CN107629988B - Bifidobacterium bifidum capable of relieving colorectal cancer and application thereof - Google Patents
Bifidobacterium bifidum capable of relieving colorectal cancer and application thereof Download PDFInfo
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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
Technical Field
The invention relates to the technical field of microorganisms, in particular to bifidobacterium bifidum capable of remarkably inhibiting colorectal cancer by regulating signal pathways of Notch1, Notch2 and VEGFR2 and application thereof.
Background
Colorectal cancer is a malignant tumor of the digestive tract. With the continuous increase of morbidity and mortality of colorectal cancer in China, the threat to the health of people in China is gradually deepened. At present, the mainstream methods for clinically treating colorectal cancer have large side effects, which greatly affect the life quality of patients. As a safe edible food, the probiotics have been proved to have the effects of alleviating and treating various diseases, such as promoting digestion and absorption, enhancing immunity, preventing infection of reproductive system, alleviating anaphylaxis, preventing and inhibiting tumor, preventing and inhibiting cardiovascular and cerebrovascular diseases, preventing and inhibiting nervous diseases, etc.
The intestine is not only the immediate diseased area of colorectal cancer, but is also the primary site for probiotics to exert their probiotic effects, and there is an increasing search for the effects of probiotics on colorectal cancer. Some studies have shown that probiotics, especially lactic acid bacteria, have the efficacy of antagonizing colon cancer cells or inhibiting colon tumorigenesis, and they can inhibit proliferation of colon cancer cells or induce apoptosis of colon cancer cells to various degrees. In addition, the antagonistic effect of probiotics on colorectal cancer is also shown in the following aspects: combining or metabolizing carcinogenic substances, inducing cancer cell apoptosis, improving immunity, inhibiting cancer cell differentiation and proliferation, protecting intestinal tracts, maintaining intestinal tract flora balance and the like, and the regulation of probiotics on the intestinal tract flora can finally influence the concentration of short-chain fatty acids in the intestinal tracts and further influence the occurrence of tumors.
Studies have shown that despite the higher concentration of butyrate in the colon, colon cancer can still develop and grow, and butyrate-rich microenvironments can select for tumor cells that can metabolize butyrate. Probiotics may also inhibit tumorigenesis through modulation of tumor-associated signaling pathways. There are many signaling pathways involved in colorectal cancer, including AKT signaling pathway, GPCR signaling pathway, MAPK signaling pathway, NF- κ B signaling pathway, JAK signaling pathway, Wnt signaling pathway, TGF- β signaling pathway, ESC signaling pathway, Apoptosis signaling pathway, CCC signaling pathway, and angiogenisis signaling pathway, among others. At present, the relationship between Wnt and NF-kB signal pathways and colorectal cancer is researched more, but the relationship between a Notch signal pathway as a signal pathway related to epithelial cell differentiation and colorectal cancer is very close. In colorectal cancer, the Notch signaling pathway plays a role in inducing and promoting tumors to increase drug resistance. Many studies in the clinic have also shown that tumor tissue in the colon has an overactivated Notch signaling pathway compared to that of paracancerous or normal tissue.
In addition to the Notch signaling pathway, numerous studies have also shown that Vascular Endothelial Growth Factor Receptors (VEGFR) exhibit high expression 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, chemotactic effects on endothelial cells, etc. Different investigators observed activation of the MAPK signaling pathway following VEGF binding to VEGFR2 in a variety of endothelial cells. VEGFR2 not only promotes the division and proliferation of vascular endothelial cells, but also induces the proliferation of tumor blood vessels and promotes the growth and metastasis of tumor cells. A number of reports also demonstrated that VEGFR2 also appears to be highly expressed in colorectal cancer tissues. Therefore, VEGFR2 can be used as a target for the treatment and diagnosis of tumor development.
The probiotics can also achieve the effects of relieving colorectal cancer or inhibiting the growth of colon cancer cells by regulating Notch and VEGFR signal pathways. Bifidobacterium bifidum is a kind of lactic acid bacteria, which are widely present in the intestinal tracts of animals and humans. There are a lot of research reports on the physiological function of bifidobacterium bifidum at home and abroad, and there are also a few reports on the inhibition of the occurrence of colorectal cancer by bifidobacterium bifidum, but the mechanism is not clear. The research on the inhibition of colorectal cancer by bifidobacterium bifidum has high value for many disciplines such as food science, preventive medicine and microbiology. Screening excellent safe bifidobacterium bifidum with the function of inhibiting the occurrence of colorectal cancer has important significance for developing functional foods and medicines.
In the published literature and patents or patent applications, there are a few patents on the inhibition of tumors by probiotics or probiotic combinations and fermentation products, but there is no clear probiotic strain that has the function of inhibiting colon cancer. For example, CN105535650A discloses a probiotic composition with an anti-tumor (liver cancer cell line subcutaneous transplantation) function, the composition relates to multiple probiotics and traditional Chinese medicine components, but the tumor inhibition function of each component is not clear, and all probiotics are not clear, and the formula does not have universality because the difference between different strains of the same kind of probiotics is significant in the probiotic function; similarly, no strains were identified in CN 104686657A and CN101711775A, and the functions of the components of the fermented product were not clear.
CN104523761A discloses that lactococcus lactis which is constructed by genetic engineering and can induce and express interleukin 12 can inhibit the growth of intestinal tumor, but the lactococcus lactis is obtained in non-natural world and can only be used for medicines but not for food. CN105441357A discloses a Lactobacillus plantarum SKT109 capable of producing exopolysaccharides, which can inhibit the growth of human colon cancer cells HT-29 in vitro and inhibit the growth of tumors in nude mice bearing tumors (axillary inoculation of colon cancer cells); CN103445068A and CN103468600A disclose a Lactobacillus plantarum Dy-1 with tumor inhibition effect, which 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 (colon cancer cells inoculated subcutaneously at the abdomen). The three patent strains are clear and have clear functions, but the related animal model adopts the colon cancer cell strain subcutaneous tumor bearing, has larger difference with the tumor environment in the intestinal tract, and has unclear effect on the regulation of tumor-related signal channels. Therefore, there is a need to screen probiotics from nature that are edible and have a clear mechanism to inhibit tumorigenesis for inhibiting the growth of tumors that occur at the colorectal site.
Disclosure of Invention
In view of the above problems in the prior art, the present application provides a bifidobacterium bifidum and uses thereof. The single strain has strong capability of regulating Notch1, Notch2 signal channels and VEGFR2 molecular expression, can obviously inhibit colorectal cancer, and has multiple functions and purposes of relieving colonic inflammation, improving the intestinal flora structure and the short-chain fatty acid level and the like.
The technical scheme of the invention is as follows:
bifidobacterium bifidum HNJ6(Bifidobacterium bifidum) is preserved in Guangdong province microorganism strain collection center in 2017, 10 and 25 months, with the preservation address of Guangzhou city Michelia Tokyo 100 # lou 59 and Guangdong province microorganism research institute, and the preservation number is GDMCC No. 60255.
A fermented food is a dairy product, a bean product and a fruit and vegetable product which are produced by using bifidobacterium bifidum HNJ6 through fermentation.
The dairy product comprises milk, sour cream or cheese; the bean product comprises soybean milk, fermented soybean or soybean paste; the fruit and vegetable product comprises cucumber, carrot, beet, celery or cabbage product.
The bifidobacterium bifidum HNJ6 or the fermented food is applied to improving intestinal flora, reducing abnormally increased short-chain fatty acid level in the intestinal tract, relieving colorectal inflammation and inhibiting colorectal cancer.
The bifidobacterium bifidum HNJ6 has the following biological properties:
(1) the characteristics of the thallus are as follows: gram-positive, non-sporulating, immotile bacteria.
(2) Colony characteristics: the anaerobic culture is carried out for 36 hours to form obvious colonies, the diameter is between 0.5 and 2mm, the front form is circular, the side form is convex, the edge is neat, the color is milky white, the color is not transparent, the surface is moist and smooth, and no pigment is generated.
(3) Growth characteristics: the cells were cultured in mMRS medium at 37 ℃ under constant temperature anaerobic conditions for about 24 hours to the end of log.
(4) The product has good tolerance to artificial simulation of gastrointestinal fluid;
(5) can regulate the expression levels of Notch1, Notch2 signaling pathway and VEGFR2 of HT-29 cells;
(6) the number of tumors in colorectal cancer tissues of a colon cancer model mouse can be obviously reduced, and the integrity of intestinal tissues of the model mouse is improved;
(7) can obviously relieve the colorectal inflammation level of a colon cancer model mouse;
(8) regulating the expression levels of Notch1, Notch2 signaling pathway and VEGFR2 in colon tissues of a colon cancer model mouse;
(9) improve the intestinal microecology and the short-chain fatty acid level of colon cancer model mice.
The extraction method of the strain comprises the following steps:
separation and screening of lactic acid bacteria
(1) Taking 1g of fresh excrement of healthy male old people from Changshoucun village in Chaihu lake of Zhongxiang city, Hubei province. After gradient dilution, the suspension is coated on mMRS solid culture medium and is placed in an anaerobic environment to be cultured for 72 hours at 37 ℃.
(2) Observing and recording colony morphology, picking colonies and streaking for purification.
(3) The colonies were gram-stained in MRS liquid medium at 37 ℃ for 48 hours, and the morphology of the colonies was recorded.
(4) Removing gram-negative bacteria strains and gram-positive cocci from the colonies, and selecting to obtain gram-positive bacilli.
(5) After catalase analysis, catalase-positive strains were discarded, and catalase-negative strains were retained.
(II) preliminary identification of Bifidobacterium bifidum: fructose-6-phosphate phosphoketolase assay
(l) Culturing the lactic acid bacteria obtained by screening in the step (I) in a liquid mMRS culture solution for 24h, and then centrifuging the lmL culture at 8000rpm for 2 min;
(2) using 0.05M KH of pH6.5 containing 0.05% (mass percent) cysteine hydrochloride2PO4Washing the solution twice;
(3) resuspending in 200. mu.L of the above phosphate buffer solution to which 0.25% (mass%) Triton X-100 was added;
(4) adding 50 mu L of mixed solution of 6mg/mL sodium fluoride and 10mg/mL sodium iodoacetate and 50 mu L of fructose-6-phosphate with the concentration of 80mg/mL, and incubating for 1h at 37 ℃;
(5) adding 300 μ L of hydroxylamine hydrochloride with concentration of 0.139g/mL and pH of 6.5, and standing at room temperature for 10 min;
(6) separately, 200. mu.L of 15% (mass percent) trichloroacetic acid and 4M HCl were added;
(7) when 200. mu.L of 0.1M HCl containing 5 mass% of ferric trichloride was added, the system rapidly turned red, which was positive for F6PPK, and it was preliminarily judged that it was a Bifidobacterium.
Molecular biological identification of Bifidobacterium bifidum
(l) Extraction of genome of single bacterium
A. Culturing the lactic acid bacteria obtained by screening in the step (II) overnight, taking the overnight-cultured bacterial suspension lmL in a 1.5mL centrifuge tube, centrifuging at 10000rpm for 2min, and removing the supernatant to obtain thalli;
B. purging the thallus with lmL sterile water, centrifuging at 10000rpm for 2min, and removing the supernatant to obtain thallus;
C. adding 200 μ LSDS lysate, and water-bathing at 80 deg.C for 30 min;
D. adding 200 μ L of phenol-chloroform solution into the thallus lysate, wherein the phenol-chloroform solution comprises Tris saturated phenol, chloroform and isoamylol at a volume ratio of 25:24:1, mixing, centrifuging at 12000rpm for 5-10min, and collecting 200 μ L of supernatant;
E. adding 400 μ L of glacial ethanol or glacial isopropanol into 200uL of supernatant, standing at-20 deg.C for 1h, centrifuging at 12000rpm for 5-10min, and discarding the supernatant;
F. adding 500 μ L70% (volume percentage) of glacial ethanol, resuspending the precipitate, centrifuging at 12000rpm for 1-3min, and discarding the supernatant;
drying in an oven at G.60 ℃ or naturally airing;
H.50μLddH2re-dissolving the precipitate with O for PCR;
(2)16S rDNA PCR
A. bacterial 16S rDNA 50 μ LPCR reaction:
10 × Taq buffer, 5 μ L; dNTP, 5. mu.L; 27F, 0.5 μ L; 1492R, 0.5 μ L; taq enzyme, 0.5. mu.L; template, 0.5 μ L; ddH2O，38μL。
PCR conditions:
95℃5min；95℃10s；55℃30s；72℃30s；step2-4 30×；72℃5min；12℃2min；
(3) preparing 1% agarose gel, mixing the PCR product with 10000 × loading buffer, loading the sample by 5 μ L, running at 120V for 30min, and performing gel imaging;
(4) sequencing the PCR product of 16S rDNA, searching and comparing the obtained sequence result in GeneBank by using BLAST, selecting the sequencing result and identifying as the lactic acid bacteria of bifidobacterium bifidum, and preserving at-80 ℃ for later use;
the beneficial technical effects of the invention are as follows:
the bifidobacterium bifidum HNJ6 has good gastric acid resistance and cholate resistance, can regulate the Notch1 and Notch2 signal pathways in colorectal tissues and the expression capacity of VEGFR2 molecules to realize the inhibition effect on colorectal cancer, obviously relieves the inflammation of the colorectal part of a colorectal cancer model mouse, and reduces the tumor number of the colorectal part of the model mouse. In addition, the composition can also improve intestinal flora and short chain fatty acid level in intestinal tract.
The strain is superior to a commercial strain Lactobacillus rhamnosus GG strain (LGG) in the effect of inhibiting the occurrence of colorectal cancer, and the mechanism of inhibiting the occurrence of colorectal cancer is different from that of the LGG. Therefore, the bifidobacterium bifidum HNJ6 can be used as an auxiliary means for clinically treating colorectal cancer, and has no toxic or side effect of medicaments. Therefore, the bifidobacterium bifidum HNJ6 can be used for preparing pharmaceutical compositions and fermented foods for relieving and preventing colorectal cancer, and has very wide application prospect.
Drawings
FIG. 1 shows the effect of the present strain on the expression levels of Notch1, Notch2 signaling pathway molecules and VEGFR2 in HT-29 cell lines;
FIG. 2 is a graph showing the effect of this strain on the expression levels of other cancer-associated signaling pathway molecules of the HT-29 cell line;
FIG. 3 is the effect of the present strain on the number of tumors in the colorectal region of colorectal cancer model mice;
FIG. 4 shows the improvement of the present strain on the tissue damage at the colorectal region of a colorectal cancer model mouse;
FIG. 5 shows the improvement of the present strain in serum IL-17 and IFN-gamma levels in mice of colorectal cancer model;
FIG. 6 shows the effect of the present strain on the expression levels of Notch1, Notch2 signaling pathway molecules and VEGFR2 in intestinal tissues of mouse model of colorectal cancer;
FIG. 7 shows the effect of the present strain on intestinal short-chain fatty acid levels in colorectal cancer model mice;
FIG. 8 shows the improvement of intestinal flora structure of colorectal cancer model mice by the strain.
Detailed Description
The present invention will be described in detail below with reference to the drawings, examples, and test examples.
Example 1: bifidobacterium bifidum HNJ6 has good tolerance to simulated gastrointestinal fluid
The method comprises the steps of streaking and inoculating HNJ6 frozen bifidobacterium bifidum into a mMRS culture medium (MRS culture medium + 0.05% cysteine hydrochloride) solid culture medium, carrying out anaerobic culture at 37 ℃ for 48h, carrying out subculture for 2-3 times by using the mMRS culture medium, taking HNJ6 culture solution of the bifidobacterium bifidum, centrifuging at 8000 Xg for 5min to collect thalli, suspending in (1:1) artificial simulated gastric juice (containing 1% pepsin and having a pH value of 2.5) and mixing, carrying out anaerobic culture at 37 ℃, sampling at the beginning of (0h), 0.5h, 1h and MRS 2h respectively, carrying out plate colony counting by using magar culture medium pouring culture, measuring the viable count and calculating the survival rate of the viable count. The survival rate is the ratio of the number of viable bacteria in the culture medium to the number of viable bacteria at 0h, and is expressed as%.
Collecting culture solution of Bifidobacterium bifidum HNJ6, centrifuging at 8000 Xg for 5min, collecting thallus, resuspending in (1:1) artificial simulated intestinal fluid (mMRS culture medium containing 0.3% ox bile salt, 1% trypsin, and pH 8.0), anaerobically culturing at 37 deg.C, sampling at 0h, 0.5h, 1h, 2h, 3h and 4h respectively, pouring and culturing with MRS agar culture medium, counting plate colony, determining viable count and calculating survival rate. The survival rate is the ratio of the number of viable bacteria at the time of sampling the culture medium to the number of viable bacteria at 0h, and is expressed as%. The results of the experiments are shown in tables 1 and 2, and 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 simulated gastric fluid
TABLE 2 Bifidobacterium bifidum HNJ6 tolerance in artificially simulated intestinal fluid
Example 2: modulation of HT-29 cell Notch1, Notch2, VEGFR2 and colon cancer-related signaling pathways by Bifidobacterium bifidum HNJ6
Resuspending the Bifidobacterium bifidum HNJ6 (or control Bifidobacterium bifidum BB37, LGG, E.coli) puree washed twice with PBS using RPMI1640 cell culture medium without antibiotics, and adjusting the cell density to about 2X 108CFU/mL; HNJ6 (or BB37, LGG, E. coli) suspension was added to a 6-well plate incubated with HT-29 (cell confluence 95%) at 2 mL/well, and 2mL of antibiotic-free cell culture medium was placed in the blank at 37 ℃ with 5% CO2Culturing for 2 hours in the cell culture box; after washing the cells 3 times with PBS, 1mL of TRIzol was added to each well (6-well plate), left to stand at room temperature for 5min, repeatedly blown with an enzyme-free gun head, transferred to an enzyme-free 1.5mL EP tube, and total RNA of the cells was extracted according to the TRIzol instructions; reverse transcription was performed with reference to the Takara RR047A instructions; fluorescent quantitative PCR was performed according to Bio-Rad iTaq Universal SYBR Green Supermix instructions using the primers shown in Table 3.
TABLE 3 qPCR primer sequences
The results are shown in FIGS. 1 and 2. Bifidobacterium bifidum HNJ6 shows down-regulation effect on the transcription levels of HT-29 cell Notch1, Notch2, Hes1 and VEGFR2 genes, while LGG only shows little down-regulation on the transcription levels of Notch1 and Hes1 genes and does not show obvious down-regulation effect on Notch2 and VEGFR 2; bifidobacterium bifidum BB37 and Escherichia coli have no significant effect on the transcription levels of Notch1, Notch2, Hes1 and VEGFR2, but the transcription level of Notch1 is improved to a certain extent. The detection results aiming at other tumor-related signal molecules show that the transcriptional regulation effect of LGG on the tumor-related signal molecules except Notch1/2 and VEGFR2 is stronger than that of Bifidobacterium bifidum HNJ 6. The demonstration shows that not all bifidobacterium bifidum can inhibit the occurrence of colorectal cancer, the function of bifidobacterium bifidum HNJ6 for inhibiting the occurrence of colorectal cancer is unique, the mechanism is different from that of LGG, and bifidobacterium bifidum HNJ6 realizes the occurrence of colorectal tumor mainly by regulating the Notch signal pathway and the level of VEGFR 2. Coli in the control group showed some down-regulation of the transcription level of cyclinD1, but had no significant effect on the transcription level of other molecules.
Example 3: bifidobacterium bifidum HNJ6 has no acute adverse side effects on mice
The Bifidobacterium bifidum HNJ6 was resuspended in 2% (w/v) sucrose solution with a cell density of 4.0X 109CFU/mL. 10 healthy male BALB/c mice weighing about 25g were subjected to intragastric administration of the suspension at this concentration once a day, observed for one week, and the death and body weight were recorded.
The results of these tests are shown in Table 4. These results show that the feed concentration was 4.0X 109The CFU/mL bifidobacterium bifidum HNJ6 has no obvious influence on mice, has no obvious change in body weight and no death phenomenon. The mice had no apparent pathological symptoms in appearance.
TABLE 4 weight change and mortality in mice
| Time (sky) | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Body 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: relieving effect of bifidobacterium bifidum HNJ6 on colorectal cancer of mice
48 healthy male BALB/c mice weighing 20-25g were randomly assigned to 6 groups: blank control group, colorectal cancer model control group, bifidobacterium bifidum HNJ6 intervention group, bifidobacterium bifidum BB37 control group, LGG control group, E.coli control group, each group contains 8 mice.
Performing colorectal cancer molding on a model control group, a bifidobacterium bifidum HNJ6 intervention group, a bifidobacterium bifidum BB37 control group, an LGG control group and an E.coli control group by using an AOM-DSS method. I.e. after the AOM is injected into the abdominal cavity (7.5mg/kg), the drinking water containing 2 percent (m/v) DSS is used for replacing the normal drinking water, and the breeding is continued for 4 days; recovering normal drinking water for 7 d; changing the normal drinking water to 2% (m/v) DSS drinking water again, and continuously feeding for 4 d; and (5) recovering normal drinking water feeding for 15 d.
Mice in the intervention groups were fed daily during the molding process with a concentration of 4.0X 10 prepared according to example 3 of the present specification90.25mL of CFU/mL bifidobacterium bifidum HNJ6 suspension, BB37 with the same amount as the gastric lavage of a BB37 control group, LGG with the same amount as the gastric lavage of an LGG control group, E.coli with the same amount as the gastric lavage of an E.coli control group, and 2 (w/v) sucrose solutions without bacteria with the same amount as the residual group are fed.
After the molding is finished, the serum, colon and rectum of the mouse are taken. Wherein, the knot field and the rectum are cut off along the axial direction, and the tumor number is calculated. Meanwhile, taking colon tissues for paraffin section operation and carrying out conventional H & E staining.
The tumor number results are shown in figure 3. As can be seen by comparing the blank control group with the model control group, the mice can be induced to suffer from colorectal cancer by using the method. The intervention group after gastric perfusion of bifidobacterium bifidum HNJ6 can obviously reduce the number of tumors, the effect is relatively better than that of the LGG group, the inhibition effect of BB37 and E.coli on tumorigenesis is not obvious, and 3 mice are killed in the E.coli group. The H & E staining results are shown in FIG. 4. It is evident from the sections that severe lesions have developed in the model group, BB37 and e. Malignant proliferating cells were significantly increased compared to normal mice, and tumors had grown in. After the administration of Bifidobacterium bifidum HNJ6, the cells infiltration, tumors or intestinal mucosa integrity were improved, and the improvement degree was stronger than that of LGG group.
Example 5: bifidobacterium bifidum HNJ6 regulating effects on colitis-related inflammatory factors in serum of colorectal cancer mice
The serum obtained in example 4 was collected, and the content of cytokines in the serum was measured by using a flow-type dot-matrix analyzer. The concentrations of the inflammatory factors IL-17 and IFN-. gamma.associated with colitis were determined according to the kit (Millipox Map kit) instructions and Luminex instructions.
The results are shown in FIG. 5. The levels of IL-17 and IFN-gamma in the serum of the colorectal cancer model mouse are obviously increased compared with that of a blank control group (the data corresponding to each column in the figure is the relative concentration of cytokines in the serum relative to the cytokines in the serum of the blank control group), the Bifidobacterium bifidum HNJ6 can obviously reduce the levels of IL-17 and IFN-gamma to normal levels, and BB37, LGG and E.coli have no obvious inhibition effect on the increase of the levels of IL-17 and IFN-gamma.
Example 6: modulation of mouse colon tissue Notch1, Notch2 signaling pathway and VEFGR2 by Bifidobacterium bifidum HNJ6
Approximately 1cm of colon tissue from example 4 was taken, 1mL of TRIzol and 3 steel beads sterilized by dry heat were added, and then disrupted at 70Hz for 30s as one cycle using a tissue disruptor, which was repeated 3 times, after which the liquid was transferred to a 1.5mL LEP tube without RNase, and total RNA was extracted, reverse transcribed and subjected to 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 FIG. 6. LGG, e.coli and bifidobacterium bifidum HNJ6, except BB37, down-regulated the abnormally elevated level of Notch1 gene transcription in colon tissue of model mice; only bifidobacterium bifidum HNJ6 has down-regulation effect on the transcription level of Notch 2; only LGG and bifidobacterium bifidum HNJ6 show obvious down-regulation effect on the transcription of a model mouse Hes1 gene, and the down-regulation effect of bifidobacterium bifidum HNJ6 is obviously stronger than that of LGG; only Bifidobacterium bifidum HNJ6 had a down-regulating effect on the transcript level of VEGFR 2.
Example 7: bifidobacterium bifidum HNJ6 downregulated butyric acid levels in intestinal tract of model mice
Taking the feces of the mouse in example 4, 500. mu.L of a saturated NaCl solution was added to a 50mg feces sample and sufficiently shaken; adding 20 mu L of 10% sulfuric acid solution, fully shaking, adding 800 mu L of diethyl ether, and shaking again; centrifuging (18000 Xg, 15min, 4 deg.C), collecting supernatant, adding 0.25g anhydrous sodium sulfate, shaking, and centrifuging again (18000 Xg, 15min, 4 deg.C); and taking the supernatant into a gas phase bottle for gas quality analysis.
The results are shown in FIG. 7. There were no significant changes in acetic acid and propionic acid in the intestine of the colon cancer model mouse, but the content of butyric acid was significantly increased compared to the blank group. Bifidobacterium bifidum HNJ6 and BB37 significantly increased the levels of intestinal acetic acid, bifidobacterium bifidum HNJ6 and LGG significantly decreased abnormally elevated butyric acid levels, and bifidobacterium bifidum HNJ6 decreased butyric acid concentrations significantly more than LGG, with no significant effect of e.
Example 8: action of Bifidobacterium bifidum HNJ6 on repairing dysbacteriosis of colon cancer model mouse intestinal flora metagenome of 0.1g mouse feces in example 4 was extracted and reference Kit (FastDNA Spin Kit for Soil) instruction was extracted with slight modification, and the specific method is as follows. Adding about 0.1g of feces to a Lysing Matrix E tube, and adding 978. mu.L of LSodium Phosphate Buffer and 122. mu.L of MT Buffer, followed by standing at room temperature for 30 min; crushing is performed using Fastprep, setting the speed to 6.0, setting the time to 40; centrifuging at 14000 Xg for 10min at 4 deg.C, collecting supernatant, adding 250 μ L PPS, and mixing; centrifuging at 14000 Xg for 10min at 4 ℃, taking the supernatant, adding 1mL Binding matrix Suspension, reversing and uniformly mixing, standing for 3min at room temperature, discarding 650 mu L of the supernatant, shaking and re-suspending, taking 650 mu L of suspension to SPIN setter, centrifuging at 14000 Xg for 2min at 4 ℃, discarding the liquid in the tube, and repeating the steps once; adding 500 μ L SEWS-M (adding 100mL anhydrous ethanol before use, and shaking thoroughly and mixing), centrifuging at 14000 × g for 1min at 4 deg.C, discarding the liquid in the tube, and centrifuging under the same conditions again; using a new liquid collecting tube, and standing for 5min at room temperature; adding 50 μ L DES, and placing in 55 deg.C metal bath for 5 min; and centrifuging at 14000 Xg for 1min at 4 ℃ to obtain a DNA solution in a liquid collecting tube, and sending the obtained DNA solution to a second-generation sequencer for sequencing and analysis.
The results are shown in FIG. 8. The number of firmicutes in the intestinal tract of the model mice was significantly reduced, while the number of verrucomicrobia and fusobacteria were abnormally increased. The intake of the bifidobacterium bifidum HNJ6 can obviously increase the abundance of firmicutes in the intestinal tract of the model mouse and inhibit abnormally increased wart microsomycota and fusobacteria; LGG can also partially restore the disordered intestinal flora structure, but has no obvious inhibition effect on abnormally increased wart microsomia, and the abundance of firmicutes is still less; col, BB37, can partially restore the proportion of firmicutes without significant restoration of the dysregulation of other bacteria.
Example 9: method for preparing soybean milk containing bifidobacterium bifidum HNJ6
Soaking soybean in soft water at 80 deg.C for 2 hr, and removing soybean hull. Then, the soaking water is drained, boiled water is added for grinding, and the temperature is kept for 12min under the condition that the temperature is higher than 80 ℃. Filtering the obtained slurry with a 150-mesh screen, performing centrifugal separation to obtain a centrifugal liquid, namely coarse soybean milk, heating the centrifugal liquid to the temperature of 140-150 ℃, and then rapidly introducing the coarse soybean milk into a vacuum cooling chamber for vacuumizing, wherein peculiar smell substances in the coarse soybean milk are rapidly discharged along with water vapor. Vacuum degassing, cooling to about 37 deg.C, inoculating Bifidobacterium bifidum HNJ6 working leaven to reach concentration of 106More than CFU/ml, and storing at 4 deg.C under refrigeration to obtain soybean milk containing live Bifidobacterium bifidum HNJ6 of the invention.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (4)
1. Bifidobacterium bifidum HNJ6 is deposited in Guangdong province microorganism culture collection center at 25.10.2017 with the collection address of Guangzhou city Michelia Tokyo 100 # 59 th building 5 th Guangdong province microorganism research institute, and the collection number is GDMCC No. 60255.
2. A fermented food is characterized in that the fermented food is a dairy product, a bean product and a fruit and vegetable product which are produced by using Bifidobacterium bifidum HNJ6 through fermentation;
the Bifidobacterium bifidum (Bifidobacterium bifidum) HNJ6 is preserved in Guangdong province microbial strain preservation center in 2017, 10 and 25 months, and the preservation address is Guangzhou city Mr. 100 Dazhou 59 th building 5 Guangdong province microbial research institute, and the preservation number is GDMCC No. 60255.
3. The fermented food product according to claim 2, wherein the dairy product comprises milk, sour cream or cheese; the bean product comprises soybean milk, fermented soybean or soybean paste; the fruit and vegetable product comprises cucumber, carrot, beet, celery or cabbage product.
4. Use of Bifidobacterium bifidum HNJ6 in the manufacture of a medicament for the inhibition of colorectal cancer according to claim 1 wherein the Bifidobacterium bifidum improves intestinal flora, reduces abnormally elevated short chain fatty acid levels in the intestine, reduces colorectal inflammation and thereby inhibits colorectal cancer.
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| CN107629987B (en) * | 2017-11-03 | 2020-05-22 | 江南大学 | Lactobacillus plantarum with colorectal cancer generation inhibiting function and application thereof |
| CN113151036A (en) * | 2018-05-11 | 2021-07-23 | 韩国亿诺生物有限公司 | Novel strain having effect of preventing or treating cancer |
| CN109055269B (en) * | 2018-08-22 | 2021-03-30 | 江南大学 | Bifidobacterium longum subspecies infantis CCFM687, fermented food thereof and application thereof |
| KR102485269B1 (en) * | 2019-10-18 | 2023-01-06 | 주식회사 지놈앤컴퍼니 | Composition for enhancing or improving immune activity comprising bifidobacterium bifidum |
| CN112458016B (en) * | 2020-11-27 | 2021-11-19 | 石家庄君乐宝乳业有限公司 | Bifidobacterium bifidum i771, and separation and purification method and application thereof |
| CN112980734B (en) * | 2021-03-24 | 2022-07-22 | 江南大学 | Bifidobacterium bifidum for relieving constipation and regulating intestinal flora disorder and application thereof |
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| CN112940980B (en) * | 2021-03-24 | 2022-07-22 | 江南大学 | Bifidobacterium bifidum for relieving constipation and fermented food and probiotic preparation prepared from same |
| CN113025530B (en) * | 2021-03-30 | 2022-11-01 | 江南大学 | Bifidobacterium bifidum for relieving laxative colon and application thereof |
| CN115011518A (en) * | 2022-06-13 | 2022-09-06 | 东北农业大学 | Lactobacillus mixture with effect of relieving colorectal cancer related to colitis and application thereof |
| CN115651856B (en) * | 2022-06-14 | 2023-04-18 | 东北农业大学 | Combined bifidobacterium capable of relieving mouse intestinal injury caused by lipopolysaccharide |
| CN116004416B (en) * | 2022-07-13 | 2024-05-10 | 四川大学 | Application of bifidobacterium bifidum from infant intestinal tract |
| CN115261426B (en) * | 2022-09-26 | 2023-01-03 | 东北农业大学 | Bifidobacterium bifidum E3 extracellular polysaccharide with antioxidant capacity |
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