WO2022048337A1 - 一株可缓解类风湿性关节炎的短双歧杆菌及其应用 - Google Patents
一株可缓解类风湿性关节炎的短双歧杆菌及其应用 Download PDFInfo
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Definitions
- the invention relates to a Bifidobacterium breve that can relieve rheumatoid arthritis and its application, and belongs to the technical field of microorganisms.
- Rheumatoid Arthritis is a complex autoimmune disease characterized by joint synovitis, progression to cartilage and bone tissue destruction, and ultimately joint stiffness and dysfunction.
- the incidence of Rheumatoid Arthritis (RA) is higher in females than in males, and higher in middle-aged and elderly people than in young children, and it shows a certain heritability.
- Th17 cells in the intestinal mucosa of rheumatoid arthritis mice are closely related to the occurrence and development of the disease (see the paper "Microbiota-Dependent Involvement of Th17 Cells in Murine Models of Inflammatory Arthritis").
- the gut contains a large number of gut microbes, which not only affect the local immune response in the gut, but also participate in the systemic immune response.
- the differentiation and development of Th17 cells requires the presence and stimulation of gut microbes. Therefore, how to target the gut to reduce Th17 cells is an important idea to prevent rheumatoid arthritis.
- an important pathological feature of rheumatoid arthritis is that synovial fibroblasts proliferate abnormally, migrate and even invade surrounding adjacent tissues, and can also secrete a variety of inflammatory cytokines. Response to external intervention is also an important evaluation indicator.
- Probiotics are a type of bacteria that change the composition of the host's intestinal flora by colonizing the human body, and then metabolize to produce beneficial metabolites such as short-chain fatty acids, so as to have a beneficial effect on the host.
- beneficial metabolites such as short-chain fatty acids
- probiotics have the advantages of high safety and low cost.
- studies have shown that a small number of probiotics can indeed prevent and/or treat some special diseases. For example, in the patent application text with publication number CN108220206A, Bifidobacterium longum YS108R can be well prevented and/or Treat colitis. Therefore, the discovery of a probiotic that can prevent and/or treat rheumatoid arthritis is critical to overcoming rheumatoid arthritis.
- Bifidobacterium bifidum KCTC13474BP can relieve rheumatoid joints
- the alleviation of rheumatoid arthritis by Bifidobacterium bifidum KCTC13474BP mainly focuses on the anti-inflammatory effect in in vitro cells and the inhibition of rheumatoid arthritis autoantibodies, and there is still a lack of research on other indicators.
- the technical problem to be solved by the present invention is to provide a strain of Bifidobacterium breve that can alleviate rheumatoid arthritis.
- the present invention provides a strain of Bifidobacterium breve CCFM1078, and the Bifidobacterium breve CCFM1078 has been preserved in Guangdong City on May 6, 2020.
- the preservation center, the preservation number is GDMCC No: 61011, and the preservation address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City.
- the Bifidobacterium breve CCFM1078 was isolated from the fecal samples of healthy infants in Wuxi area. The strain was analyzed by sequencing, and its 16S rDNA sequence was shown in SEQ ID NO.1. The sequences were compared in NCBI, and the results showed that the strain was Bifidobacterium breve, named Bifidobacterium breve CCFM1078.
- the colony of the Bifidobacterium breve CCFM1078 on the MRS medium showed milky white, smooth surface and round protrusions.
- the present invention also provides a method for preparing a product for preventing and/or treating rheumatoid arthritis by using the above-mentioned Bifidobacterium breve CCFM1078.
- the viable count of the above-mentioned Bifidobacterium breve CCFM1078 is not less than 1 ⁇ 10 5 CFU/mL or 1 ⁇ 10 5 CFU/g.
- the product comprises a food product or a pharmaceutical product.
- the medicine contains the above-mentioned Bifidobacterium breve CCFM1078, a pharmaceutical carrier and/or a pharmaceutical excipient.
- the food includes a health food containing the above-mentioned Bifidobacterium breve CCFM1078.
- the food includes dairy products, soy products, meat products or fruit and vegetable products produced by using the above-mentioned starter containing Bifidobacterium breve CCFM1078.
- the product comprises a freeze-dried bacterial powder or a starter containing the Bifidobacterium breve.
- the preparation method of the starter is to inoculate the above-mentioned Bifidobacterium breve CCFM1078 into the medium according to the inoculum amount of 2-4% of the total mass of the medium, and at 37 Cultivate at °C for 18 hours to obtain a culture solution; centrifuge the culture solution to obtain bacterial cells; resuspend the bacterial cells with physiological saline to obtain a starter.
- the medium is MRS medium.
- described freeze-dried bacterial powder is prepared according to the following method:
- the present invention also provides a product for preventing and/or treating rheumatoid arthritis, the product containing the above-mentioned Bifidobacterium breve CCFM1078.
- the prevention and/or treatment of rheumatoid arthritis includes at least one of the following effects:
- the viable count of the above-mentioned Bifidobacterium breve CCFM1078 is not less than 1 ⁇ 10 5 CFU/mL or 1 ⁇ 10 5 CFU/g.
- the product comprises a food product or a pharmaceutical product.
- the medicine contains the above-mentioned Bifidobacterium breve CCFM1078, a pharmaceutical carrier and/or a pharmaceutical excipient.
- the food comprises a health food containing the above-mentioned Bifidobacterium breve CCFM1078; or the food comprises a starter produced by using the above-mentioned Bifidobacterium breve CCFM1078 dairy, soy, meat or fruit and vegetable products.
- the preparation method of the starter is to inoculate the above-mentioned Bifidobacterium breve CCFM1078 into the medium according to the inoculum amount of 2-4% of the total mass of the medium, and at 37 Cultivate at °C for 18 hours to obtain a culture solution; centrifuge the culture solution to obtain bacterial cells; resuspend the bacterial cells with physiological saline to obtain a starter.
- the medium is MRS medium.
- the present invention also provides the use of the Bifidobacterium breve or the product containing the Bifidobacterium breve in preventing and/or treating rheumatoid arthritis.
- the use is the ingestion of the Bifidobacterium breve or a product containing the Bifidobacterium breve into the gastrointestinal tract of a mammal.
- the present invention has screened out a strain of Bifidobacterium breve CCFM1078, and this Bifidobacterium breve CCFM1078 has the effect of relieving rheumatoid arthritis, which is embodied in:
- Bifidobacterium breve CCFM1078 has great application prospects in the preparation of products (such as food or medicine) for preventing and/or treating rheumatoid arthritis.
- Bifidobacterium breve (Bifidobacterium breve) is a kind of probiotic bacteria, which has been included in the "List of Bacteria that Can Be Used in Food" issued by the Ministry of Health. Therefore, the Bifidobacterium breve (Bifidobacterium breve) screened by the present invention is obtained.
- CCFM1078 does not present any potential safety concerns for patients with Rheumatoid Arthritis (RA).
- Figure 1 Effect of Bifidobacterium breve CCFM1078 on secretion of anti-inflammatory factor IL-10 by RAW264.7 cells.
- Figure 2 Effects of Bifidobacterium breve CCFM1078 on body weight in rheumatoid arthritis rats.
- Figure 3 Effects of Bifidobacterium breve CCFM1078 on paw joint thickness in rheumatoid arthritis rats.
- Figure 4 Effect of Bifidobacterium breve CCFM1078 on the clinical score of rheumatoid arthritis rats.
- Figure 5 Effect of Bifidobacterium breve CCFM1078 on serum IL-1 ⁇ levels in rheumatoid arthritis rats.
- Figure 6 Effect of Bifidobacterium breve CCFM1078 on the content of TNF ⁇ in serum of rheumatoid arthritis rats.
- Figure 7 Effect of Bifidobacterium breve CCFM1078 on serum IL-10 content in rheumatoid arthritis rats.
- Figure 8 Effect of Bifidobacterium breve CCFM1078 on acetic acid content in feces of rheumatoid arthritis rats.
- Figure 9 Effect of Bifidobacterium breve CCFM1078 on the content of propionic acid in feces of rheumatoid arthritis rats.
- Figure 10 Effect of Bifidobacterium breve CCFM1078 on the content of butyric acid in feces of rheumatoid arthritis rats.
- Figure 11 Effect of Bifidobacterium breve CCFM1078 on the content of isobutyric acid in feces of rheumatoid arthritis rats.
- Figure 12 Effect of Bifidobacterium breve CCFM1078 on valeric acid content in feces of rheumatoid arthritis rats.
- Figure 13 Effect of Bifidobacterium breve CCFM1078 on the proportion of Th17 cells in the mesentery of rheumatoid arthritis rats.
- Figure 14 Effect of Bifidobacterium breve CCFM1078 on IL-6 secretion by synovial cells of rheumatoid arthritis rats.
- Pepsin (product number: A610411), trypsin (product number: A610629), bile salts (product number: A600225) involved in the following examples were purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.; in the following examples
- the involved Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) was purchased from the American Type Culture Collection (ATCC); the RAW264.7 cells involved in the following examples were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai); the following The fetal bovine serum and DEME complete medium involved in the examples were purchased from Life technologies; the endotoxin (LPS) involved in the following examples was purchased from Sigma; the methotrexate involved in the following examples was purchased from Shengyi Bioengineering (Shanghai) Co., Ltd.; the bovine type II collagen solution and Freund's incomplete adjuvant involved in the following examples were purchased from Chondrex; the detection of IL-1 ⁇
- MRS solid medium peptone 10g/L, beef extract 10g/L, glucose 20g/L, sodium acetate 2g/L, yeast powder 5g/L, diammonium hydrogen citrate 2g/L, K 2 PO 4 3H 2 O 2.6 g/L, MgSO 4 ⁇ 7H 2 O 0.1 g/L, MnSO 4 0.05 g/L, Tween 80 1 mL/L, agar 20 g/L, cysteine salt 0.5 g/L.
- MRS liquid medium peptone 10g/L, beef extract 10g/L, glucose 20g/L, sodium acetate 2g/L, yeast powder 5g/L, diammonium hydrogen citrate 2g/L, K 2 PO 4 3H 2 O 2.6 g/L, MgSO 4 ⁇ 7H 2 O 0.1 g/L, MnSO 4 0.05 g/L, Tween 80 1 mL/L, and cysteine 0.5 g/L.
- Example 1 Screening and strain identification of Bifidobacterium breve CCFM1078
- Fecal samples from healthy infants from the Wuxi area were stored in a -80°C freezer in 30% glycerol. After taking out the sample and thawed at low temperature, mix the sample, draw 0.5mL of the sample and add it to 4.5mL of normal saline, and carry out gradient dilution with 0.9g/100mL normal saline containing 0.05g/100mL cysteine.
- the solution was spread on MRS solid medium supplemented with 0.05g/100mL cysteine, cultivated at 37°C for 48h, picked typical colonies on MRS solid medium for streak purification, picked a single colony and transferred to MRS liquid
- the cells were cultured in a medium (containing 0.05 g/100 mL of cysteine), and the cultured cells were stored in 30% glycerol to obtain strain CCFM1078.
- the genome of CCFM1078 is extracted, the 16S rDNA of CCFM1078 is amplified and sequenced (by Huada Gene Technology Co., Ltd., wherein the nucleotide sequence amplified by the 16S rDNA of CCFM1078 is shown in SEQ ID NO.1), the The sequences were compared in NCBI, and the results showed that the strain was Bifidobacterium breve, named Bifidobacterium breve CCFM1078.
- the genome of CCFM1078 was extracted and sequenced on the Illumina Hiseq sequencing platform to obtain the original data of the whole genome draft.
- the genome of Bifidobacterium breve CCFM1078 is 2.36Mb, and the content of G+C% is 58.90%.
- the Bifidobacterium breve CCFM1078 obtained in Example 1 was inserted into the MRS solid medium (containing 0.05g/100mL cysteine) and cultivated in an anaerobic incubator at 37°C for 48 hours.
- the colonies are milky white, with smooth surface and rounded protrusions.
- the Bifidobacterium breve CCFM1078 obtained in Example 1 was inserted into MRS liquid medium (containing 0.05g/100mL cysteine) at 37°C for anaerobic culture for 24h, and then transferred to fresh MRS liquid medium (containing 0.05g/100mL cysteine), cultured under the same conditions for 24h, centrifuged the cells at 6000g for 15min, washed the cells with 0.9g/100mL saline, and then centrifuged them again at 6000g for 10min to obtain the cells, which were resuspended in 30% sucrose solution , frozen at -80°C for later use.
- MRS liquid medium containing 0.05g/100mL cysteine
- Example 3 Tolerance of Bifidobacterium breve CCFM1078 to simulated gastrointestinal fluids
- the Bifidobacterium breve CCFM1078 obtained in Example 1 was inserted into the MRS liquid medium for anaerobic culture at 37°C for 18 hours, and the cells were collected by centrifugation. The collected cells were washed with physiological saline, and after the washing was completed, centrifuged again. Cells were collected, and the collected cells were resuspended in physiological saline containing 3 g/L pepsin with pH 3 (pH adjusted by HCl), and 0.1 mL of bacterial solution was taken to count the viable bacteria on the plate by pouring method as the bacterial solution.
- survival rate (%) after tolerance to gastric juice (viable number of Bifidobacterium breve in bacterial fluid after tolerance to simulated gastric juice/original viable number of Bifidobacterium breve in bacterial fluid) ⁇ 100%.
- the calculation result is: the survival rate of Bifidobacterium breve CCFM1078 after tolerance to gastric juice is as high as 67%.
- the Bifidobacterium breve CCFM1078 obtained in Example 1 was inserted into the MRS liquid medium for anaerobic culture at 37°C for 24 hours, and the cells were collected by centrifugation. The collected cells were washed with physiological saline, and after the washing was completed, centrifuged again.
- survival rate (%) after tolerance to intestinal fluid (viable number of Bifidobacterium breve in bacterial fluid after tolerance to simulated intestinal fluid/original viable number of Bifidobacterium breve in bacterial fluid) ⁇ 100%.
- the calculation result is: the survival rate of Bifidobacterium breve CCFM1078 after tolerance to intestinal fluid is as high as 11.78%.
- Bifidobacterium breve CCFM1078 has strong tolerance to artificial simulated gastric juice and artificial simulated intestinal juice.
- Example 4 Effect of Bifidobacterium breve CCFM1078 on the secretion of anti-inflammatory factor IL-10 by RAW264.7 cells
- the RAW264.17 cell line stored in liquid nitrogen was taken out, thawed at 37°C, and the cryopreserved solution was transferred to 5 mL of DEME complete medium to obtain a mixed solution; the mixed solution was centrifuged at 1000 g for 5 min, and the supernatant was discarded to obtain a precipitate; The pellet was added to 10 mL of DEME complete medium containing 10% (v/v) fetal bovine serum, pipetted evenly, transferred to a cell culture dish, and cultured in a cell culture incubator at 37 °C. During the culture process, the cells were observed under a microscope.
- cell suspension B to obtain cell suspension B; transfer cell suspension B to a new petri dish, and after 24 h in a cell incubator at 37°C, carefully aspirate the medium, and wash the cells with sterile PBS buffer to remove non-adherent cells cells to obtain adherent RAW264.17 cells.
- Bifidobacterium breve CCFM1078 obtained in Example 1 and Lactobacillus rhamnosus LGG were respectively inserted into MRS liquid medium (containing 0.05g/100mL cysteine) and cultured at 37°C for 24h anaerobic, respectively. Transferred to fresh MRS liquid medium (containing 0.05g/100mL cysteine), cultured under the same conditions for 24h, centrifuged the cells at 6000g for 15min, washed the cells with 0.9g/100mL normal saline and centrifuged them again for 10min at 6000g to obtain bacteria.
- MRS liquid medium containing 0.05g/100mL cysteine
- Bifidobacterium breve CCFM1078 and Lactobacillus rhamnosus LGG cells were resuspended in DEME complete medium to a cell concentration of 5 ⁇ 10 6 cells/mL to obtain Bifidobacterium breve cells/mL.
- the adherent RAW264.17 cells were divided into four groups, namely blank group, LPS positive group, CCFM1078 group and LGG group; 1 mL of DEME complete medium was added to adherent RAW264.17 cells in the blank group, and adherent in the LPS positive group Add 1 mL of DEME complete medium to RAW264.17 cells, add 1 mL of DEME complete medium containing Bifidobacterium breve CCFM1078 to adherent RAW264.17 cells in CCFM1078 group, and add adherent RAW264.17 cells to LGG group After adding 1mL of DEME complete medium containing Lactobacillus rhamnosus LGG, it was placed in a cell incubator (37°C, 5% CO 2 ) for 4 hours, and adhered to RAW264.17 cells in the LPS positive group, CCFM1078 group and LGG group.
- the culture medium was centrifuged to take the supernatant, and the content of IL-10 in the supernatant was determined. Each group was repeated six times (see Figure 1 for the determination results).
- CIA Collagen-induced arthritis
- RA Rheumatoid Arthritis
- Lactobacillus rhamnosus GG Lactobacillus rhamnosus GG
- RA Rheumatoid Arthritis
- the preparation method of Bifidobacterium breve CCFM1078 and Lactobacillus rhamnosus LGG bacterial liquid is as follows:
- Bifidobacterium breve CCFM1078 obtained in Example 1 and Lactobacillus rhamnosus LGG were respectively inserted into MRS liquid medium (containing 0.05g/100mL cysteine) and cultured at 37°C for 24h anaerobic, respectively. Transferred to fresh MRS liquid medium (containing 0.05g/100mL cysteine), cultured under the same conditions for 24h, centrifuged the cells at 6000g for 15min, washed the cells with 0.9g/100mL normal saline and centrifuged them again for 10min at 6000g to obtain bacteria.
- MRS liquid medium containing 0.05g/100mL cysteine
- Bifidobacterium breve CCFM1078 and Lactobacillus rhamnosus LGG were resuspended to a cell concentration of 5 ⁇ 10 9 CFU/mL with sterile normal saline with a concentration of 0.9 g/100 mL, respectively, to obtain Bifidobacterium breve CCFM1078 and Lactobacillus rhamnosus LGG broth.
- Example 5 Effects of Bifidobacterium breve CCFM1078 on body weight, joint thickness, clinical score and incidence of rheumatoid arthritis in rats
- 60 7-week-old SPF Specific Pathogen Free female Wistar rats were reared at room temperature of 22-24°C, humidity of 40-60%, alternating day and night for 12h/12h, and free food and water for 1 day. A week later, they were randomly divided into 5 groups with 12 animals in each group. The 5 groups were: normal group, model group, drug group administered with methotrexate, and LGG administered with Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG). Group, CCFM1078 group of Bifidobacterium breve CCFM1078 by gavage.
- the experiment lasted for eight weeks: the experiment started after one week of adaptive feeding of the animals.
- the normal group, the drug group and the model group were each given 1.5 mL of sterile saline with a concentration of 0.9 g/100 mL per day, and the LGG group was given 1.5 mL of sterile saline per day.
- Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) with a concentration of 5 ⁇ 10 9 CFU/mL in mL, and each animal in CCFM1078 group was given 1.5 mL of Bifidobacterium breve (Bifidobacterium breve) with a concentration of 5 ⁇ 10 9 CFU/mL every day.
- the third to fourth week is the modeling period, on the first day of modeling, the same volume of bovine type II collagen solution (Chondrex, 20022) and incomplete Freund's adjuvant (Chondrex, 7002) Mix and emulsify to form a complete emulsion (the emulsion is prepared and used immediately, and the prepared emulsion is used within 1 hour), the rats are anesthetized with isoflurane, the rats are immobilized, and the rats are treated with 75% (v/v) alcohol. After the entire tail base was sterilized, the rats were immunized for the first time.
- 0.15 mL of the complete emulsion was accurately drawn and injected subcutaneously at a distance of 1.5 cm from the tail base of the rat.
- the same treatment method was used for boosting immunization, that is, accurate suction.
- 0.15mL of complete emulsion was subcutaneously injected at 2.0cm from the base of the tail of the rats, and the rats in the normal group were injected with the same volume of sterile saline in the same way; during the modeling period, it continued until the end of the experiment.
- Each animal in the group was given 1.5 mL of sterile saline with a concentration of 0.9 g/100 mL per day, and the drug group was given a dose of 7.6 mg/kg of methotrexate according to body weight.
- mL of sterile saline with a concentration of 0.9 g/100 mL each animal in the LGG group was given 1.5 mL of Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) bacterial solution with a concentration of 5 ⁇ 10 9 CFU/mL every day
- each animal in the CCFM1078 group 1.5 mL of Bifidobacterium breve CCFM1078 bacterial solution with a concentration of 5 ⁇ 10 9 CFU/mL was administered by gavage every day.
- the average paw thickness of the rats in the CCFM1078 group was only 87.5%, 90.1% and 88.6% of the rats in the model group; Thickness was lower than that of LGG group rats.
- the clinical scores of the rats in the CCFM1078 group were only 53.3%, 75.0% and 74.2% of those in the model group; at the same time point, the clinical scores of the rats in the CCFM1078 group were lower than those in the LGG group but higher than those in the drug group.
- Bifidobacterium breve CCFM1078 can treat and prevent rheumatoid arthritis, and has a better effect in relieving weight loss, paw swelling and clinical score in rheumatoid arthritis, although these effects are weaker than the drug methotrexate However, it is stronger than the control strain LGG, but the long-term use of the drug methotrexate has side effects, such as gastrointestinal reactions (diarrhea, nausea, etc.), liver and kidney damage, etc.
- Example 6 The effect of Bifidobacterium breve CCFM1078 on the content of IL-1 ⁇ and TNF ⁇ in the serum of rheumatoid arthritis rats
- 60 7-week-old SPF Specific Pathogen Free female Wistar rats were reared at room temperature of 22-24°C, humidity of 40-60%, alternating day and night for 12h/12h, and free food and water for 1 day. A week later, they were randomly divided into 5 groups with 12 animals in each group. The 5 groups were: normal group, model group, drug group administered with methotrexate, and LGG administered with Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG). Group, CCFM1078 group of Bifidobacterium breve CCFM1078 by gavage.
- the experiment lasted for eight weeks: the experiment started after one week of adaptive feeding of the animals.
- the normal group, the drug group and the model group were each given 1.5 mL of sterile saline with a concentration of 0.9 g/100 mL per day, and the LGG group was given 1.5 mL of sterile saline per day.
- Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) with a concentration of 5 ⁇ 10 9 CFU/mL in mL, and each animal in CCFM1078 group was given 1.5 mL of Bifidobacterium breve (Bifidobacterium breve) with a concentration of 5 ⁇ 10 9 CFU/mL every day.
- the third to fourth week is the modeling period, on the first day of modeling, the same volume of bovine type II collagen solution (Chondrex, 20022) and incomplete Freund's adjuvant (Chondrex, 7002) Mix and emulsify to form a complete emulsion (the emulsion is prepared and used immediately, and the prepared emulsion is used within 1 hour), the rats are anesthetized with isoflurane, the rats are immobilized, and the rats are treated with 75% (v/v) alcohol. After the entire tail base was sterilized, the rats were immunized for the first time.
- 0.15 mL of the complete emulsion was accurately drawn and injected subcutaneously at a distance of 1.5 cm from the tail base of the rat.
- the same treatment method was used for boosting immunization, that is, accurate suction.
- 0.15mL of complete emulsion was subcutaneously injected at 2.0cm from the base of the tail of the rats, and the rats in the normal group were injected with the same volume of sterile saline in the same way; during the modeling period, it continued until the end of the experiment.
- Each animal in the group was given 1.5 mL of sterile saline with a concentration of 0.9 g/100 mL per day, and the drug group was given a dose of 7.6 mg/kg of methotrexate according to body weight.
- mL of sterile saline with a concentration of 0.9 g/100 mL each animal in the LGG group was given 1.5 mL of Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) bacterial solution with a concentration of 5 ⁇ 10 9 CFU/mL every day
- each animal in the CCFM1078 group 1.5 mL of Bifidobacterium breve CCFM1078 bacterial solution with a concentration of 5 ⁇ 10 9 CFU/mL was administered by gavage every day.
- the average concentration of IL-1 ⁇ in the serum of the rats in the model group was 835ng/L, which was significantly higher than that in the normal group (143ng/L).
- the rats in the LGG group and the CCFM1078 group The level of IL-1 ⁇ in the serum of the rats was significantly decreased, which were 17.37% and 9.94% of the model group, respectively; the content of IL-1 ⁇ in the serum of the rats in the drug group (159ng/L) was also significantly lower than that in the model group.
- the serum TNF ⁇ content of the model group was 14ng/L, which was significantly higher than that of the normal group (9.8ng/L); the serum TNF ⁇ content of the LGG group and CCFM1078 group were 12ng respectively /L and 13ng/L, both were lower than those in the model group; the content of TNF ⁇ in the serum of the rats in the drug group was 12ng/L.
- Bifidobacterium breve CCFM1078 can reduce the levels of pro-inflammatory factors IL-1 ⁇ and TNF ⁇ in serum of rheumatoid arthritis rats, especially the effect of inhibiting IL-1 ⁇ is stronger than that of control bacteria LGG and drugs, while CCFM1078, LGG and the drug were comparable in their ability to inhibit TNF ⁇ production.
- Example 7 Effect of Bifidobacterium breve CCFM1078 on the content of anti-inflammatory factor IL-10 in serum of rheumatoid arthritis rats
- 60 7-week-old SPF Specific Pathogen Free female Wistar rats were reared at room temperature of 22-24°C, humidity of 40-60%, alternating day and night for 12h/12h, and free food and water for 1 day. A week later, they were randomly divided into 5 groups with 12 animals in each group. The 5 groups were: normal group, model group, drug group administered with methotrexate, and LGG administered with Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG). Group, CCFM1078 group of Bifidobacterium breve CCFM1078 by gavage.
- the experiment lasted for eight weeks: the experiment started after one week of adaptive feeding of the animals.
- the normal group, the drug group and the model group were each given 1.5 mL of sterile saline with a concentration of 0.9 g/100 mL per day, and the LGG group was given 1.5 mL of sterile saline per day.
- Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) with a concentration of 5 ⁇ 10 9 CFU/mL in mL, and each animal in CCFM1078 group was given 1.5 mL of Bifidobacterium breve (Bifidobacterium breve) with a concentration of 5 ⁇ 10 9 CFU/mL every day.
- the third to fourth week is the modeling period, on the first day of modeling, the same volume of bovine type II collagen solution (Chondrex, 20022) and incomplete Freund's adjuvant (Chondrex, 7002) Mix and emulsify to form a complete emulsion (the emulsion is prepared and used immediately, and the prepared emulsion is used within 1 hour), the rats are anesthetized with isoflurane, the rats are immobilized, and the rats are treated with 75% (v/v) alcohol. After the entire tail base was sterilized, the rats were immunized for the first time.
- 0.15 mL of the complete emulsion was accurately drawn and injected subcutaneously at a distance of 1.5 cm from the tail base of the rat.
- the same treatment method was used for boosting immunization, that is, accurate suction.
- 0.15mL of complete emulsion was subcutaneously injected at 2.0cm from the base of the tail of the rats, and the rats in the normal group were injected with the same volume of sterile saline in the same way; during the modeling period, it continued until the end of the experiment.
- Each animal in the group was given 1.5 mL of sterile saline with a concentration of 0.9 g/100 mL per day, and the drug group was given a dose of 7.6 mg/kg of methotrexate according to body weight.
- mL of sterile saline with a concentration of 0.9 g/100 mL each animal in the LGG group was given 1.5 mL of Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) bacterial solution with a concentration of 5 ⁇ 10 9 CFU/mL every day
- each animal in the CCFM1078 group 1.5 mL of Bifidobacterium breve CCFM1078 bacterial solution with a concentration of 5 ⁇ 10 9 CFU/mL was administered by gavage every day.
- the average concentration of IL-10 in the serum of the rats in the model group was 1.64 ng/L, which was close to that of the rats in the normal group (1.59 ng/L);
- the serum IL-10 level of the CCFM1078 group was significantly increased (1.75ng/L);
- the serum IL-10 concentration of the drug group and the LGG group were 1.62ng/L and 1.56ng/L, respectively.
- Bifidobacterium breve CCFM1078 can promote the production of anti-inflammatory factors, which is significantly stronger than that of Lactobacillus rhamnosus LGG and the drug methotrexate.
- Example 8 Effect of Bifidobacterium breve CCFM1078 on the content of short-chain fatty acids in the feces of rheumatoid arthritis rats
- 60 7-week-old SPF Specific Pathogen Free female Wistar rats were reared at room temperature of 22-24°C, humidity of 40-60%, alternating day and night for 12h/12h, and free food and water for 1 day. A week later, they were randomly divided into 5 groups with 12 animals in each group. The 5 groups were: normal group, model group, drug group administered with methotrexate, and LGG administered with Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG). Group, CCFM1078 group of Bifidobacterium breve CCFM1078 by gavage.
- the experiment lasted for eight weeks: the experiment started after one week of adaptive feeding of the animals.
- the normal group, the drug group and the model group were each given 1.5 mL of sterile saline with a concentration of 0.9 g/100 mL per day, and the LGG group was given 1.5 mL of sterile saline per day.
- Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) with a concentration of 5 ⁇ 10 9 CFU/mL in mL, and each animal in CCFM1078 group was given 1.5 mL of Bifidobacterium breve (Bifidobacterium breve) with a concentration of 5 ⁇ 10 9 CFU/mL every day.
- the third to fourth week is the modeling period, on the first day of modeling, the same volume of bovine type II collagen solution (Chondrex, 20022) and incomplete Freund's adjuvant (Chondrex, 7002) Mix and emulsify to form a complete emulsion (the emulsion is prepared and used immediately, and the prepared emulsion is used within 1 hour), the rats are anesthetized with isoflurane, the rats are immobilized, and the rats are treated with 75% (v/v) alcohol. After the entire tail base was sterilized, the rats were immunized for the first time.
- 0.15 mL of the complete emulsion was accurately drawn and injected subcutaneously at a distance of 1.5 cm from the tail base of the rat.
- the same treatment method was used for boosting immunization, that is, accurate suction.
- 0.15mL of complete emulsion was subcutaneously injected at 2.0cm from the base of the tail of the rats, and the rats in the normal group were injected with the same volume of sterile saline in the same way; during the modeling period, it continued until the end of the experiment.
- Each animal in the group was given 1.5 mL of sterile saline with a concentration of 0.9 g/100 mL per day, and the drug group was given a dose of 7.6 mg/kg of methotrexate according to body weight.
- mL of sterile saline with a concentration of 0.9 g/100 mL each animal in the LGG group was given 1.5 mL of Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) bacterial solution with a concentration of 5 ⁇ 10 9 CFU/mL every day
- each animal in the CCFM1078 group 1.5 mL of Bifidobacterium breve CCFM1078 bacterial solution with a concentration of 5 ⁇ 10 9 CFU/mL was administered by gavage every day.
- the rats Four weeks after the booster immunization, the rats were fasted for 12 hours, and the feces were collected and placed in liquid nitrogen, and then transferred to a -80 °C refrigerator, taken out before the detection of short-chain fatty acid content, vacuum freeze-dried, and accurately weighed 0.05g
- the lyophilized fecal samples were dissolved in 0.5 mL of saturated sodium chloride solution, soaked for 30 min, homogenized by a tissue homogenizer, added with 0.02 mL of 10% sulfuric acid, shaken for 30 s, and accurately added 0.8 to the fecal solution in a fume hood.
- the content of acetic acid, propionic acid, isobutyric acid, butyric acid and valeric acid in the feces of the rats in the model group decreased to 76.4%, 86.6%, 52.1%, 69.6% and 76.6% of those in the normal group, respectively.
- the contents of acetic acid, propionic acid, isobutyric acid, butyric acid and valeric acid in the feces of the CCFM1078 group were significantly up-regulated, which were 13.16, 7.476, 0.857, 19.23 and 1.152 ⁇ mol/g, which were 1.8, 2.2, 1.8, 3.3 and 1.7 times of the model group, respectively;
- the contents of acetic acid, propionic acid, isobutyric acid, butyric acid and valeric acid in the feces of the LGG group rats were The contents of acetic acid, propionic acid, isobutyric acid, butyric acid and valeric acid in the feces of the rats in the drug group were 1.1, 1.2, 1.3, 1.3, 1 times that of the model group, respectively. times.
- Bifidobacterium breve CCFM1078 can generally increase the content of short-chain fatty acids in the feces of rheumatoid arthritis rats, Lactobacillus rhamnosus LGG can mainly increase the content of butyric acid in short-chain fatty acids, and the drug's effect on short-chain fatty acids. Restoration is limited.
- Example 9 Effects of Bifidobacterium breve CCFM1078 on the proportion of Th17 cells in the mesenteric lymph nodes of rheumatoid arthritis rats
- 60 7-week-old SPF Specific Pathogen Free female Wistar rats were reared at room temperature of 22-24°C, humidity of 40-60%, alternating day and night for 12h/12h, and free food and water for 1 day. A week later, they were randomly divided into 5 groups with 12 animals in each group. The 5 groups were: normal group, model group, drug group administered with methotrexate, and LGG administered with Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG). Group, CCFM1078 group of Bifidobacterium breve CCFM1078 by gavage.
- the experiment lasted for eight weeks: the experiment started after one week of adaptive feeding of the animals.
- the normal group, the drug group and the model group were each given 1.5 mL of sterile saline with a concentration of 0.9 g/100 mL per day, and the LGG group was given 1.5 mL of sterile saline per day.
- Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) with a concentration of 5 ⁇ 10 9 CFU/mL in mL, and each animal in CCFM1078 group was given 1.5 mL of Bifidobacterium breve (Bifidobacterium breve) with a concentration of 5 ⁇ 10 9 CFU/mL every day.
- the third to fourth week is the modeling period, on the first day of modeling, the same volume of bovine type II collagen solution (Chondrex, 20022) and incomplete Freund's adjuvant (Chondrex, 7002) Mix and emulsify to form a complete emulsion (the emulsion is prepared and used immediately, and the prepared emulsion is used within 1 hour), the rats are anesthetized with isoflurane, the rats are immobilized, and the rats are treated with 75% (v/v) alcohol. After the entire tail base was sterilized, the rats were immunized for the first time.
- 0.15 mL of the complete emulsion was accurately drawn and injected subcutaneously at a distance of 1.5 cm from the tail base of the rat.
- the same treatment method was used for boosting immunization, that is, accurate suction.
- 0.15mL of complete emulsion was subcutaneously injected at 2.0cm from the base of the tail of the rats, and the rats in the normal group were injected with the same volume of sterile saline in the same way; during the modeling period, it continued until the end of the experiment.
- Each animal in the group was given 1.5 mL of sterile saline with a concentration of 0.9 g/100 mL per day, and the drug group was given a dose of 7.6 mg/kg of methotrexate according to body weight.
- mL of sterile saline with a concentration of 0.9 g/100 mL each animal in the LGG group was given 1.5 mL of Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) bacterial solution with a concentration of 5 ⁇ 10 9 CFU/mL every day
- each animal in the CCFM1078 group 1.5 mL of Bifidobacterium breve CCFM1078 bacterial solution with a concentration of 5 ⁇ 10 9 CFU/mL was administered by gavage every day.
- the rats were anesthetized with isoflurane and blood was collected from the heart of the rats.
- the sacrificed rats were immersed in 75% alcohol for 15 minutes and then taken out.
- the single cell suspension of 2 ⁇ 10 6 CFU/mL was added to the stimulator and incubated in a 37°C cell incubator for stimulation for 6 hours.
- the stimulated cells were stained with CD4-FITC antibody, fixed and permeabilized.
- IL -17A-PE was intracellularly stained, and the proportion of Th17 cells was detected by flow cytometry. The detection results are shown in Figure 13.
- the proportion of Th17 cells in the mesenteric lymph nodes of the rats in the model group was significantly increased, which was 13.97 times that of the normal group.
- the proportion of Th17 cells in the mesenteric lymph nodes of the CCFM107 group and the LGG group was significantly higher. decreased to 22.2% and 10.0% of the model group respectively; the proportion of Th17 cells in the mesenteric lymph nodes of the rats in the drug group decreased to 12.7% of the model group.
- rheumatoid arthritis can lead to an imbalance in the proportion of Th17 cells in the mesentery of rats.
- Bifidobacterium breve CCFM1078, Lactobacillus rhamnosus LGG and the drug methotrexate can significantly reduce Th17 in the mesentery of rheumatoid arthritis rats.
- Example 10 Effects of Bifidobacterium breve CCFM1078 on the secretion of pro-inflammatory factor IL-6 by synovial cells
- the synovial tissue at the knee joint of the rat was taken and placed in a 10 cm cell culture dish, washed five times with sterile PBS, and chopped with sterile dissecting scissors, and 3 mL of collagenase-containing DMEM complete medium (DEME) was used.
- DEME collagenase-containing DMEM complete medium
- Type II collagenase 25:1, v/v) to completely infiltrate the tissue, place it in a cell incubator (37°C, 5% CO 2 ) for digestion for 6 hours, and after 6 hours, add 3 mL of trypsin to stop the digestion by pipetting the cells to obtain the original Passage cells; filter the primary cells through a 70 ⁇ m pore size cell strainer, collect the filtrate, centrifuge at 1500 rpm for 5 min, resuspend the cells in DEME complete medium containing 10% (v/v) fetal bovine serum, and transfer to a 9 cm cell culture dish (37°C, 5% CO 2 ). During the culture process, observe the cell state under a microscope.
- the culture medium in the culture dish is sucked away, and sterile PBS buffer is used. Rinse three times, add 2 mL of 0.25g/100 mL trypsin to digest at 37°C for 2 min, gently pipet the cells, and finally add 2 mL of DMEM complete medium containing 5% (v/v) fetal bovine serum to terminate the digestion to obtain cell suspension A ; Centrifuge the cell suspension A at 1000 rpm for 5 min, discard the supernatant to obtain bacterial cells; add 10 mL of DEME complete medium containing 10% (v/v) fetal bovine serum to the bacterial cells by pipetting evenly for counting, and use 10% (v/v) /v) The DEME complete medium of fetal bovine serum was adjusted to a cell concentration of 2 ⁇ 10 6 cells/mL to obtain a cell suspension B.
- Bifidobacterium breve CCFM1078 obtained in Example 1 and Lactobacillus rhamnosus LGG were respectively inserted into MRS liquid medium (containing 0.05g/100mL cysteine) and cultured at 37°C for 24h anaerobic, respectively. Transferred to fresh MRS liquid medium (containing 0.05g/100mL cysteine), cultured under the same conditions for 24h, centrifuged the cells at 6000g for 15min, washed the cells with 0.9g/100mL normal saline and centrifuged them again for 10min at 6000g to obtain bacteria.
- MRS liquid medium containing 0.05g/100mL cysteine
- Bifidobacterium breve CCFM1078 and Lactobacillus rhamnosus LGG cells were resuspended in DEME complete medium to a cell concentration of 5 ⁇ 10 6 cells/mL to obtain Bifidobacterium breve ( DEME complete medium of Bifidobacterium breve) CCFM1078 and Lactobacillus rhamnosus LGG.
- the cell suspension B was divided into four groups, namely blank group, positive control group, CCFM1078 group and LGG group; 1 mL DEME complete medium was added to the cell suspension B of the blank group and positive control group, and 1 mL of DEME complete medium was added to the cell suspension B of the CCFM1078 group.
- the culture medium was centrifuged to take the supernatant, and the content of IL-6 in the supernatant was determined. Each group was repeated six times (see Figure 14 for the determination results).
- Example 11 Preparation of freeze-dried bacterial agent by Bifidobacterium breve CCFM1078
- Bifidobacterium breve CCFM1078 was inserted into MRS liquid medium (containing 0.05% cysteine) and cultured anaerobic at 37°C for 24 hours, and then transferred to fresh MRS liquid medium (containing 0.05% cysteine). acid), cultured under the same conditions for 24h, centrifuged the cells at 6000g for 15min, washed the cells with 0.9% saline, and then centrifuged them again for 10min at 6000g to obtain the cells; the cells were washed three times with a phosphate buffer with a pH of 7.2 to 7.4.
- Example 12 Preparation of food by Bifidobacterium breve CCFM1078
- Bifidobacterium breve CCFM1078 can be used to ferment and prepare other fermented foods, and the fermented foods include solid foods, liquid foods and semi-solid foods.
- the fermented food includes dairy products, soy products, fruit and vegetable products, and the dairy products include milk, sour cream, and cheese; the fruit and vegetable products include cucumber, carrot, beet, celery, and cabbage products.
- Bifidobacterium breve CCFM1078 can be used to prepare liquid preparations, powders or tablets, and the specific preparation process is as follows:
- the Bifidobacterium breve liquid preparation prepared by the aforementioned method is freeze-dried to obtain Bifidobacterium breve powder (powder).
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Abstract
提供了一株可缓解类风湿性关节炎的短双歧杆菌及其应用,该短双歧杆菌CCFM1078具有缓解类风湿性关节炎的作用,能够促进RAW264.7细胞分泌抑炎因子IL-10;降低类风湿性关节炎大鼠的关节厚度、临床评分以及发病率;降低类风湿性关节炎大鼠血清中促炎性细胞因子TNFα和IL-1β的含量;调节类风湿性关节炎大鼠粪便中短链脂肪酸的含量;降低类风湿性关节炎大鼠肠系膜淋巴结中Th17细胞的比例;降低滑膜细胞分泌促炎因子IL-6。
Description
本发明涉及一株可缓解类风湿性关节炎的短双歧杆菌及其应用,属于微生物技术领域。
类风湿性关节炎(Rheumatoid Arthritis,RA)是一种复杂的自身免疫性疾病,病理特征主要为关节滑膜炎、恶化至软骨及骨组织破坏,最终导致关节强直及功能障碍。类风湿性关节炎(Rheumatoid Arthritis,RA)的发病率女性高于男性,中老年高于幼儿,且呈现出一定的遗传性。
现阶段,在治疗类风湿性关节炎方面,常使用如抗风湿药物、糖皮质激素、非甾类抗炎药等传统药物,以及一些新型生物制剂。这些药物均存在局限性,例如,在疾病初始阶段,临床上使用传统药物来限制病情,但这些药物会对肝肾、胃肠道有损伤;在传统药物治疗效果下降或者出现不良应答时,常会采用一些以炎性因子IL-6或TNFα等为靶标的生物制剂,这些生物制剂在临床短期应用中效果良好,但其造价昂贵、保存条件苛刻,另外,生物制剂属于强行阻止体内免疫炎症的联级反应,长期使用会导致患者的免疫系统被过度抑制,会有诱发其他疾病的风险。
在预防类风湿性关节炎方面,临床上则尚不存在针对类风湿性关节炎的具有明确预防作用的药物,这是因为类风湿性关节炎的发病过程错综复杂,不仅涉及免疫、遗传,还涉及到机体共生菌。其中,免疫应答的异常既存在于关节病变部位,在身体的其他免疫器官中也有体现。例如,近年来对类风湿性关节炎患者关节腔滑液样本进行分析发现促炎因子水平和Th17细胞比例显著高于健康人,而Th17细胞对促炎性因子的产生起着关键的作用。在动物试验中也证实,类风湿性关节炎小鼠的肠粘膜中Th17细胞与疾病的发生、发展密切相关(参见论文《Microbiota-Dependent Involvement of Th17 Cells in Murine Models of Inflammatory Arthritis》)。
肠道作为一个重要的免疫位点,存在大量的肠道微生物,这些微生物不仅影响肠道局部的免疫应答,还会参与系统性免疫反应。Th17细胞的分化发育需要肠道微生物的存在及刺激,因此,如何以肠道为靶点降低Th17细胞是预防类风湿性关节炎的一个重要思路。此外,类风湿性关节炎在病理学中的一个重要特征是滑膜成纤维细胞增殖异常、迁移甚至是侵染周围邻近组织,还可分泌多种炎症性细胞因子,所以滑膜成纤维细胞对外界干预的应答也是一个重要的评估指标。
益生菌是通过定殖在人体内,改变宿主肠道菌群组成,进而代谢产生如短链脂肪酸等有益代谢物,以对宿主产生有益影响的一类菌。研究发现,短链脂肪酸能够影响免疫细胞的发育分化,促进抑炎性细胞及细胞因子的形成。并且,与普通的药物相比,益生菌具有安全性高和成本低的优势。另外,研究表明,少数益生菌确实可对一些特殊的疾病起到预防和/或治疗作用,例如,公开号为CN108220206A的专利申请文本中,长双歧杆菌YS108R就可很好的预防和/或治疗结肠炎。因此,发现一株可预防和/或治疗风湿性关节炎的益生菌对攻克类风湿性关节炎这一疾病十分关键。
目前,关于益生菌应用在预防和/或治疗类风湿性关节炎方面,已经取得了一定的进展,例如,公开号为US10617725B2的专利申请文本中,两歧双歧杆菌KCTC13474BP可缓解类风湿性关节炎,但是,该两歧双歧杆菌KCTC13474BP缓解类风湿性关节炎主要集中于体外细胞中的抑炎作用以及对类风湿性关节炎自抗体的抑制,仍缺乏对其他指标的研究。
发明内容
本发明要解决的技术问题是提供一株可缓解类风湿性关节炎的短双歧杆菌(Bifidobacterium breve)。
[技术方案]
为解决本发明的技术问题,本发明提供了一株短双歧杆菌(Bifidobacterium breve)CCFM1078,所述短双歧杆菌(Bifidobacterium breve)CCFM1078已于2020年05月06日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61011,保藏地址为广州市先烈中路100号大院59号楼5楼。
所述短双歧杆菌(Bifidobacterium breve)CCFM1078是从来源于无锡地区的健康婴儿粪便样本中分离得到的,该菌株经测序分析,其16S rDNA序列如SEQ ID NO.1所示,将测序得到的序列在NCBI中进行核酸序列比对,结果显示菌株为短双歧杆菌,命名为短双歧杆菌(Bifidobacterium breve)CCFM1078。
所述短双歧杆菌(Bifidobacterium breve)CCFM1078在MRS培养基上的菌落呈现乳白色、表面光滑、圆形凸起。
本发明还提供了一种制备预防和/或治疗类风湿性关节炎的产品的方法,所述方法为使用上述短双歧杆菌(Bifidobacterium breve)CCFM1078。
在本发明的一种实施方式中,所述产品中,上述短双歧杆菌(Bifidobacterium breve)CCFM1078的活菌数为不低于1×10
5CFU/mL或1×10
5CFU/g。
在本发明的一种实施方式中,所述产品包括食品或药品。
在本发明的一种实施方式中,所述药品含有上述短双歧杆菌(Bifidobacterium breve)CCFM1078、药物载体和/或药用辅料。
本发明的一种实施方式中,所述食品包括含有上述短双歧杆菌(Bifidobacterium breve)CCFM1078的保健食品。
本发明的一种实施方式中,所述食品包括使用含有上述短双歧杆菌(Bifidobacterium breve)CCFM1078的发酵剂生产得到的乳制品、豆制品、肉制品或果蔬制品。
在一种实施方式中,所述产品包括含所述短双歧杆菌的冻干菌粉或发酵剂。
在本发明的一种实施方式中,所述发酵剂的制备方法为将上述短双歧杆菌(Bifidobacterium breve)CCFM1078按照占培养基总质量2~4%的接种量接种到培养基中,于37℃下培养18h,得到培养液;将培养液离心,得到菌体;将菌体用生理盐水重悬,得到发酵剂。
在本发明的一种实施方式中,所述培养基为MRS培养基。
在一种实施方式中,所述冻干菌粉是按照如下方法制备的:
(1)将短双歧杆菌接入MRS液体培养基中,于30~37℃厌氧培养,收集菌体细胞;
(2)将菌体细胞用洗涤后,用含海藻糖的冻干保护剂重悬至浓度≥5×10
10CFU/mL,得到重悬液;
(3)将重悬液采用真空冷冻法进行冻干,得到短双歧杆菌冻干粉。
本发明还提供了一种用于预防和/或治疗类风湿性关节炎的产品,所述产品含有上述短双歧杆菌(Bifidobacterium breve)CCFM1078。
本发明的一种实施方式中,所述预防和/或治疗类风湿性关节炎包括如下至少一种作用:
(1)促进细胞分泌抑炎因子IL-10;
(2)降低类风湿性关节炎个体的关节厚度、临床评分以及发病率;
(3)降低类风湿性关节炎个体血清中促炎性细胞因子TNFα和IL-1β的含量;
(4)调节类风湿性关节炎个体粪便中短链脂肪酸的含量;
(5)降低类风湿性关节炎个体肠系膜淋巴结中Th17细胞的比例;
(6)降低类风湿性关节炎个体滑膜细胞分泌促炎因子IL-6。
在本发明的一种实施方式中,所述产品中,上述短双歧杆菌(Bifidobacterium breve)CCFM1078的活菌数为不低于1×10
5CFU/mL或1×10
5CFU/g。
在本发明的一种实施方式中,所述产品包括食品或药品。
在本发明的一种实施方式中,所述药品含有上述短双歧杆菌(Bifidobacterium breve)CCFM1078、药物载体和/或药用辅料。
在本发明的一种实施方式中,所述食品包含含有上述短双歧杆菌(Bifidobacterium breve)CCFM1078的保健食品;或所述食品包含使用上述短双歧杆菌(Bifidobacterium breve)CCFM1078的发酵剂生产得到的乳制品、豆制品、肉制品或果蔬制品。
在本发明的一种实施方式中,所述发酵剂的制备方法为将上述短双歧杆菌(Bifidobacterium breve)CCFM1078按照占培养基总质量2~4%的接种量接种到培养基中,于37℃下培养18h,得到培养液;将培养液离心,得到菌体;将菌体用生理盐水重悬,得到发酵剂。
在本发明的一种实施方式中,所述培养基为MRS培养基。
本发明还提供所述短双歧杆菌或含所述短双歧杆菌的产品在预防和/或治疗类风湿性关节炎中的应用。
在一种实施方式中,所述应用是将所述短双歧杆菌或含所述短双歧杆菌的产品摄入哺乳动物胃肠道中。
1、本发明筛选出了一株短双歧杆菌(Bifidobacterium breve)CCFM1078,此短双歧杆菌(Bifidobacterium breve)CCFM1078具有缓解类风湿性关节炎的作用,具体体现在:
(1)显著促进RAW264.7细胞分泌抑炎因子IL-10;
(2)显著降低类风湿性关节炎大鼠的关节厚度、临床评分以及发病率;
(3)显著降低类风湿性关节炎大鼠血清中促炎性细胞因子TNFα和IL-1β的含量;
(4)显著调节类风湿性关节炎大鼠粪便中短链脂肪酸的含量;
(5)显著降低类风湿性关节炎大鼠肠系膜淋巴结中Th17细胞的比例;
(6)显著降低滑膜细胞分泌促炎因子IL-6,
因此,短双歧杆菌(Bifidobacterium breve)CCFM1078在制备预防和/或治疗类风湿性关节炎的产品(如食品或药品等)中,具有巨大的应用前景。
2、短双歧杆菌(Bifidobacterium breve)是益生菌的一种,目前已被纳入卫生部下发的《可用于食品的菌种名单》,因此,本发明筛选得到的短双歧杆菌(Bifidobacterium breve)CCFM1078不会给类风湿性关节炎(Rheumatoid Arthritis,RA)患者带来任何潜在的安全隐患。
3、短双歧杆菌(Bifidobacterium breve)的培育过程仅需培养基以及一些培养条件的控制,成本相对低廉,与造价昂贵的生物制剂相比,不会对类风湿性关节炎(Rheumatoid Arthritis,RA)患者带来太大的经济负担。
生物材料保藏
一株短双歧杆菌(Bifidobacterium breve)CCFM1078,分类学命名为Bifidobacterium breve,已于2020年05月06日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61011,保藏地址为广州市先烈中路100号大院59号楼5楼。
图1:短双歧杆菌(Bifidobacterium breve)CCFM1078对RAW264.7细胞分泌抑炎因子IL-10的影响。
图2:短双歧杆菌(Bifidobacterium breve)CCFM1078对类风湿性关节炎大鼠体重的影响。
图3:短双歧杆菌(Bifidobacterium breve)CCFM1078对类风湿性关节炎大鼠足爪关节厚度的影响。
图4:短双歧杆菌(Bifidobacterium breve)CCFM1078对类风湿性关节炎大鼠临床评分的影响。
图5:短双歧杆菌(Bifidobacterium breve)CCFM1078对类风湿性关节炎大鼠血清中IL-1β含量的影响。
图6:短双歧杆菌(Bifidobacterium breve)CCFM1078对类风湿性关节炎大鼠血清中TNFα 含量的影响。
图7:短双歧杆菌(Bifidobacterium breve)CCFM1078对类风湿性关节炎大鼠血清中IL-10含量的影响。
图8:短双歧杆菌(Bifidobacterium breve)CCFM1078对类风湿性关节炎大鼠粪便中乙酸含量的影响。
图9:短双歧杆菌(Bifidobacterium breve)CCFM1078对类风湿性关节炎大鼠粪便中丙酸含量的影响。
图10:短双歧杆菌(Bifidobacterium breve)CCFM1078对类风湿性关节炎大鼠粪便中丁酸含量的影响。
图11:短双歧杆菌(Bifidobacterium breve)CCFM1078对类风湿性关节炎大鼠粪便中异丁酸含量的影响。
图12:短双歧杆菌(Bifidobacterium breve)CCFM1078对类风湿性关节炎大鼠粪便中戊酸含量的影响。
图13:短双歧杆菌(Bifidobacterium breve)CCFM1078对类风湿性关节炎大鼠肠系膜处Th17细胞比例的影响。
图14:短双歧杆菌(Bifidobacterium breve)CCFM1078对类风湿性关节炎大鼠滑膜细胞分泌IL-6的影响。
下面结合具体实施例和附图对本发明进行进一步的阐述。
下述实施例中涉及的胃蛋白酶(产品编号:A610411)、胰蛋白酶(产品编号:A610629)、胆盐(产品编号:A600225)购自生工生物工程(上海)股份有限公司;下述实施例中涉及的鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)购自美国典型培养物菌种保藏中心(ATCC);下述实施例中涉及的RAW264.7细胞购自中国科学院细胞库(上海);下述实施例中涉及的胎牛血清、DEME完全培养基购自Life technologies公司;下述实施例中涉及的内毒素(LPS)购自Sigma公司;下述实施例中涉及的甲氨喋呤购自生工生物工程(上海)股份有限公司;下述实施例中涉及的牛II型胶原蛋白溶液以及氟氏不完全佐剂购自Chondrex公司;下述实施例中涉及的检测IL-1β(货号:DY501)、IL-10(货号:DY522)、TNFα(货号:DY510)和IL-6(货号:DY506)的ELISA试剂盒购自R&D公司。
下述实施例中涉及的培养基如下:
MRS固体培养基:蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K
2PO
4·3H
2O 2.6g/L、MgSO
4·7H
2O 0.1g/L、MnSO
4 0.05g/L、吐温80 1mL/L、琼脂20g/L、半胱氨酸氨酸盐0.5g/L。
MRS液体培养基:蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K
2PO
4·3H
2O 2.6g/L、MgSO
4·7H
2O 0.1g/L、MnSO
4 0.05g/L、吐温80 1mL/L、半胱氨酸氨酸盐0.5g/L。
实施例1:短双歧杆菌CCFM1078的筛选及菌种鉴定
1、筛选
来源于无锡地区的健康婴儿粪便样本于30%的甘油中保存于-80℃冰箱。将样本取出低温解冻后,混匀样本,吸取0.5mL样本加到4.5mL的生理盐水中,以含有0.05g/100mL半胱氨酸的0.9g/100mL生理盐水进行梯度稀释,选择合适的梯度稀释液涂布在加了0.05g/100mL半胱氨酸的MRS固体培养基上,于37℃培养48h,挑取典型菌落至MRS固体培养基上划线纯化,挑取单菌落转接至MRS液体培养基(含0.05g/100mL半胱氨酸)中进行培养,将培养得到的菌体于30%的甘油中保存,得到菌株CCFM1078。
2、鉴定
提取CCFM1078的基因组,将CCFM1078的16S rDNA进行扩增和测序(由华大基因科技有限公司进行,其中,CCFM1078的16S rDNA扩增的核苷酸序列如SEQ ID NO.1所示), 将该序列在NCBI中进行核酸序列比对,结果显示菌株为短双歧杆菌,命名为短双歧杆菌(Bifidobacterium breve)CCFM1078。
3、基因组草图信息
提取CCFM1078的基因组,在Illumina Hiseq测序平台进行基因组序列测序,得到全基因组草图原始数据。短双歧杆菌(Bifidobacterium breve)CCFM1078基因组为2.36Mb,G+C%的含量为58.90%。
实施例2:短双歧杆菌CCFM1078的培养
将实施例1获得的短双歧杆菌(Bifidobacterium breve)CCFM1078接入MRS固体培养基(含0.05g/100mL半胱氨酸)中于37℃厌氧培养箱中培养48h后,观察其菌落,发现其菌落呈现乳白色、表面光滑、圆形凸起。
将实施例1获得的短双歧杆菌(Bifidobacterium breve)CCFM1078接入MRS液体培养基(含0.05g/100mL半胱氨酸)中于37℃厌氧培养24h后,转入新鲜的MRS液体培养基(含0.05g/100mL半胱氨酸)中,同样条件培养24h,6000g离心菌体15min,0.9g/100mL生理盐水洗涤菌体后6000g再次离心10min,得到菌体,用30%蔗糖溶液重悬,冻存在-80℃待用。
实施例3:短双歧杆菌CCFM1078对模拟胃肠液的耐受性
1、短双歧杆菌CCFM1078对模拟胃液的耐受性
将实施例1获得的短双歧杆菌(Bifidobacterium breve)CCFM1078接入MRS液体培养基中于37℃厌氧培养18h后,离心收集细胞,将收集到细胞经生理盐水洗涤,洗涤结束后,再次离心收集细胞,将收集到的细胞分别重悬于pH为3(pH由HCl调节)的含有3g/L胃蛋白酶的生理盐水中,取0.1mL菌液通过倾注法进行平板活菌计数作为菌液中短双歧杆菌(Bifidobacterium breve)CCFM1078的原始活菌数,将剩余菌液置于37℃厌氧培养3h后,取0.1mL菌液通过倾注法进行平板活菌计数作为耐受模拟胃液后菌液中短双歧杆菌(Bifidobacterium breve)CCFM1078的活菌数,计算短双歧杆菌(Bifidobacterium breve)CCFM1078耐受胃液后的存活率;
其中,耐受胃液后的存活率(%)=(耐受模拟胃液后菌液中短双歧杆菌的活菌数/菌液中短双歧杆菌的原始活菌数)×100%。
计算结果为:短双歧杆菌(Bifidobacterium breve)CCFM1078耐受胃液后的存活率高达67%。
2、短双歧杆菌CCFM1078对模拟肠液的耐受性
将实施例1获得的短双歧杆菌(Bifidobacterium breve)CCFM1078接入MRS液体培养基中于37℃厌氧培养24h后,离心收集细胞,将收集到细胞经生理盐水洗涤,洗涤结束后,再次离心收集细胞,将收集到细胞分别重悬于pH为8(pH由NaOH调节)的含有1g/L胰蛋白酶和0.3g/L胆盐的生理盐水中,取0.1mL菌液进行平板活菌计数作为菌液中短双歧杆菌的原始活菌数,将剩余菌液置于37℃培养4h后,取0.1mL菌液通过倾注法进行平板活菌计数作为耐受模拟肠液后菌液中短双歧杆菌的活菌数,计算短双歧杆菌(Bifidobacterium breve)CCFM1078耐受肠液后的存活率;
其中,耐受肠液后的存活率(%)=(耐受模拟肠液后菌液中短双歧杆菌的活菌数/菌液中短双歧杆菌的原始活菌数)×100%。
计算结果为:短双歧杆菌(Bifidobacterium breve)CCFM1078耐受肠液后的存活率高达11.78%。
综上,短双歧杆菌(Bifidobacterium breve)CCFM1078对人工模拟胃液、人工模拟肠液的耐受能力均较强。
实施例4:短双歧杆菌CCFM1078对RAW264.7细胞分泌抑炎因子IL-10的影响
将保存在液氮中的RAW264.17细胞株取出,37℃解冻,将冻存液转移到5mL的DEME完全培养基中,得到混合液;将混合液1000g离心5min后弃上清,得到沉淀;将沉淀加入 10mL含有10%(v/v)胎牛血清的DEME完全培养基吹打均匀后,转移到细胞培养皿中,在37℃的细胞培养箱中培养,培养过程中,在显微镜下观察细胞状态,待培养皿中细胞贴壁生长,形态良好,且长满培养皿80%左右时,小心吸去培养基,在培养皿中加入0.25g/100mL的胰蛋白酶消化3min,消化结束后,在培养皿中加入2mL的DEME完全培养基终止消化,并轻轻吹打细胞使得细胞脱壁,收集细胞悬液A;将细胞悬液A 1000g离心5min后弃上清,得到细胞沉淀物;将沉淀用10mL含有10%(v/v)胎牛血清的DEME完全培养基吹打均匀进行计数,并用10%(v/v)胎牛血清的DEME完全培养基调整细胞浓度为5×10
6个细胞/mL,得到细胞悬液B;将细胞悬液B转移到新的培养皿中,在37℃的细胞培养箱中24h后,小心吸去培养基,使用无菌PBS缓冲液清洗细胞以去除未贴壁的细胞,得到贴壁RAW264.17细胞。
将实施例1获得的短双歧杆菌(Bifidobacterium breve)CCFM1078和鼠李糖乳杆菌LGG分别接入MRS液体培养基(含0.05g/100mL半胱氨酸)中于37℃厌氧培养24h后,分别转入新鲜的MRS液体培养基(含0.05g/100mL半胱氨酸)中,同样条件培养24h,6000g离心菌体15min,0.9g/100mL生理盐水洗涤菌体后6000g再次离心10min,得到菌体;用DEME完全培养基将短双歧杆菌(Bifidobacterium breve)CCFM1078和鼠李糖乳杆菌LGG的菌体分别重悬至菌体浓度为5×10
6个细胞/mL,得到含有短双歧杆菌(Bifidobacterium breve)CCFM1078和鼠李糖乳杆菌LGG的DEME完全培养基。
将贴壁RAW264.17细胞分为四组,分别为空白组、LPS阳性组、CCFM1078组和LGG组;在空白组贴壁RAW264.17细胞中加入1mL DEME完全培养基,在LPS阳性组贴壁RAW264.17细胞中加入1mL DEME完全培养基,在CCFM1078组贴壁RAW264.17细胞中加入1mL含有短双歧杆菌(Bifidobacterium breve)CCFM1078的DEME完全培养基,在LGG组贴壁RAW264.17细胞中加入1mL含有鼠李糖乳杆菌LGG的DEME完全培养基后,置于细胞培养箱(37℃,5%CO
2)中培养4h,在LPS阳性组、CCFM1078组和LGG组贴壁RAW264.17细胞中加入1mL浓度为200ng/mL的LPS溶液,在空白组加入1mL DEME完全培养基后,置于细胞培养箱(37℃,5%CO
2)中继续培养48h,得到培养液。
将培养液离心取上清,测定上清中IL-10的含量,每个组别重复六次(测定结果见图1)。
由图1可知,经LPS刺激后,RAW264.17细胞分泌IL-10的分泌量显著升高(升高至1.8ng/mL),而短双歧杆菌(Bifidobacterium breve)CCFM1078和鼠李糖乳杆菌LGG均可进一步提高RAW264.17细胞分泌IL-10的分泌量(分别提高至1.9ng/mL和2.2ng/mL),表明在体外,短双歧杆菌(Bifidobacterium breve)CCFM1078和鼠李糖乳杆菌LGG均可促进抑炎因子的分泌。
II胶原诱导的关节炎模型(Collagen-induced arthritis,CIA)是一类经典的RA动物模型,CIA的发作与类风湿性关节炎(Rheumatoid Arthritis,RA)有许多相似之处,如滑膜增生、血管翳形成以及软骨破坏等。目前,在RA临床上主要使用鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)治疗类风湿性关节炎(Rheumatoid Arthritis,RA)。因此,实施例5~9中,以II胶原诱导的关节炎模型(Collagen-induced arthritis,CIA)作为RA动物模型,以鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)作为对照。
实施例5~9中,短双歧杆菌(Bifidobacterium breve)CCFM1078和鼠李糖乳杆菌LGG菌液的制备方法如下:
将实施例1获得的短双歧杆菌(Bifidobacterium breve)CCFM1078和鼠李糖乳杆菌LGG分别接入MRS液体培养基(含0.05g/100mL半胱氨酸)中于37℃厌氧培养24h后,分别转入新鲜的MRS液体培养基(含0.05g/100mL半胱氨酸)中,同样条件培养24h,6000g离心菌体15min,0.9g/100mL生理盐水洗涤菌体后6000g再次离心10min,得到菌体;用浓度为0.9g/100mL的无菌生理盐水将短双歧杆菌(Bifidobacterium breve)CCFM1078和鼠李糖乳杆菌LGG的菌体分别重悬至细胞浓度为5×10
9CFU/mL,得到短双歧杆菌 (Bifidobacterium breve)CCFM1078和鼠李糖乳杆菌LGG菌液。
实施例5:短双歧杆菌CCFM1078对类风湿性关节炎大鼠体重、关节厚度、临床评分以及发病率的影响
取7周龄的SPF(Specific Pathogen Free)级雌性Wistar大鼠60只,于饲养室温为22~24℃,湿度为40~60%,12h/12h昼夜交替,自由进食及饮水的条件下饲养1周后,随机分为5组,每组12只,5组分别为:正常组、模型组、灌胃甲氨喋呤的药物组、灌胃鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)的LGG组、灌胃短双歧杆菌(Bifidobacterium breve)CCFM1078的CCFM1078组。
实验共八周:动物适应性饲养一周后开始实验。从造模前两周开始,一直持续到造模开始,正常组、药物组和模型组每只每天灌胃1.5mL浓度为0.9g/100mL的无菌生理盐水,LGG组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)菌液,CCFM1078组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的短双歧杆菌(Bifidobacterium breve)CCFM1078菌液;第三周到第四周为造模期,造模第一天时,将牛II型胶原蛋白溶液(Chondrex,20022)与氟氏不完全佐剂(Chondrex,7002)等体积混合、乳化,形成完全乳化液(乳化液现配现用,配制好的乳化液在1h内使用),使用异氟烷麻醉大鼠,固定大鼠并用75%(v/v)酒精对大鼠尾根部整体进行消毒后,对大鼠进行初次免疫,初次免疫精确吸取0.15mL完全乳化液在距离大鼠尾根部1.5cm处进行皮下注射,一周后采用相同的处理方法进行加强免疫,即准确吸取0.15mL完全乳化液在距离大鼠尾根部2.0cm处进行皮下注射,正常组大鼠仅采用同样的方法注射相同体积的无菌生理盐水;造模期间,一直持续到实验结束,正常组和模型组每只每天灌胃1.5mL浓度为0.9g/100mL的无菌生理盐水,药物组按照体重计算灌胃剂量为7.6mg/kg的甲氨喋呤,分两次灌胃,其余时间灌胃1.5mL浓度为0.9g/100mL的无菌生理盐水,LGG组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)菌液,CCFM1078组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的短双歧杆菌(Bifidobacterium breve)CCFM1078菌液。
造模期间以及造模结束后,通过体重计测定每组大鼠体重,通过螺旋测微器测定每组大鼠关节厚度,并且,通过观察踝和指关节的肿胀、红肿程度、僵直程度测定每组大鼠临床评分(具体可见参考文献:Shan,J.,et al.,Integrated Serum and Fecal Metabolomics Study of Collagen-Induced Arthritis Rats and the Therapeutic Effects of the Zushima Tablet.Front Pharmacol,2018.9:p.891.),测定结果分别见图2~4。
由图2可知,在加强免疫后,模型组大鼠的体重增加率在前两周下降,在第六周时体重接近于造模前(第3周)的体重(体重分别为初始体重的1.292和1.249倍),最后两周开始恢复体重增加,但体重增加率显著低于同期正常组大鼠;药物组大鼠体重变化不显著;LGG组和CCFM1078组大鼠的体重变化趋势与模型组相似,但CCFM1078组大鼠的体重增加率与药物组相当。
由图3可知,在加强免疫后1周后,模型组大鼠的关节、足爪开始肿胀,在随后的10天内模型组大鼠的足爪厚度显著高于正常组大鼠;药物组大鼠足爪厚度明显低于模型组在最后一周恢复至正常;CCFM1078组大鼠的足爪关节肿胀变化趋势与模型组大鼠的变化情况相一致,但CCFM1078组大鼠的平均足爪厚度低于模型组大鼠,其中,在实验第5、6和6.5周,CCFM1078组大鼠的平均足爪厚度仅为模型组大鼠的87.5%、90.1%和88.6%;同一时期CCFM1078组大鼠的足爪厚度是低于LGG组大鼠。
由图4可知,每组大鼠临床评分的变化与足爪关节厚度的变化曲线相似;在加强免疫1周后,模型组大鼠的临床评分显著高于正常组大鼠和药物组大鼠;CCFM1078组大鼠的临床评分低于模型组大鼠,从实验第6周开始,CCFM1078组大鼠的临床评分开始趋于稳定且低于LGG组,其中,在实验第5、6和6.5周,CCFM1078组大鼠的临床评分仅为模型组的53.3%、75.0%和74.2%;在同一时间点,CCFM1078组大鼠的临床评分低于LGG组但高于药物组。
可见,短双歧杆菌CCFM1078可治疗以及预防类风湿性关节炎,在缓解类风湿性关节炎 的体重降低、足爪肿胀和临床评分有较好的效果,这些效果虽然弱于药物甲氨喋呤但是强于对照菌LGG,但药物甲氨喋呤长期使用有副作用,如胃肠道反应(腹泻、恶心等)、肝肾损伤等。
实施例6:短双歧杆菌CCFM1078对类风湿性关节炎大鼠血清中IL-1β和TNFα含量的影响
取7周龄的SPF(Specific Pathogen Free)级雌性Wistar大鼠60只,于饲养室温为22~24℃,湿度为40~60%,12h/12h昼夜交替,自由进食及饮水的条件下饲养1周后,随机分为5组,每组12只,5组分别为:正常组、模型组、灌胃甲氨喋呤的药物组、灌胃鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)的LGG组、灌胃短双歧杆菌(Bifidobacterium breve)CCFM1078的CCFM1078组。
实验共八周:动物适应性饲养一周后开始实验。从造模前两周开始,一直持续到造模开始,正常组、药物组和模型组每只每天灌胃1.5mL浓度为0.9g/100mL的无菌生理盐水,LGG组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)菌液,CCFM1078组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的短双歧杆菌(Bifidobacterium breve)CCFM1078菌液;第三周到第四周为造模期,造模第一天时,将牛II型胶原蛋白溶液(Chondrex,20022)与氟氏不完全佐剂(Chondrex,7002)等体积混合、乳化,形成完全乳化液(乳化液现配现用,配制好的乳化液在1h内使用),使用异氟烷麻醉大鼠,固定大鼠并用75%(v/v)酒精对大鼠尾根部整体进行消毒后,对大鼠进行初次免疫,初次免疫精确吸取0.15mL完全乳化液在距离大鼠尾根部1.5cm处进行皮下注射,一周后采用相同的处理方法进行加强免疫,即准确吸取0.15mL完全乳化液在距离大鼠尾根部2.0cm处进行皮下注射,正常组大鼠仅采用同样的方法注射相同体积的无菌生理盐水;造模期间,一直持续到实验结束,正常组和模型组每只每天灌胃1.5mL浓度为0.9g/100mL的无菌生理盐水,药物组按照体重计算灌胃剂量为7.6mg/kg的甲氨喋呤,分两次灌胃,其余时间灌胃1.5mL浓度为0.9g/100mL的无菌生理盐水,LGG组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)菌液,CCFM1078组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的短双歧杆菌(Bifidobacterium breve)CCFM1078菌液。
加强免疫四周后,取血并处死大鼠,取大鼠血清,通过ELISA试剂盒测定每组大鼠血清中IL-1β和TNFα的含量,检测结果见图5-6。
如图5所示,模型组大鼠血清中IL-1β的平均浓度为835ng/L,比正常组(143ng/L)显著升高;相比于模型组大鼠,LGG组大鼠和CCFM1078组大鼠血清中IL-1β水平显著降低,分别为模型组的17.37%和9.94%;药物组大鼠血清中IL-1β的含量(159ng/L)也显著低于模型组。
如图6所示,模型组大鼠血清中TNFα的含量为14ng/L,比正常组(9.8ng/L)显著升高;LGG组大鼠和CCFM1078组大鼠血清中TNFα的含量分别为12ng/L和13ng/L,均较模型组大鼠的含量有所降低;药物组大鼠血清中TNFα的含量为12ng/L。
可见,短双歧杆菌(Bifidobacterium breve)CCFM1078可降低类风湿性关节炎大鼠血清中促炎因子IL-1β和TNFα的水平,尤其是抑制IL-1β的效果强于对照菌LGG和药物,而CCFM1078、LGG和药物抑制TNFα产生的能力相当。
实施例7:短双歧杆菌CCFM1078对类风湿性关节炎大鼠血清中抑炎因子IL-10含量的影响
取7周龄的SPF(Specific Pathogen Free)级雌性Wistar大鼠60只,于饲养室温为22~24℃,湿度为40~60%,12h/12h昼夜交替,自由进食及饮水的条件下饲养1周后,随机分为5组,每组12只,5组分别为:正常组、模型组、灌胃甲氨喋呤的药物组、灌胃鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)的LGG组、灌胃短双歧杆菌(Bifidobacterium breve)CCFM1078的CCFM1078组。
实验共八周:动物适应性饲养一周后开始实验。从造模前两周开始,一直持续到造模开 始,正常组、药物组和模型组每只每天灌胃1.5mL浓度为0.9g/100mL的无菌生理盐水,LGG组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)菌液,CCFM1078组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的短双歧杆菌(Bifidobacterium breve)CCFM1078菌液;第三周到第四周为造模期,造模第一天时,将牛II型胶原蛋白溶液(Chondrex,20022)与氟氏不完全佐剂(Chondrex,7002)等体积混合、乳化,形成完全乳化液(乳化液现配现用,配制好的乳化液在1h内使用),使用异氟烷麻醉大鼠,固定大鼠并用75%(v/v)酒精对大鼠尾根部整体进行消毒后,对大鼠进行初次免疫,初次免疫精确吸取0.15mL完全乳化液在距离大鼠尾根部1.5cm处进行皮下注射,一周后采用相同的处理方法进行加强免疫,即准确吸取0.15mL完全乳化液在距离大鼠尾根部2.0cm处进行皮下注射,正常组大鼠仅采用同样的方法注射相同体积的无菌生理盐水;造模期间,一直持续到实验结束,正常组和模型组每只每天灌胃1.5mL浓度为0.9g/100mL的无菌生理盐水,药物组按照体重计算灌胃剂量为7.6mg/kg的甲氨喋呤,分两次灌胃,其余时间灌胃1.5mL浓度为0.9g/100mL的无菌生理盐水,LGG组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)菌液,CCFM1078组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的短双歧杆菌(Bifidobacterium breve)CCFM1078菌液。
加强免疫四周后,取血并处死大鼠,取大鼠血清,通过ELISA试剂盒测定每组大鼠血清中IL-1β和TNFα的含量,检测结果见图7。
如图7所示,模型组大鼠血清中IL-10的平均浓度为1.64ng/L,与正常组大鼠(1.59ng/L)相接近;相比于模型组、正常组和LGG组大鼠,CCFM1078组大鼠血清中IL-10水平显著升高(1.75ng/L);药物组和LGG组大鼠血清中IL-10的浓度分别为1.62ng/L和1.56ng/L。
可见,短双歧杆菌(Bifidobacterium breve)CCFM1078可促进抑炎因子的产生,这种促进作用显著强于鼠李糖乳杆菌LGG和药物甲氨喋呤。
实施例8:短双歧杆菌CCFM1078对类风湿性关节炎大鼠粪便中短链脂肪酸含量的影响
取7周龄的SPF(Specific Pathogen Free)级雌性Wistar大鼠60只,于饲养室温为22~24℃,湿度为40~60%,12h/12h昼夜交替,自由进食及饮水的条件下饲养1周后,随机分为5组,每组12只,5组分别为:正常组、模型组、灌胃甲氨喋呤的药物组、灌胃鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)的LGG组、灌胃短双歧杆菌(Bifidobacterium breve)CCFM1078的CCFM1078组。
实验共八周:动物适应性饲养一周后开始实验。从造模前两周开始,一直持续到造模开始,正常组、药物组和模型组每只每天灌胃1.5mL浓度为0.9g/100mL的无菌生理盐水,LGG组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)菌液,CCFM1078组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的短双歧杆菌(Bifidobacterium breve)CCFM1078菌液;第三周到第四周为造模期,造模第一天时,将牛II型胶原蛋白溶液(Chondrex,20022)与氟氏不完全佐剂(Chondrex,7002)等体积混合、乳化,形成完全乳化液(乳化液现配现用,配制好的乳化液在1h内使用),使用异氟烷麻醉大鼠,固定大鼠并用75%(v/v)酒精对大鼠尾根部整体进行消毒后,对大鼠进行初次免疫,初次免疫精确吸取0.15mL完全乳化液在距离大鼠尾根部1.5cm处进行皮下注射,一周后采用相同的处理方法进行加强免疫,即准确吸取0.15mL完全乳化液在距离大鼠尾根部2.0cm处进行皮下注射,正常组大鼠仅采用同样的方法注射相同体积的无菌生理盐水;造模期间,一直持续到实验结束,正常组和模型组每只每天灌胃1.5mL浓度为0.9g/100mL的无菌生理盐水,药物组按照体重计算灌胃剂量为7.6mg/kg的甲氨喋呤,分两次灌胃,其余时间灌胃1.5mL浓度为0.9g/100mL的无菌生理盐水,LGG组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)菌液,CCFM1078组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的短双歧杆菌(Bifidobacterium breve)CCFM1078菌液。
加强免疫四周后,大鼠禁食不禁水12h,收集粪便置于液氮中,后转移至-80℃冰箱,在进行短链脂肪酸含量的检测前取出,进行真空冷冻干燥,准确称取0.05g冻干后的粪便样品 溶解于0.5mL饱和氯化钠溶液中,浸泡30min,组织匀浆机匀浆,加入0.02mL浓度为10%的硫酸,震荡30s,在通风橱内向粪便溶液中准确加入0.8mL乙醚溶液,震荡30s后离心15min(8000g、4℃),移取上清液至含有0.3g无水硫酸钠的离心管中,震荡均匀,离心15min(8000g、4℃),取上清至气质容量瓶中,通过GCMS检测短链脂肪酸含量,检测结果见图8~12。
如图8~12所示,模型组大鼠粪便中乙酸、丙酸、异丁酸、丁酸和戊酸的含量分别下降到正常组大鼠的76.4%、86.6%、52.1%、69.6%和84.4%,其中,异丁酸含量的下降最为显著;CCFM1078组大鼠粪便中乙酸、丙酸、异丁酸、丁酸和戊酸的含量与模型组相比显著上调,分别是13.16、7.476、0.857、19.23和1.152μmol/g,分别为模型组的1.8、2.2、1.8、3.3、1.7倍;LGG组大鼠粪便中乙酸、丙酸、异丁酸、丁酸和戊酸的含量则分别是模型组的1.1、1.2、1.1、2.0、1倍;药物组大鼠粪便中乙酸、丙酸、异丁酸、丁酸和戊酸的含量分别为模型组的1.1、1.2、1.3、1.3、1倍。
可见,短双歧杆菌CCFM1078可普遍提高类风湿性关节炎大鼠粪便中短链脂肪酸的含量,鼠李糖乳杆菌LGG主要可提高短链脂肪酸中丁酸的含量,而药物对短链脂肪酸的恢复效果有限。
实施例9:短双歧杆菌CCFM1078对类风湿性关节炎大鼠肠系膜淋巴结中Th17细胞比例的影响
取7周龄的SPF(Specific Pathogen Free)级雌性Wistar大鼠60只,于饲养室温为22~24℃,湿度为40~60%,12h/12h昼夜交替,自由进食及饮水的条件下饲养1周后,随机分为5组,每组12只,5组分别为:正常组、模型组、灌胃甲氨喋呤的药物组、灌胃鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)的LGG组、灌胃短双歧杆菌(Bifidobacterium breve)CCFM1078的CCFM1078组。
实验共八周:动物适应性饲养一周后开始实验。从造模前两周开始,一直持续到造模开始,正常组、药物组和模型组每只每天灌胃1.5mL浓度为0.9g/100mL的无菌生理盐水,LGG组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)菌液,CCFM1078组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的短双歧杆菌(Bifidobacterium breve)CCFM1078菌液;第三周到第四周为造模期,造模第一天时,将牛II型胶原蛋白溶液(Chondrex,20022)与氟氏不完全佐剂(Chondrex,7002)等体积混合、乳化,形成完全乳化液(乳化液现配现用,配制好的乳化液在1h内使用),使用异氟烷麻醉大鼠,固定大鼠并用75%(v/v)酒精对大鼠尾根部整体进行消毒后,对大鼠进行初次免疫,初次免疫精确吸取0.15mL完全乳化液在距离大鼠尾根部1.5cm处进行皮下注射,一周后采用相同的处理方法进行加强免疫,即准确吸取0.15mL完全乳化液在距离大鼠尾根部2.0cm处进行皮下注射,正常组大鼠仅采用同样的方法注射相同体积的无菌生理盐水;造模期间,一直持续到实验结束,正常组和模型组每只每天灌胃1.5mL浓度为0.9g/100mL的无菌生理盐水,药物组按照体重计算灌胃剂量为7.6mg/kg的甲氨喋呤,分两次灌胃,其余时间灌胃1.5mL浓度为0.9g/100mL的无菌生理盐水,LGG组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的鼠李糖乳杆菌LGG(Lactobacillus rhamnosus GG)菌液,CCFM1078组每只每天灌胃1.5mL浓度为5×10
9CFU/mL的短双歧杆菌(Bifidobacterium breve)CCFM1078菌液。
加强免疫四周后,异氟烷麻醉大鼠并对大鼠进行心脏采血,将处死后的大鼠在75%的酒精中浸泡15min后取出,无菌取肠系膜淋巴结,将肠系膜淋巴结制备成细胞浓度为2×10
6CFU/mL的单细胞悬浮液,将单细胞悬浮液加入刺激剂在37℃细胞培养箱中培养刺激6h,将刺激后的细胞经CD4-FITC抗体表面染色,固定破膜,IL-17A-PE胞内染色,通过流式细胞仪检测Th17细胞比例,检测结果见图13。
如图13所示,与正常组大鼠相比,模型组大鼠肠系膜淋巴结中Th17细胞的比例显著升高,是正常组的13.97倍,CCFM107组和LGG组大鼠肠系膜淋巴结中Th17细胞比例显著下降,分别低至模型组的22.2%、10.0%;药物组大鼠肠系膜淋巴结中Th17细胞比例降低至模 型组的12.7%。
可见,类风湿性关节炎会导致大鼠肠系膜处Th17细胞的比例失衡,短双歧杆菌CCFM1078、鼠李糖乳杆菌LGG和药物甲氨喋呤则可显著降低风湿性关节炎大鼠肠系膜处Th17细胞的比例,使之趋于正常水平。
实施例10:短双歧杆菌CCFM1078对滑膜细胞分泌促炎因子IL-6的影响
取大鼠膝关节处的滑膜组织放置于10cm的细胞培养皿中,使用无菌PBS清洗五次,用无菌解剖剪将组织剪碎之后,用3mL含胶原酶的DMEM完全培养基(DEME:II型胶原酶=25:1,v/v)完全浸润组织,置于细胞培养箱(37℃,5%CO
2)消化6h,6h后,加入3mL的胰酶吹打细胞终止消化,得到原代细胞;将原代细胞经70μm孔径的细胞滤网过滤,收集滤液,1500rpm离心5min,使用含10%(v/v)胎牛血清的DEME完全培养基重悬细胞,转移至9cm细胞培养皿(37℃,5%CO
2)中培养,培养过程中,在显微镜下观察细胞状态,待培养皿中原代细胞贴壁80%,将培养皿中的培养基吸走,用无菌PBS缓冲液冲洗三次,加入2mL 0.25g/100mL的胰酶在37℃消化2min,轻轻吹打细胞,最后加入2mL含5%(v/v)胎牛血清的DMEM完全培养基终止消化,得到细胞悬液A;将细胞悬液A 1000rpm离心5min,弃上清,得到菌体;在菌体中加入10mL含有10%(v/v)胎牛血清的DEME完全培养基吹打均匀进行计数,并用10%(v/v)胎牛血清的DEME完全培养基调整细胞浓度为2×10
6个细胞/mL,得到细胞悬液B。
将实施例1获得的短双歧杆菌(Bifidobacterium breve)CCFM1078和鼠李糖乳杆菌LGG分别接入MRS液体培养基(含0.05g/100mL半胱氨酸)中于37℃厌氧培养24h后,分别转入新鲜的MRS液体培养基(含0.05g/100mL半胱氨酸)中,同样条件培养24h,6000g离心菌体15min,0.9g/100mL生理盐水洗涤菌体后6000g再次离心10min,得到菌体;用DEME完全培养基将短双歧杆菌(Bifidobacterium breve)CCFM1078和鼠李糖乳杆菌LGG的菌体分别重悬至细胞浓度为5×10
6个细胞/mL,得到含有短双歧杆菌(Bifidobacterium breve)CCFM1078和鼠李糖乳杆菌LGG的DEME完全培养基。
将细胞悬液B分为四组,分别为空白组、阳性对照组、CCFM1078组和LGG组;在空白组和阳性对照组细胞悬液B中加入1mL DEME完全培养基,在CCFM1078组细胞悬液B中加入1mL含有短双歧杆菌(Bifidobacterium breve)CCFM1078的DEME完全培养基,在LGG组细胞悬液B中加入1mL含有鼠李糖乳杆菌LGG的DEME完全培养基后,置于细胞培养箱(37℃,5%CO
2)中培养4h,在阳性对照组、CCFM1078组和LGG组细胞悬液B中加入1mL浓度为200ng/mL的LPS溶液,在空白组加入1mL DEME完全培养基后,置于细胞培养箱(37℃,5%CO
2)中继续培养48h,得到培养液。
将培养液离心取上清,测定上清中IL-6的含量,每个组别重复六次(测定结果见图14)。
由图14可知,滑膜细胞经LPS刺激后分泌IL-6的水平显著升高,是未刺激组的1.77倍;经短双歧杆菌CCFM1078或LGG预处理之后,滑膜细胞产生IL-6的水平降低(分别降低至0.72ng/mL和0.94ng/mL)。表明在体外,短双歧杆菌(Bifidobacterium breve)CCFM1078可抑制滑膜细胞分泌促炎因子IL-6,这种抑制作用强于LGG。
实施例11:短双歧杆菌CCFM1078制备冻干菌剂
将短双歧杆菌(Bifidobacterium breve)CCFM1078接入MRS液体培养基(含0.05%半胱氨酸)中于37℃厌氧培养24h后,转入新鲜的MRS液体培养基(含0.05%半胱氨酸)中,同样条件培养24h,6000g离心菌体15min,0.9%生理盐水洗涤菌体后6000g再次离心10min,得到菌体;将菌体用pH为7.2~7.4的磷酸盐缓冲液清洗3次后用海藻糖浓度为100g/L的海藻糖冻干保护剂(冻干保护剂和菌体的质量比为2:1)重悬至浓度为5×10
10CFU/mL,得到重悬液;将重悬液采用真空冷冻法进行冻干,得到短双歧杆菌(Bifidobacterium breve)CCFM1078的冻干粉。
取1g上述冻干粉每天灌胃类风湿性关节炎大鼠,连续五周,可有效缓解大鼠类风湿性关节炎的症状,在预防和/或治疗类风湿性关节炎上有极好的效果。
实施例12:短双歧杆菌CCFM1078制备食品
短双歧杆菌CCFM1078制备果蔬饮料:
选用新鲜蔬菜洗净后榨汁,接着进行高温瞬间灭菌,在温度140℃下高温热杀菌2秒后,立即降温至37℃,再接种实施例11制备的短双歧杆菌CCFM1078发酵菌剂,使蔬菜汁中的短双歧杆菌CCFM1078的浓度达到10
6CFU/mL以上,在温度4℃下冷藏保存,于是得到含有本发明短双歧杆菌CCFM1078活菌的果蔬饮料。
利用本发明能够使用短双歧杆菌CCFM1078发酵生产制备其他发酵食品,所述发酵食品包括固态食品、液态食品、半固态食品。所述发酵食品包括乳制品、豆制品、果蔬制品,所述乳制品包括牛奶、酸奶油、干酪;所述果蔬制品包括黄瓜、胡萝卜、甜菜、芹菜、圆白菜制品。
实施例13:短双歧杆菌CCFM1078制备药物
短双歧杆菌CCFM1078可用于制备液体制剂、粉剂或片剂,具体制备过程如下:
挑取实施例1获得的短双歧杆菌(Bifidobacterium breve)CCFM1078的单菌落接入MRS液体培养基中,于37℃培养24h,得到活化液;将活化液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃培养24h,得到一级种子液;将一级种子液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃培养24h,得到二级种子液;将二级种子液按照1%(v/v)的接种量接入MRS液体培养基中,于37℃培养24h,得到菌液;将菌液6000g离心15min,收集沉淀;将沉淀用pH为7.4的PBS缓冲液洗涤两次后,6000g再次离心10min,得到菌体;用含有130g/L脱脂乳、20g/L海藻糖和20g/L蔗糖的保护剂溶液将短双歧杆菌菌体重悬至细胞浓度为1×10
10CFU/mL,得到短双歧杆菌的液体制剂。
将按前述方法制备的短双歧杆菌液体制剂冷冻干燥,得到短双歧杆菌菌粉(粉剂)。
向按前述方法制备的短双歧杆菌菌粉中加入占短双歧杆菌菌菌粉总重计2%的硬脂酸作润滑剂、3%的CMC-Na作粘合剂后进行压片,得到片剂。
取1mL上述液体制剂或1g上述片剂每天灌胃类风湿性关节炎大鼠,连续五周,可有效缓解大鼠类风湿性关节炎的症状,在预防和/或治疗类风湿性关节炎上有极好的效果。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (15)
- 一株短双歧杆菌(Bifidobacterium breve),其特征在于,所述短双歧杆菌(Bifidobacterium breve)已于2020年05月06日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61011。
- 一种制备预防和/或治疗类风湿性关节炎的产品的方法,其特征在于,使用权利要求1所述短双歧杆菌;所述产品包含食品或药品。
- 如权利要求2所述的一种制备预防和/或治疗类风湿性关节炎的产品的方法,其特征在于,所述产品中,权利要求1所述短双歧杆菌的活菌数为不低于1×10 5CFU/mL或1×10 5CFU/g。
- 如权利要求2或3所述的一种制备预防和/或治疗类风湿性关节炎的产品的方法,其特征在于,将权利要求1所述的短双歧杆菌作为发酵菌株用于食品的发酵。
- 如权利要求2或3所述的一种制备预防和/或治疗类风湿性关节炎的产品的方法,其特征在于,将权利要求1所述的短双歧杆菌作为有效成分加入至药物中。
- 如权利要求5所述的一种制备预防和/或治疗类风湿性关节炎的产品的方法,其特征在于,所述药品含有权利要求1所述短双歧杆菌、药物载体和/或药用辅料。
- 一种用于预防和/或治疗类风湿性关节炎的产品,其特征在于,所述产品含有如权利要求1所述的短双歧杆菌。
- 如权利要求7所述的一种用于预防和/或治疗类风湿性关节炎的产品,其特征在于,所述产品中,权利要求1所述短双歧杆菌的活菌数为不低于1×10 5CFU/mL或1×10 5CFU/g。
- 如权利要求7或8所述的一种用于预防和/或治疗类风湿性关节炎的产品,其特征在于,所述产品为含权利要求1所述的短双歧杆菌的冻干菌粉。
- 如权利要求9所述的一种用于预防和/或治疗类风湿性关节炎的产品,其特征在于,所述冻干菌粉是按照如下方法制备的:将短双歧杆菌接入MRS液体培养基中,于30~37℃厌氧培养,收集菌体细胞;将菌体细胞用洗涤后,用含海藻糖的冻干保护剂重悬至浓度≥5×10 10CFU/mL,得到重悬液;将重悬液采用真空冷冻法进行冻干,得到短双歧杆菌冻干粉。
- 如权利要求7或8所述的一种用于预防和/或治疗类风湿性关节炎的产品,其特征在于,所述产品为食品或药品。
- 如权利要求11所述的一种用于预防和/或治疗类风湿性关节炎的产品,其特征在于,所述药品含有权利要求1所述短双歧杆菌、药物载体和/或药用辅料。
- 如权利要求11所述的一种用于预防和/或治疗类风湿性关节炎的产品,其特征在于,所述食品包含含有权利要求1所述短双歧杆菌的保健食品。
- 如权利要求11所述的一种用于预防和/或治疗类风湿性关节炎的产品,其特征在于,所述食品包含使用权利要求1所述短双歧杆菌的发酵剂生产得到的乳制品、豆制品、肉制品或果蔬制品。
- 一种预防和/或治疗类风湿性关节炎的方法,其特征在于,对哺乳动物施用权利要求1所述的短双歧杆菌或权利要求7~14任一所述的产品。
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