CN115820458B

CN115820458B - Bifidobacterium longum 050101 with ulcerative colitis relieving effect and application thereof

Info

Publication number: CN115820458B
Application number: CN202210922944.9A
Authority: CN
Inventors: 谢新强; 杜明珠; 蔡淑珍; 柏建玲; 张菊梅; 吴清平; 杨双红; 李滢; 赵辉; 李芬发; 梁婷婷; 蒋同; 杨娟; 李龙岩; 吴磊
Original assignee: Guangdong Kehuan Biotechnology Co ltd; Institute of Microbiology of Guangdong Academy of Sciences; Guangdong Huankai Biotechnology Co Ltd
Current assignee: Guangdong Kehuan Biotechnology Co ltd; Institute of Microbiology of Guangdong Academy of Sciences; Guangdong Huankai Biotechnology Co Ltd
Priority date: 2022-08-02
Filing date: 2022-08-02
Publication date: 2023-06-30
Anticipated expiration: 2042-08-02
Also published as: CN115820458A

Abstract

The invention discloses bifidobacterium longum 050101 with an effect of relieving ulcerative colitis and application thereof. Bifidobacterium longum 050101, accession number GDMCCNo:62550. the bifidobacterium longum 050101 provided by the invention can obviously reduce the disease activity index of mice during the period of inducing colitis by DSS, reduce the weight loss and shorten colon. (2) The observation result of colon tissue pathological tissue section shows that the muscular layer and the villus structure of the modeling group mice are damaged, and the muscular layer structure of the group mice is complete and the villus is tidy by intervention of bifidobacterium longum 050101. (3) Bifidobacterium longum 050101 intervention significantly reduced the content of pro-inflammatory cytokines IL-1 beta and IL-6 in colon tissue.

Description

Bifidobacterium longum 050101 with ulcerative colitis relieving effect and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to bifidobacterium longum 050101 with an ulcerative colitis relieving effect and application thereof.

Background

Ulcerative colitis (Ulcerative colitis, UC) is a typical disorder of intestinal barrier dysfunction, and the clinical symptoms of patients with UC are manifested by damage to the colon, rectum and even ileum, with non-specific, non-infectious characteristics.

The clinical treatment of UC is mainly dependent on the severity of the disease and its evolution over time. For non-severe UC patients, 5-aminosalicylic acid is generally used to relieve the condition, and depending on the therapeutic effect, corticosteroids, thiopurine or higher therapies such as tumor necrosis factor therapy, vedolizumab, you-tec-mab, tofacitinib, tacrolimus and cyclosporine are selected. For patients with acute severe UC, no radical cure and specific treatment means exist at present. Treatment of patients with acute severe UC generally requires a first diagnosis by hospital specialists, with conservative treatment or surgery being selected according to the severity of the disease. When the use of drug-conservative treatment is not effective, the patient is required to undergo a colectomy, but the surgery may cause a series of adverse reactions, such as intestinal dysfunction, tumor, metabolic abnormality and other complications. At present, the clinically adopted treatment strategies mainly adhere to the conservation treatment principle, follow the doctor's advice to take medicine, and reduce the negative effect caused by treatment to the greatest extent.

According to the problems of poor treatment effect, postoperative complication risk and poor treatment pertinence existing in the current UC treatment, it is important to find a dietary supplement which has wider application and huge potential and has the effect of relieving colonitis.

Disclosure of Invention

The invention aims to provide bifidobacterium longum 050101 and application thereof in preparing products for preventing and/or treating colonitis, and the bifidobacterium longum 050101 can effectively improve intestinal barrier and relieve colonitis.

In order to achieve the above object, the present invention adopts the following technical measures:

the first object of the present invention is to provide a bifidobacterium longum strainBifidobacterium longum) 050101, which was deposited at the Guangdong province microbiological bacterial collection center (GDMCC) at month 17 of 2022, address: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070 with accession number GDMCC No:62550.

a second object of the present invention is to provide the use of the above bifidobacterium longum 050101 for the preparation of a product for the prevention and/or treatment of colitis.

A third object of the present invention is to provide a product for preventing and/or treating colitis, which contains the above bifidobacterium longum 050101 as an active ingredient.

The product for preventing and/or treating the colonitis can be a medicament for preventing and/or treating colonitis.

In one embodiment of the invention, the colitis is ulcerative colitis.

In one embodiment of the present invention, the viable count of bifidobacterium longum 050101 in the product is not less than 1×10 ¹⁰ CFU/mL。

In one embodiment of the present invention, the bifidobacterium longum 050101 is prepared by inoculating a seed solution of bifidobacterium longum 050101 into a TPY liquid culture medium according to an inoculum size of 2-4% (v/v) for culturing, wherein the culturing conditions are as follows: 37. anaerobic culture at 36deg.C for 36 h, centrifuging at 4000 rpm for 30 min, collecting bacterial mud, washing with physiological saline for 2 times, and re-suspending.

In one embodiment of the present invention, the TPY liquid medium is a TPY liquid medium supplemented with vitamin K and hemin.

In one embodiment of the invention, the vitamin K and the hemin are added in an amount of 0.1% (by volume) in a vitamin K1 solution with a mass fraction of 0.1% and 5mg/mL of hemin.

It is a fourth object of the present invention to provide a target nucleotide sequence specifically recognizing bifidobacterium longum 050101 as shown in SEQ ID No. 1. The target is characterized in that it operates according to an embodiment, which is capable of specifically distinguishing the strain from other bifidobacteria isolates.

A fifth object of the present invention is to provide a primer set for identifying bifidobacterium longum 050101, specifically: 5'-AGCTCAAAGATGAGTCCGACA-3' and 5'-GTCCCGGAGAATAAAGCAATC-3'.

A sixth object of the present invention is to provide a method for identifying Bifidobacterium longum 050101, wherein the primer set is used as an amplification primer for PCR amplification of a bacterium to be detected, if 1253bp of the product is amplified, it is Bifidobacterium longum 050101, and if 1253bp of the product is not amplified, it is not Bifidobacterium longum 050101.

A seventh object of the present invention is to provide an application of the bifidobacterium longum 050101 in producing thioredoxin reductase, wherein the amino acid sequence of the thioredoxin reductase is as follows:

MFHVKHNVIIIGSGPAGYTAAIYLGRAGLNPVMVTGALSPGGQLVNTTEVENFPGFPEGILGPDLMDRMKEQAKRFGTTYITDDVSSIEACESDSVKPTYRVTLSDDSQLEASSLIIATGSSFRKLGVPGEQELSGHGVSYCATCDGFFFRNKPIVVVGGGDSAFEEALFLTRFGSSVTLIHRRDSFRASQIMVDRAKANPTITLLTNTVVTSITGTSSPTQNTGAPIAIPGLTIKRPAVAPASVSSIAVRNVVTGEESTLDTNAVFVAIGHTPATDFAAGVVDRDDDGYVVVQGASTVTSAPGIFAAGDCVDRTYRQAISAAGMGCRAALDTQAYLTD。

compared with the prior art, the invention has the following advantages:

1. the bifidobacterium longum provided by the inventionBifidobacterium longum) 050101 isolated from fecal samples of healthy donors transplanted with fecal bacteria is an excellent strain of human origin.

2. The bifidobacterium longum provided by the inventionBifidobacterium longum) 050101 has good effect of relieving colonitis, and is specifically expressed as follows:

(1) The bifidobacterium longum provided by the inventionBifidobacterium longum) 050101, it is capable of significantly reducing the disease activity index, weight loss and colon shortening in mice during DSS-induced colitis.

(2) The observation result of colon tissue pathological tissue section shows that the muscle layer and villus structure of the modeling module mice are destroyed, and bifidobacterium longum is treatedBifidobacterium longum) 050101 the muscle layer structure of the mice in the intervention group is complete and the villus is neat.

(3) Bifidobacterium longumBifidobacterium longum) 050101 intervention significantly reduced the levels of the pro-inflammatory cytokines IL-1 beta and IL-6 in colon tissue.

3. The detection method provided by the invention has short time, and can accurately know whether the bifidobacterium longum 050101 strain exists in the sample only by carrying out agarose gel electrophoresis on the PCR product, so that the detection cost is reduced; the invention is based on whole genome sequencing data, and the strain specificity target of bifidobacterium longum 050101 obtained by pan-gene analysis has more reliable detection result; meanwhile, the invention is a specific detection target for bifidobacterium longum 050101, can distinguish the strain bifidobacterium longum 050101 from bifidobacterium, and is a novel detection method.

Bifidobacterium longum050101 was deposited at the Guangdong province microbiological bacterial collection center (GDMCC) on month 6 and 17 of 2022 at the address: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070 with accession number GDMCC No:62550.

drawings

FIG. 1 is a graph showing the body weight and disease activity score index of each group of mice according to the present invention;

FIG. 2 shows the liver, kidney and spleen conditions of mice in each group according to the invention;

FIG. 3 shows colon length of each group of mice according to the present invention;

FIG. 4 is a view of colon pathological tissue pathological section of each group of mice according to the present invention;

FIG. 5 shows colon inflammatory factors of mice in each group according to the present invention;

FIG. 6 shows the antioxidant effect of thioredoxin reductase produced by Bifidobacterium longum 050101 in accordance with the present invention;

FIG. 7 shows the results of detection of 69 bifidobacteria using the molecular targets of the bifidobacterium longum 050101 strain of the present invention (in the figure, the reference numeral +is bifidobacterium longum 050101, the reference numeral C is a negative control, the rest are other strains of bifidobacterium, and the target band size is 1253 bp).

Detailed Description

The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.

It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The following examples relate to the following media:

TPY agar medium (g/L): 10.0 g/L of hydrolyzed casein, 5.0 g/L of phytone, 2.0 g/L of yeast powder, 5.0 g/L, L-cysteine 0.5 g/L of glucose, 2.0 g/L of dipotassium hydrogen phosphate, 0.5 g/L of magnesium chloride, 0.25 g/L of zinc sulfate, 0.15 g/L of calcium chloride, 0.0001 g/L of ferric chloride, 1.0 g/L of tween 80 and 15 g/L of agar, and the solvent is water.

TPY liquid medium (g/L): 10.0 g/L of hydrolyzed casein, 5.0 g/L of phytone, 2.0 g/L of yeast powder, 5.0 g/L, L-cysteine 0.5 g/L of glucose, 2.0 g/L of dipotassium hydrogen phosphate, 0.5 g/L of magnesium chloride, 0.25 g/L of zinc sulfate, 0.15 g/L of calcium chloride, 0.0001 g/L of ferric chloride and 1.0 g/L of tween 80, and the solvent is water.

1mL of a vitamin K1 solution with the mass fraction of 0.1% and 1mL of hemin (5 mg/mL) are added into each 1000mL of culture medium, and the mixture is uniformly mixed for standby. The preparation method of the hemin solution comprises the following steps: 0.5g of hemin is weighed, dissolved in 1mL of 1mol/L sodium hydroxide solution, distilled water is added to 100mL, the mixture is autoclaved at 121 ℃ for 15min-20min, and the mixture is stored in a refrigerator.

EXAMPLE 1 isolation and identification of Bifidobacterium longum 050101

1. Isolation and preservation of bifidobacterium longum 050101

Collecting a fecal fungus transplanting healthy donor sample as a sample, adding 0.1 g g fecal into 10mL of TPY liquid culture medium under a sterile environment, shaking and uniformly mixing, carrying out enrichment culture at 37 ℃ under anaerobic condition for 48 h, and absorbing 0.5g mL bacterial liquid for gradient dilution. Adding physiological saline to make into 10 ^-1 To 10 ^-8 Diluting gradient bacterial suspension, selecting 10 ^-6 、10 ^-7 、10 ^-8 Three gradient bacterial suspensions were aspirated 100 μl to TPY agar medium, spread evenly using a spreading bar, and cultured under anaerobic conditions at 37 ℃ for 48 h. And (3) picking a typical colony on a flat plate, performing streak purification on the colony on a TPY agar culture medium, picking a single colony after purification, inoculating the single colony into the TPY liquid culture medium, performing anaerobic culture for 48 hours at 37 ℃, and preserving 30% glycerol in a-80 ℃ ultralow temperature refrigerator, thereby obtaining the strain 050101.

2. Identification of Bifidobacterium longum 050101

Bacterial identification with MALDI-TOF: the single colony of the strain 050101 is picked and evenly coated on a MALDI target plate, 1 mu L of 70% formic acid aqueous solution is added on the single colony coating after natural drying, 1 mu L of matrix solution (prepared by evenly mixing 10 mg alpha-cyano-4-hydroxy cinnamic acid, 500 mu L of acetonitrile, 475 mu L of deionized water and 20 mu L of trifluoroacetic acid) is added after natural drying, and the mixture is put into a mass spectrometer for identification after the surface is dried. The result shows that the homology of the strain and bifidobacterium longum is highest, and the strain is named as bifidobacterium longum [ (longum) ]Bifidobacterium longum) 050101, which was deposited at the Guangdong province microbiological bacterial collection center (GDMCC) on month 17 of 2022, at the address: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070 and accession number is GDMCC 62550.

Example 2

1. Acute ulcerative colitis mouse model establishment and grouping

(1) And (3) preparing bacterial liquid. Before animal experiment, bifidobacterium longum 050101 is streaked, separated and purified for 1 time, and then spread and cultured for 2 times by using TPY culture medium liquid (37 ℃/48 h), and bifidobacterium longum 050101 bacterial liquid which is activated for 3 times is prepared according to a volume ratio of 3% inoculum size is inoculated to TPY culture medium, after fermentation 48 and h, the supernatant is removed by centrifugation to collect the bacterial cells, and the bacterial cells are washed with sterile physiological saline for 2 times and diluted to obtain 1X 10 ¹⁰ CFU/mL of the gastric lavage bacterial suspension, and viable count was measured by plate decanting.

(2) Establishment of a mouse model of acute ulcerative colitis. Animal origin: animal species in Guangdong province medical laboratory animal center: c57BL/6 mice, male mice; age and quality of animals: the animals were kept in the laboratory of the microbiological institute of Guangdong university (ethical examination number: GT-IACUC 202106243) at a age of 6 weeks and had a weight of about 18 g to 20 g, and were subjected to experiments after 2 weeks of adaptive feeding with light and dark alternate environments of 12 h/12 h and with appropriate temperature and humidity, free feeding and drinking water. Wherein, the modeling method of ulcerative colitis comprises the following steps: dextran Sodium Sulfate (DSS) induction. Animals were grouped and treated as shown in table 1.

2. Collection of colon tissue samples from mice

On the last day of the experiment, mice fasted 12 h (normal drinking water), were blood taken from eyeballs, left to stand 2 h, and the supernatant was centrifuged to obtain serum (3500 r/min,15 min), which was brought back to the laboratory and placed in a-80 ℃ refrigerator for subsequent physiological and biochemical, inflammatory factor index detection. After blood is taken from an experimental mouse, colon is collected and split charging is carried out, and a part of tissue slices are fixed in formaldehyde reagent prepared in advance and used for observing histopathological sections; and (3) quick freezing a part of the tissue slices by liquid nitrogen, and transporting dry ice to a laboratory, and preserving the tissue slices by a refrigerator at-80 ℃ for subsequent physiological and biochemical index analysis.

3. Mouse body weight, disease activity score index and inflammatory factor detection analysis

(1) Measurement of the change in the body weight of the mice, the body weight of the mice per day was recorded. As shown in fig. 1 (a), there was no significant change in body weight in the mice of each group at end 3, d, which was drinking 2.5% DSS, and at 4, d, there was a slight decrease in body weight in the mice of the model group (DSS) and bifidobacterium longum 050101-interfered group (DBL). Group of miceBody weight at end 7 d compared to end 0 d, the rates of change of body weight were: 1.0002 0.0286 (control), 0.9982+ -0.0471 (model), 0.9999+ -0.0331 (bifidobacterium longum 050101 intervention group); the results of body weight at end 7, d of each group of mice showed that the model group mice had very significantly reduced body weight compared to normal mice (P<0.0001 While the mice in the bifidobacterium longum 050101 intervention group have a slow weight decrease trend, which is obviously different from the model groupP < 0.01）。

(2) Determination of the disease activity score index in mice. During the experiment, the body weight of the mice is weighed and recorded daily, the feces of the mice are collected, and the properties of the feces and the hematochezia of the feces are observed. The weight loss percentage (weight is not changed to 0, weight loss percentage is 1 percent between 1% and 5%, weight loss percentage is 2 percent between 5% and 10%, weight loss percentage is 3 percent between 11% and 15%, weight loss percentage is 4 percent higher than 15%), stool characteristics (normal weight is 0 percent, loose weight is 2 percent, loose stool is 4 percent) and hematochezia condition (normal weight is 0 percent, occult blood positive weight is 2 percent, dominant bleeding is 4 percent) are scored, the total sum of the weight loss percentage, the loose weight and the loose stool is 2 percent, the dominant bleeding is 4 percent) is divided by three, and the total sum is the disease activity index (Disease activity index, DAI) of each group of mice, the DAI can reflect the disease condition of the mice, and meanwhile, the method can also be used for judging whether the UC model is modeled successfully. Faeces from the model group (DSS) and bifidobacterium longum 050101 intervention group (DBL) were normal in shape and colour with 2.5% DSS at end 3 d. At the end of 4 d, individual model mice appeared black, burnt, with no obvious bloody stool, but 4 mice appeared fecal occult blood. Until 7 th d, the feces of the mice in the model group became yellow and thin, and as shown in fig. 1 (b), blood stains were visually observed at the anus of the mice in the model group; mice were treated with bifidobacterium 050101 for reduced stool and reduced bleeding. Since DAI is a comprehensive score indicator of weight change, rare bowel movement and fecal hemorrhage in mice, the results shown in fig. 1 (c) were obtained in combination with the above cases: DAI scores of 4 d-7 d mice in the model group and mice in the bifidobacterium longum 050101 intervention group after DSS treatment are gradually increased; up to 7 th d, the DAI values for each group of mice were: 0.0333 0.1000 (control), 2.6000.+ -. 1.4048 (model), 1.9333.+ -. 1.5040 (Bifidobacterium longum 050101 intervention group); body weight of mice of group 7/7 dThe results of (2) show that the DAI score of the mice in the model group is extremely obviously increased compared with that of the mice in the normal groupP <0.0001 After the intervention of bifidobacterium longum 050101, the DAI of the ulcerative colitis mice is reducedP <0.05 Bifidobacterium longum 050101 is shown to have the effect of alleviating weight loss, runny stool and colon bleeding in UC mice.

(3) Liver, kidney, spleen of mice. From fig. 2 (a), the organ indexes of the liver, kidney and spleen of each group of mice were not significantly changed, but the images were taken according to the anatomical day, that is, fig. 2 (b), it was found that the organ colors of the mice in the model group (DSS) were significantly different from those of the mice in the control group (NC), the organs of the mice in the model group were light red or even yellow, and the organs of the mice in the control group were dark red. According to the severe hematochezia phenomenon of mice in the model group, we speculate that insufficient blood supply and erythrocyte reduction occur in the mice in the model group, so that the organs of the mice in the model group are lighter than those of normal healthy mice, and the abnormal liver, kidney and spleen functions of the mice can be caused by the insufficient blood supply in the body, thereby further aggravating the severity of the diseases. Whereas after feeding mice with bifidobacterium longum 050101, fig. 2 (b) shows that the organ color of the mice in the intervention group (DBL) is closer to that of the mice in the control group, demonstrating that bifidobacterium longum 050101 has the ability to alleviate bleeding in ulcerative colitis mice. Previous studies have shown that ulcerative colitis bleeding is closely related to vascular rupture of mucosal and submucosal layers and intestinal wall ulceration, thus demonstrating the potential of bifidobacterium longum 050101 to alleviate colonic mucus layer and intestinal wall damage.

(4) Determination of colon length in mice. In UC patients, the colon is obviously shortened, the exacerbation of the disease is positively correlated with the colon shortening condition, and in UC animal experiments, the inflammation condition of ulcerative colitis mice can be judged by observing the colon length of the mice. As shown in fig. 3 (a) and (b), the colon of the control group (NC) mice is smooth, no edema occurs, the feces in the colon are distributed in a granular form, and no blood water occurs; compared with the mice in the control group, the DSS causes significant colon shortening of the colon of the UC miceP <0.0001 Furthermore, faeces in the colon are mostly present in the form of yellow mucopurulent stool; when ulcerative colitisAfter the mice eat bifidobacterium longum 050101, the colon shortening trend of the mice is relievedP <0.01 Blood sample in the colon of the mice is reduced, and the feces are formed in a small amount, which indicates that the bifidobacterium longum 050101 has the functions of relieving the colon shortening and the stool dilution of the ulcerative colitis mice.

(5) Pathological section of colon pathology tissue of mice. Clinically, it is generally necessary to diagnose UC by enteroscopy or colon biopsy pathology. As shown in fig. 4 (a), the colon muscle layer of the control group (NC) mice is complete, the epithelial cells are complete, the villi are well-aligned and well-defined; the colon tissue fibrosis of the model group (DSS) mice is obvious, the colon villi is seriously damaged and arranged sparsely, part of the crypt disappears, and the epithelial layer is incomplete; meanwhile, as can be seen from FIG. 4 (b), the colon histopathological score of the mice in the model group is greatly increased compared with that of the control groupP <0.0001 Indicating successful establishment of the ulcerative colitis model. In addition, fig. 4 (a) also shows that after the long bifidobacterium 050101 gastric lavage treatment, the inflammation of the colon mucous layer of the mouse is relieved, the integrity of epithelial cells and tissue injury are obviously improved, the villus infiltration is relieved, and the recess deficiency condition is inhibited; meanwhile, as shown in FIG. 4 (b), bifidobacterium longum 050101 reduced the colon injury score (P)<0.05). The above results demonstrate that bifidobacterium longum 050101 has the function of improving colon tissue damage in ulcerative colitis mice, so we demonstrate that bifidobacterium longum 050101 participates in the recovery of the intestinal barrier and thereby relieves intestinal damage in ulcerative colitis mice.

(6) Measurement of inflammatory factors in the colon of mice. The content of inflammatory factors (IL-6, IL-1. Beta.) in the colon of the mice was determined by ELISA kit (Dogesce), and the specific procedures were performed according to the instructions. The colon tissue immune inflammation level of each group of mice is shown in figure 5, and the IL-6 of the colon of the model group of miceP <0.001 Protein expression amount and IL-1 beta%P <0.01 Protein expression level is significantly higher than that of control group, and IL-6 in colon of mice after treatment with Bifidobacterium longum 050101P <0.001 IL-1 beta%P <0.01 Reduced and restored to normal mouse levels, indicating that bifidobacterium longum 050101 pair promotesThe level of inflammatory cytokines has a good improving effect, so that it is also proved that the bifidobacterium longum 050101 can reduce the protein expression amount of pro-inflammatory factors in the colon by inhibiting the expression of cytokine-receptor pathways, relieve the abnormal oxidative stress state of the colon environment of mice and relieve the inflammation of UC.

Example 3 genome excavation and in vitro validation of bifidobacterium longum 050101 antioxidant actives.

Bifidobacterium longum 050101 was subjected to whole genome sequencing using Illumina Nextseq 550 second generation sequencing platform. Bacterial genomic DNA was extracted using the kit, and second generation sequencing was performed using AMT Rapid DNA-Seq Kit for Illumina (CISTRO, CHINA) and using the High Output v2.5 kit (Illumina, USA). The machine-down data are respectively controlled by adopting Trimmomatic (v 0.39) and filtering (v 0.2.0) software, and then are assembled by adopting Unicycler (v 0.4.8) software. The genome of bifidobacterium longum 050101 after assembly was assessed for genome quality control using the quick (v 5.0.2) software.

Sequencing found that the genome data of bifidobacterium longum 050101 contained the complete gene sequence of thioredoxin reductase (Thioredoxin reductase). The molecular weight (Mw) of the thioredoxin reductase was 35.42 kDa.

The specific sequence of thioredoxin reductase is:

MFHVKHNVIIIGSGPAGYTAAIYLGRAGLNPVMVTGALSPGGQLVNTTEVENFPGFPEGILGPDLMDRMKEQAKRFGTTYITDDVSSIEACESDSVKPTYRVTLSDDSQLEASSLIIATGSSFRKLGVPGEQELSGHGVSYCATCDGFFFRNKPIVVVGGGDSAFEEALFLTRFGSSVTLIHRRDSFRASQIMVDRAKANPTITLLTNTVVTSITGTSSPTQNTGAPIAIPGLTIKRPAVAPASVSSIAVRNVVTGEESTLDTNAVFVAIGHTPATDFAAGVVDRDDDGYVVVQGASTVTSAPGIFAAGDCVDRTYRQAISAAGMGCRAALDTQAYLTD（SEQ ID NO.2）。

1, 1-Diphenyl-2-trinitrophenylhydrazine (1, 1-Diphenyl-2-picrylhydrazyl radical, DPPH) is a very stable radical of nitrogen centers and is therefore widely used for measuring the antioxidant capacity of substances. 100. Mu.L of the bifidobacterium fermentation supernatant was added to 100. Mu.L of 0.2 mM DPPH methanol solution and incubated at room temperature for 30 minutes in the absence of light, and finally absorbance was measured at 517 nm using a microplate reader, distilled water being used as a control in the test. DPPH radical scavenging activity was calculated using the following formula:

in the above formula, AC represents absorbance value of bifidobacterium fermentation supernatant, and AS represents absorbance value of control group.

As a result of measurement, as shown in FIG. 6, among 13 bifidobacterium longum isolates, bifidobacterium longum 050101 had the highest DPPH clearance (77.02% + -1.08%); of the 5 bifidobacterium bifidum isolates, the highest DPPH radical scavenging rate was bifidobacterium bifidum 070305 (72.24% ± 1.40%), and the lowest was bifidobacterium bifidum 070202 (63.01% ± 0.95%); the DPPH radical scavenging rate of the bifidobacterium pseudocatenulatum 070108 is 65.74% + -4.90%. Based on the above results, it was demonstrated that bifidobacterium longum 050101 has good antioxidant capacity at a seed level.

EXAMPLE 4 excavation of Bifidobacterium longum 050101 specific molecular targets

The specific molecular target of bifidobacterium longum 050101 is mainly obtained according to the genome-wide analysis result. Wherein, total genome data of 67 bifidobacteria longum strains are selected, 65 bifidobacteria longum strains are obtained from NCBI database, 2 bifidobacteria are obtained from the laboratory, the genome is analyzed by adopting an MP method in prokaryotic genome automation analysis software (an-Genomics Analysis Pipeline, PGAP), and analysis results are processed through a local Perl script to obtain core gene information and non-core gene information of all strains. And selecting a gene specific to bifidobacterium longum 050101, removing a non-specific sequence through local Blast comparison, and obtaining a bifidobacterium longum 050101 specific detection target after PCR amplification verification (the types and the number of strains used in genome-wide analysis are shown in Table 4). Thus, 1 specific detection target of bifidobacterium longum 050101 is obtained through screening, and the nucleotide sequence is shown as SEQ ID NO. 1:

AGCTCAAAGATGAGTCCGACATGGATGAGACGGATCTCAAGCGTGTACTTGCAGCAGAAAATTGGAATACGCCAATTGTGGTCACTACTGCAGTGCAATTCTTCGAGAGCCTGTACTCGAATAAAACATCACGGTGTCGCAAGTTGCATAATATAGCAAATTCCGTCATCGTTTTTGACGAGGCGCAAACGTTGCCCGTACCTTATCTGCGGCCATGCATACGAGCAATTACCGAACTCGTGGAGCGATATGGCTCTACGGCAGTATTATGCACTGCCACACAACCAGAACTTCAACCGTTGTTCGACGAGTCTTTCAACAGTGGCTCTGTTCAAATTCCGGAGATATCACCATTCACAAATGACGATCGAGACTCCTTCCGCCGTGTGACCATTAAACGTATCGGAGACGTTGCATTGGATGCGCTTGCAGATCAGCTAGGAAATCATGAACAGGTTTTATGCGTAGTAAACACGCGCAAAAAAGCCCAGTACCTCTATGATCGTCTCGCACAAGACGACGCTGAAGGTAGCTTCTGCCTGACCACGTTGCAGTGCGCGGCTGATCGTCAACGTCTACTCAGTGGAATCCGCAATCGTCTTGATGGCGGAAAAACGTGTCGTGTAGTATCTACTTCATTGATTGAAGCCGGCGTAGACATTGATTTTCCTACTGCATACCGCGAAGAAACCGGACTGGATTCTATTATCCAGACAGCTGGACGTTGCAATCGAGAGGGTCATCATTCTGCATCAGAAAGCATGGTTTATGTGTTCTCCACAGAAGGGGGTTGCGCTCCTTTTCTCCAGCAAAGCATTGCAGCGTTCCGGACAACGGCAAACAGTCACTCTGATTTGACTGCAGATGACGCGATTCGCACATATTTCAAAGAGATCTTATGCCTGCGAGATGGTAACACTGCCAGCAAACTCACCGGCAATGATGCGCTTGATAAGTACCGCATCCTACCGTTACACAGGTTTGATGAAAACATGCCCTTTGCGACCATTGCCAAACGATTCAAGCTTATTGATACACCAACTGTACCGGTGTACATTCCACTACCCGATGAAGGCGCACGCCTGTGCAAACAACTCGAACATGGCGATATAGACCGTACGCTGTTCCGGAAGCTGGGTAAATATGCAGTTAACGTATGGCCTCGTCATCTTGAAAAACTGCAATCGGTGGGAGCGATACTAGCGGTTGGGCGGGGGAAAAACGACGAAGAGGATTGCTTTATTCTCCGGGAC (SEQ ID NO. 1)

Example 5 establishment of method for rapid detection of Bifidobacterium longum 050101 specific target

A pair of specific amplification primers is designed according to the sequence SEQ ID NO.1, and the sequences of the primers are as follows:

forward primer F:5'-AGCTCAAAGATGAGTCCGACA-3'

Reverse primer R:5'-GTCCCGGAGAATAAAGCAATC-3';

PCR was performed using genomic DNA of bifidobacteria (including 44 strains of bifidobacterium longum) isolated from stool and food samples in the laboratory as a template.

The PCR amplification reaction system was 12.5. Mu.L, which included: 2X Taq PCR MasterMix II 6.25. Mu.L, 0.25. Mu. L, DNA template for each of the forward and reverse primers, 0.5. Mu.L and 5.25. Mu.L of sterile double distilled water.

The PCR reaction conditions were: pre-denaturation at 94℃for 3 min; denaturation at 94℃for 30 s, annealing at 67℃for 30 s, elongation at 72℃for 90 s for 30 cycles; finally, the extension is carried out for 10 min at 72 ℃.

Detecting PCR products by agarose gel electrophoresis (agarose concentration is 1.0%), judging whether single amplification bands exist in 1253bp of the amplification products detected by the agarose gel electrophoresis, and if so, indicating that the samples contain bifidobacterium longum 050101; if no corresponding single amplified band is present, it is indicated that the sample does not contain bifidobacterium longum 050101. The test results are shown in Table 5 and FIG. 7, and "+" in the test result column indicates positive and "-" indicates negative.

The gel results of the PCR amplification products are shown in FIG. 7, wherein 1-44 are other bifidobacterium longum strains, 45-68 are non-target bifidobacterium strains, + is target positive bifidobacterium longum 050101, C is a negative control group, and the template of the control group is an aqueous solution without genome. As can be seen from FIG. 7, the detection result of the primer set only shows a specific amplified band for the bifidobacterium longum 050101 strain, and NO specific band exists for the non-bifidobacterium longum 050101 strain, indicating that the sequence SEQ ID NO.1 is a specific molecular target of the bifidobacterium longum 050101 strain.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. Bifidobacterium longumBifidobacterium longum) 050101 it is deposited under the accession number GDMCC No:62550.

2. use of bifidobacterium longum 050101 as claimed in claim 1 in the manufacture of a product for the prophylaxis and/or treatment of ulcerative colitis.

3. Use according to claim 2, characterized in that the product is a medicament for the prevention and/or treatment of ulcerative colitis.

4. The use according to claim 2, wherein the viable count of bifidobacterium longum 050101 in the product is not less than 1 x 10 ¹⁰ CFU/mL。

5. The use according to claim 2, wherein the bifidobacterium longum 050101 is cultivated by: inoculating the bifidobacterium longum 050101 seed liquid into a TPY liquid culture medium according to the inoculum size of 2-4% by volume ratio for culturing, wherein the culture conditions are as follows: 37. anaerobic culture at 36deg.C for 36 h, centrifuging at 4000 rpm for 30 min, collecting bacterial mud, washing with physiological saline for 2 times, and re-suspending.

6. The use according to claim 5, wherein the TPY liquid medium is a TPY liquid medium supplemented with vitamin K and hemin.

7. The use according to claim 6, wherein the vitamin K, hemin is added in an amount of 0.1% by volume of 0.1% vitamin K1 solution and 5mg/mL hemin.

8. A product for preventing and/or treating ulcerative colitis, characterized by comprising bifidobacterium longum 050101 as an active ingredient according to claim 1.

9. A method for identifying bifidobacterium longum 050101 as claimed in claim 1, characterised in that the primer sets 5'-AGCTCAAAGATGAGTCCGACA-3' and 5'-GTCCCGGAGAATAAAGCAATC-3' are used as amplification primers and the genomic DNA of the bacteria to be identified is used as a template for amplification by a PCR reaction, if 1253bp of product is amplified, bifidobacterium longum 050101 and if 1253bp of product is not amplified, bifidobacterium longum 050101.

10. Use of bifidobacterium longum 050101 as claimed in claim 1 for DPPH radical scavenging in vitro.