amount of E.coli, and the other two groups were given the same amount of 2% (w/v) sucrose solution without bacteria.

After modeling, the serum, colon and rectum of mice were collected. Among them, the nodal field and rectum were cut along the axial direction, and the number of tumors was calculated. At the same time, colon tissue was taken for paraffin section operation and routine H&E staining.

The tumor number results are shown in Figure 3. By comparing the blank control group with the model control group, it can be seen that using this method can induce colorectal cancer in mice. However, the intervention group treated with Bifidobacterium bifidum HNJ6 by gavage of the present invention can significantly reduce the number of tumors, and the effect is relatively better than that of the LGG group. BB37 and E.coli have no obvious inhibitory effect on tumorigenesis, and the E.coli group died. 3 mice. The results of H&E staining are shown in Figure 4. It is obvious from the slices that severe lesions have occurred in the model group, BB37 and E.coli intervention groups. Compared to normal mice, malignant proliferative cells were significantly increased, and tumors had grown inward. After intragastric administration of Bifidobacterium bifidum HNJ6, the cell infiltration, tumor and intestinal mucosa integrity were improved, and the improvement was stronger than that in the LGG group.

Example 5: Regulation of Bifidobacterium bifidum HNJ6 on colitis-related inflammatory factors in serum of colorectal cancer mice

The serum obtained in Example 4 was taken, and the content of cytokines in the serum was determined by a flow array analyzer. The concentrations of inflammatory factors IL-17 and IFN-γ associated with colitis were determined according to the instructions of the kit (Milliplex Map kit) and the operation instructions of Luminex.

The results are shown in Figure 5. The levels of IL-17 and IFN-γ in the serum of colorectal cancer model mice were significantly higher than those of the blank control group (the data corresponding to each column in the figure is the relative concentration of cytokines in serum relative to the serum cytokines of the blank control group) , Bifidobacterium bifidum HNJ6 can significantly reduce the levels of IL-17 and IFN-γ to normal levels, while BB37, LGG and E.coli have no obvious inhibitory effect on the increase of IL-17 and IFN-γ levels.

Example 6: Regulation of Bifidobacterium bifidum HNJ6 on Notch1, Notch2 signaling pathway and VEFGR2 in mouse colon tissue

Take the colon tissue of about 1 cm in Example 4, add 1 mL of TRIzol and 3 steel balls sterilized by dry heat, and then use a tissue disrupter to disrupt at 70 Hz for 30 s as a cycle, repeat 3 times, and then move the liquid to RNA-free enzyme in a 1.5 mL EP tube, and extracted total RNA, reverse transcribed and performed q-PCR according to the method of Example 2. The primers used are shown in Table 5.

Table 5 qPCR primer sequences

The results are shown in Figure 6. Except for BB37, LGG, E.coli and Bifidobacterium bifidum HNJ6 all down-regulated the abnormally elevated Notch1 gene transcription level in colon tissue of model mice; only Bifidobacterium bifidum HNJ6 down-regulated the transcription level of Notch2 only LGG and Bifidobacterium bifidum HNJ6 showed significant down-regulation of Hes1 gene transcription in model mice, and the down-regulation of Bifidobacterium bifidum HNJ6 was significantly stronger than that of LGG; only Bifidobacterium bifidum HNJ6 had a significant down-regulation effect on VEGFR2 The transcription level is down-regulated.

Example 7: Bifidobacterium bifidum HNJ6 down-regulates the level of butyrate in the intestinal tract of model mice

Take the feces of the mice in Example 4, add 500 μL of saturated NaCl solution to the 50 mg feces sample and fully shake; add 20 μL of 10% sulfuric acid solution, and after sufficient shaking, add 800 μL of ether and shake again; centrifuge (18000 × g, 15 min, 4°C), take the supernatant, add 0.25 g of anhydrous sodium sulfate, fully shake and centrifuge again (18000×g, 15min, 4°C); take the supernatant to a gas phase bottle for gas analysis.

The results are shown in Figure 7. There were no significant changes in acetic acid and propionic acid in the intestinal tract of colon cancer model mice, but the content of butyric acid increased significantly compared with the blank group. Bifidobacterium bifidum HNJ6 and BB37 can significantly increase the level of acetic acid in the intestine, Bifidobacterium bifidum HNJ6 and LGG can significantly reduce the level of abnormally elevated butyric acid, and Bifidobacterium bifidum HNJ6 reduces the level of butyric acid. The magnitude was significantly larger than that of LGG, and E.coli had no significant effect on the levels of the three acids.

Example 8: Repairing effect of Bifidobacterium bifidum HNJ6 on intestinal flora imbalance in colon cancer model mice The metagenome of 0.1 g of mouse feces in Example 4 was taken and extracted with reference to the instructions of the FastDNA Spin Kit for Soil , slightly modified, the specific method is as follows. Add about 0.1 g of feces to the Lysing Matrix E tube, add 978 μL of Sodium Phosphate Buffer and 122 μL of MT Buffer, and let stand for 30 min at room temperature; use Fastprep, set the speed to 6.0, set the time to 40, and perform crushing; at 4°C Centrifuge at 14,000 × g for 10 min, take the supernatant, add 250 μL of PPS, and mix by inversion; centrifuge at 14,000 × g for 10 min at 4°C, take the supernatant, add 1 mL of Binding Matrix Suspension, invert and mix, and let stand at room temperature 3 min, and discard 650 μL of supernatant. After shaking and resuspending, take 650 μL of suspension to SPIN Fitter, centrifuge at 14,000 × g for 2 min at 4°C, discard the liquid in the custody and repeat the above steps once; add 500 μL SEWS- M (add 100 mL of absolute ethanol before use, and shake and mix well), centrifuge at 14,000 × g for 1 min at 4°C, discard the liquid in the tube, and centrifuge again under the same conditions; Let stand for 5 min at room temperature; add 50 μL of DES, and place it in a metal bath at 55 °C for 5 min; then centrifuge at 14,000 × g for 1 min at 4 °C, the collection tube is the DNA solution, and the obtained DNA solution is sent to the second Generation sequencer sequenced and analyzed.

The results are shown in Figure 8. The Firmicutes in the intestinal tract of the model mice were significantly reduced, while the Verrucomicrobia and Fusobacterium were abnormally increased. The intake of Bifidobacterium bifidum HNJ6 can significantly increase the abundance of Firmicutes in the intestinal tract of model mice, and inhibit the abnormally increased Verrucomicrobia and Fusobacterium; LGG can also partially restore the dysregulated intestinal bacteria However, it has no significant inhibitory effect on the abnormally increased Verrucomicrobia, and the abundance of Firmicutes is still low; BB37 and E.coli can partially restore the proportion of Firmicutes, which is not conducive to the imbalance of other bacteria. There was no significant recovery effect.

Example 9: Utilize Bifidobacterium bifidum HNJ6 of the present invention to manufacture soymilk containing the bacteria

The soybeans were soaked in soft water, soaked at a temperature of 80 °C for 2 hours, and then the soybean hulls were removed. Next, drain the soaking water, add boiling water to refine the slurry, and keep it at a temperature higher than 80° C. for 12 minutes. The obtained slurry is filtered with a 150-mesh sieve, followed by centrifugation, and the obtained centrifuge is crude soy milk, which is then heated to a temperature of 140-150 ° C, and then the crude soy milk is quickly introduced into a vacuum cooling chamber for vacuuming, The odorous substances in the crude soy milk are quickly discharged with the water vapor. After vacuum degassing, the temperature was lowered to about 37°C, and then the Bifidobacterium bifidum HNJ6 working starter of the present invention was connected to make its concentration reach 10 ⁶ CFU/ml or more, and the temperature was 4°C for refrigerated storage. Thus, the soybean milk containing the live bacteria of Bifidobacterium bifidum HNJ6 of the present invention is obtained.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (4)

1. A bifidobacteria (Bifidobacterium bifidum) HNJ6, preserved in the Guangdong Provincial Microbial Culture Collection Center on October 25, 2017, and the preservation address is Guangdong Province, Guangdong Province, 5th Floor, Building 59, No. 100, Middle Xianlie Road, Guangzhou Institute of Microbiology, the deposit number is GDMCC No.60255. 2. A fermented food, characterized in that the fermented food is a dairy product, a soy product and a fruit and vegetable product produced by using Bifidobacterium bifidum (Bifidobacterium bifidum) HNJ6 fermentation; The Bifidobacterium bifidum HNJ6 was deposited in the Guangdong Provincial Microbial Culture Collection Center on October 25, 2017, and the preservation address is Guangdong Province Microbiological Research on the 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City Institute, the deposit number is GDMCC No.60255. 3. The fermented food according to claim 2, wherein the dairy product comprises milk, sour cream or cheese; the soy product comprises soy milk, tempeh or soybean paste; the fruit and vegetable product comprises cucumber, carrot , beet, celery or cabbage products. 4. the application of bifidobacterium bifidum (Bifidobacterium bifidum) HNJ6 described in claim 1 in the preparation of medicine for inhibiting rectal cancer, it is characterized in that described bifidobacteria improves intestinal flora, reduces abnormally elevated short-term in intestinal tract. Chain fatty acid levels, reduce colorectal inflammation, thereby inhibiting colorectal carcinogenesis.

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