WO2023246674A1 - 治疗糖尿病及相关病症的两歧双歧杆菌 - Google Patents
治疗糖尿病及相关病症的两歧双歧杆菌 Download PDFInfo
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- WO2023246674A1 WO2023246674A1 PCT/CN2023/100940 CN2023100940W WO2023246674A1 WO 2023246674 A1 WO2023246674 A1 WO 2023246674A1 CN 2023100940 W CN2023100940 W CN 2023100940W WO 2023246674 A1 WO2023246674 A1 WO 2023246674A1
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- bifidobacterium bifidum
- ibiome001
- diabetes
- bifidum
- bifidobacterium
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention belongs to the field of microbial technology, and specifically relates to Bifidobacterium bifidum ( Bifidobacterium bifidum ) for treating diabetes and related diseases.
- the vast intestinal flora has formed an inseparable structural and functional connection through long-term co-evolution with the host. It digests nutrients, provides the host with vitamins and energy, and participates in normal activities. A series of functions such as immune construction are involved in host homeostasis. Dysbiosis of intestinal flora has been linked to more than 50 diseases. In recent years, studies have found that metabolic diseases, especially obesity and diabetes, are closely related to intestinal flora, and intestinal flora can regulate the host's fat accumulation and insulin sensitivity. Clinical FMT studies have shown that insulin resistance was significantly improved in obese patients after 6 weeks of intestinal FMT from normal individuals. Another study studied the intestinal flora of 171 Chinese adults with type 2 diabetes and 174 healthy volunteers and identified a total of 52,484 intestinal bacterial genes related to type 2 diabetes. Therefore, probiotics have great potential in treating obesity and diabetes.
- GPR120 is a long-chain unsaturated free fatty acid receptor that has multiple physiological functions such as regulating gastrointestinal hormone secretion and adipocyte development and differentiation.
- Animal experiments show that high-fat diet-induced GPR120-deficient mice develop more severe obesity, insulin resistance, and hepatic steatosis.
- a series of preclinical studies have shown that GPR120 agonists can regulate glucose and energy homeostasis, including improving chronic inflammation and insulin resistance caused by obesity, regulating adipocyte thermogenesis and regulating appetite.
- Human cohort studies have also shown that the R270H mutation in the GPR120L amino acid sequence is significantly associated with obesity. Therefore, GPR120 is a potentially important target for the treatment of metabolic syndromes such as obesity and diabetes.
- triglyceride lipases in adipose tissue
- ATGL adipose triglyceride lipase
- HSL hormone-sensitive lipase
- the former is active without hormone activation, so it is very important in basic lipolysis and is also the most important lipolytic enzyme.
- the latter needs to be activated by lipolytic hormones to be active.
- the hydrolyzed triglyceride (TG) of the two accounts for about 95% of the total hydrolysis.
- HSL was discovered in 1962 and was named because its lipase activity is greatly affected by hormones. Studies have shown that HSL activators can phosphorylate HSL through PKA and translocate it into lipid droplets, thus promoting the lipolysis process. Insulin is its most important inhibitor.
- ATGL was discovered in 2004. Its C-terminus contains a hydrophobic lipid droplet binding region, so it is mainly located on the surface of lipid droplets. ATGL can specifically hydrolyze the first ester bond of TG and is considered to be the rate-limiting enzyme in the TG hydrolysis process. Its reduced expression will lead to a large accumulation of TG in adipocytes and other tissues, causing obesity and other metabolic complications.
- the object of the present invention is to provide Bifidobacterium bifidum ( Bifidobacterium bifidum ) for treating diabetes and related diseases.
- Bifidobacterium bifidum ( Bifidobacterium bifidum ) ibiome001, the Bifidobacterium bifidum strain is preserved in the Guangdong Provincial Microbial Culture Collection Center, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province Institute of Microbiology, Academy of Sciences, with a deposit date of May 17, 2022, and a deposit number of GDMCC No. 62473. The full genome sequence is shown in SEQ ID NO.2.
- Bifidobacterium bifidum ( Bifidobacterium bifidum ) ibiome001
- the Bifidobacterium bifidum strain contains at least one specific gene fragment in SEQ ID NO. 2-5 or its complementary fragment.
- the present invention also protects the use of the Bifidobacterium bifidum ibiome001 and its metabolites, or the mixture containing the bacterium and/or its metabolites in the preparation of functional bacterial agents or medicines, wherein the function Antibacterial agents or drugs for the prevention or treatment of one or more of the following diseases and conditions (a) to (j) in mammals:
- the present invention also protects the use of the Bifidobacterium bifidum ibiome001 and its metabolites, or the mixture containing the bacterium and/or its metabolites in the preparation of functional bacterial agents or drugs that activate GPR120.
- the present invention also protects the Bifidobacterium bifidum ( Bifidobacterium bifidum ) ibiome001 and its metabolites, or the mixture containing the bacterium and/or its metabolites in the preparation of functional bacteria that improve the expression of lipolysis genes ATGL and/or HSL. Application in agents or medicines.
- the diabetes is type 2 diabetes.
- the mammal is a mammal on a high-fat diet.
- the present invention also protects the use of the Bifidobacterium bifidum ibiome001 and its metabolites, or the mixture containing the bacterium and/or its metabolites in the preparation of food or dietary supplements.
- the present invention also protects a composition, which includes the Bifidobacterium bifidum ( Bifidobacterium bifidum ) ibiome001 or its metabolites, and a pharmaceutically acceptable carrier.
- a composition which includes the Bifidobacterium bifidum ( Bifidobacterium bifidum ) ibiome001 or its metabolites, and a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier includes one of fillers, binders, wetting agents, disintegrants, lubricants, flavoring agents, diluents, and absorption enhancers commonly used in medicine. Or two or more.
- Bifidobacterium bifidum Bifidobacterium bifidum
- ibiome001 of the present invention can significantly reduce the body weight and white fat weight of high-fat feed-induced obesity and diabetic mice, increase the expression of lipolysis genes ATGL and HSL, and significantly reduce the weight of mice. Fasting blood glucose, area under the oral glucose tolerance curve, plasma glycated hemoglobin, and insulin levels.
- Bifidobacterium bifidum ( Bifidobacterium bifidum ) ibiome001 is a potential functional strain for treating obesity, diabetes and other metabolic syndromes, and its effect is better than the combination with other Bifidobacterium bifidum.
- Bifidobacterium bifidum ( Bifidobacterium bifidum ) ibiome001, deposited on May 17, 2022, deposited at the Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Republic Institute of Microbiology, Provincial Academy of Sciences, preservation number is GDMCC No. 62473.
- Figure 1 is a smear microscopic image (40X) of ibiome001 of the present invention.
- Figure 2 shows the colony morphology of ibiome001 of the present invention on solid culture medium.
- Figure 3 is a gene comparison diagram between ibiome001 of the present invention and other bifidobacteria.
- Figures 4-7 are enlarged views of the gene alignment between ibiome001 of the present invention and other Bifidobacterium bifidum at SEQ ID NO. 2-5.
- Figure 8 shows the results of GPR120 activation by different strains of Bifidobacterium bifidum.
- Figure 9 shows the effects of ibiome001 of the present invention and other three groups of controls on the body weight of obese and diabetic mice, where a is the weight change rate, b is the body weight value; * represents P ⁇ 0.05; ** represents P ⁇ 0.01.
- Figure 10 shows the effects of ibiome001 of the present invention and other three groups of controls on the blood sugar levels of obese and diabetic mice, where * indicates P ⁇ 0.05.
- Figure 11 shows the effects of ibiome001 of the present invention and other three groups of controls on oral glucose tolerance (OGTT) and area under the curve (AUC of OGTT) in obese and diabetic mice, where * indicates P ⁇ 0.05.
- Figure 12 shows the effects of ibiome001 of the present invention and other three groups of controls on the fat content of obese and diabetic mice, where * represents P ⁇ 0.05; ** represents P ⁇ 0.01.
- Figure 13 shows the effects of ibiome001 of the present invention and other three groups of controls on lipolysis gene expression in white fat of obese and diabetic mice, where * indicates P ⁇ 0.05.
- Figure 14 shows the effects of ibiome001 of the present invention and other three groups of controls on the glycated hemoglobin content of obese and diabetic mice, where ** indicates P ⁇ 0.01.
- Figure 15 shows the effects of ibiome001 of the present invention and other three groups of controls on plasma insulin content in obese and diabetic mice, where * represents P ⁇ 0.05 and ** represents P ⁇ 0.01.
- MRS broth medium purchased from Solarbio, product number M8540
- upstream primer 27F AGAGTTTG ATCCTGGCTCAG
- downstream primer 1492R GGTTA CCTTGTTACGACTT
- the amplified product was sent for sequencing, and Bifidobacterium bifidum was selected for in vitro screening through 16s rRNA gene sequence comparison and was designated as Bifidobacterium bifidum ibiome001.
- PCR system (20 ⁇ L): 2 ⁇ Taq Master Mix: 10 ⁇ L; Primer 1 (341F): 1 ⁇ L; Primer 2 (1492R): 1 ⁇ L; ddH 2 O: 6 ⁇ L; bacterial liquid: 2 ⁇ L.
- the PCR product of the 16S rRNA gene was sequenced, and the results are shown in SEQ ID NO.1.
- ibiome001 is Gram-stained positive, is short rod-shaped, slender rod-shaped or spherical, can form various branches or bifurcations, and has no spores and no power.
- Catalase test Add 2-3 drops of catalase reaction reagent (purchased from Qingdao Haibo Biotechnology, product number HB8650) to the colony of ibiome001. If the result is free of bubbles, it is negative.
- Sugar alcohol fermentation biochemical reaction detection Use a sterilized pipette tip to pick a single bacterial colony into a commercial ampoule for bacterial biochemical detection (purchased from Qingdao Haibo Biotech, product number GB057, GB102-1, GB104-1, GB176, GB178, GB188, GB189, GB193, GB195, GB196, GB197, GB199, GB200, GB201, GB202, GB203, GB204, GB206, GB207), place it in anaerobic culture at 37°C for 48 hours after inoculation, and judge the test results according to the kit instructions, such as Table 1 shows:
- step (3) After culturing the HEK293 cells in step (1) for two days (reaching approximately 90% confluence), add the transfection mixture in step (2) to the HEK293 cells in step (1);
- ibiome001 has the strongest activation effect on GPR120, which is 4 times that of the culture medium control.
- the activation effects of other B. bifidum strains are less than 2 times, proving that ibiome001 has the strongest activation effect on GPR120.
- the activation effect is significantly better than that of other Bifidobacterium bifidum strains.
- mice C57BL/6J (10 weeks old, male) mice, SPF grade, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. After the mice adapted for one week, they were given 60% high-fat feed (purchased from Medison, Cat. No. MD12033) and administered at the same time. The experiment was divided into 4 groups:
- Control group In the control group, 0.2 mL of PBS solution was administered orally;
- Group Bb 10 9 CFU of Bifidobacterium bifidum ibiome001 was administered orally;
- Bb+BI group Bifidobacterium bifidum ibiome001 + Bifidobacterium longum were administered orally (the strains were mixed at a ratio of 1:1, and the total number of colonies was 10 9 CFU);
- mice were divided into groups according to their body weight. After the start of the experiment, the body weight of the mice was recorded every week for a total of 13 weeks. The weight change rate and absolute value of body weight of mice are shown in Figure 9.
- OGTT experimental method Animals were fasted for 12 hours, blood was taken from the tip of the tail of the mouse, and the fasting blood glucose value of the mouse was detected with blood glucose test paper (at time 0). At the same time, glucose solution was administered to the mice at a dose of 2g/kg, and then glucose was administered to the mice respectively. Blood glucose was measured from the tip of the tail at 30, 60, and 120 minutes after the test, and a blood glucose change curve with time was drawn, and the area under the curve was calculated.
- mice were fasted for 12 hours, sacrificed, and collected plasma and adipose tissue (mesenteric white fat, subcutaneous white fat, epididymal white fat, and brown fat), and weighed the adipose tissue respectively.
- Oral administration of Bifidobacterium bifidum ibiome001 (Bb) can significantly reduce the weight of subcutaneous white fat and mesenteric white fat in mice.
- Gavage administration of a combination of B. bifidum and B. longum (Bb+BI) was only able to significantly reduce subcutaneous white fat weight in mice.
- Intragastric administration of a mixture of 5 bifidobacteria had no effect on fat in any part of the mice.
- the specific methods are as follows:
- Extraction of tissue RNA Use the animal tissue total RNA extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., product number DP424) to extract RNA from the tissue. For specific operations, refer to the kit instructions. Then, use NanoDrop and Agarose gel electrophoresis was used to determine RNA concentration and purity.
- Reverse transcription Add 2 ⁇ g of total RNA in tissue to 2 ⁇ L of 5 ⁇ g DNA Buffer, make up to 10 ⁇ L with RNase-Free ddH 2 O, centrifuge briefly, place at 42°C, incubate for 3 minutes, and place on ice for 10 min, then add 2 ⁇ L 10 ⁇ Fast RT Buffer 2 ⁇ L , RT Enzyme Mix 1 ⁇ L , FQ-RT Primer Mix 2 ⁇ L , make up to 20 ⁇ L with RNase-Free ddH 2 O, and incubate in a 42 oC water bath for 15 After 3 minutes, react at 95 oC for 3 minutes and store the product at -80 oC.
- qPCR 2 ⁇ g cDNA, 10 ⁇ L SYBR dye solution, 0.8 ⁇ L primers, and the remaining volume is made up with ddH 2 O (total reaction system: 20 ⁇ L ).
- Reaction conditions denaturation at 95 oC for 10 minutes, amplification (95 oC for 15 seconds, 60 oC for 1 minute, a total of 40 cycles).
- Two accessory wells were set up for each gene, and 2 - ⁇ CT was used for data processing for relative quantitative analysis.
- test kit purchased from Huamei Biotech, product number CSB-E08141m and CSB-E05071m
- the level of glycated hemoglobin is the gold standard for measuring blood sugar control.
- intragastric administration of B. bifidum ibiome001 (Bb) significantly reduced the glycated hemoglobin content in the plasma of obese and diabetic mice induced by high-fat feed.
- Bb B. bifidum ibiome001
- B. bifidum ibiome001 (Bb) significantly reduced the insulin content in the plasma of obese and diabetic mice induced by high-fat feed, while oral administration of the combination of B. bifidum and B. longum (Bb+BI) has no effect on plasma insulin levels.
- Bifidobacterium bifidum ibiome001 can significantly reduce the body weight and white fat weight of high-fat feed-induced obesity and diabetic mice, increase the expression of lipolysis genes ATGL and HSL; and significantly reduce the expression of lipolytic genes ATGL and HSL. Rat fasting blood glucose, area under the oral glucose tolerance curve, plasma glycosylated hemoglobin and insulin content.
- Bifidobacterium bifidum ibiome001 is a potential functional strain for treating obesity, diabetes and other metabolic syndromes, and its effect is better than the combination with other Bifidobacterium bifidum.
- Bifidobacterium bifidum Bifidobacterium bifidum
- ibiome001 can significantly reduce the body weight and white fat weight of high-fat feed-induced obesity and diabetic mice, increase the expression of lipolysis genes ATGL and HSL, and significantly reduce the weight of mice. Fasting blood glucose, area under the oral glucose tolerance curve, plasma glycated hemoglobin, and insulin levels.
- Bifidobacterium bifidum ( Bifidobacterium bifidum ) ibiome001 is a potential functional strain for treating obesity, diabetes and other metabolic syndromes. The effect is better than the combination with other Bifidobacterium bifidum, and it has obvious industrial practicability.
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Abstract
提供了一种两歧双歧杆菌(Bifidobacterium bifidum)菌株ibiome001,能够显著降低高脂饲料诱导的肥胖、糖尿病小鼠的体重、白色脂肪重量,提高脂解基因ATGL和HSL的表达;并且显著降低小鼠禁食血糖、口服葡萄糖耐量曲线下面积、血浆糖化血红蛋白和胰岛素含量。该菌株是潜在的治疗肥胖、糖尿病等代谢综合症的功能菌株,效果优于与其他两歧双歧杆菌的组合。
Description
本发明属于微生物技术领域,具体涉及治疗糖尿病及相关病症的两歧双歧杆菌(
Bifidobacterium bifidum)。
随着社会的发展,人们生活水平的不断提高和饮食结构的调整,饮食呈富余化趋势,且多大鱼大肉,高脂饮食已成为一种重要的日常膳食方式,然而由此导致代谢类疾病的发病率不断提高,其中以肥胖、糖尿病等代谢综合症最为常见,严重影响人们的身心健康。
在肠道中约有1.5 kg的细菌,数量浩瀚的肠道菌群通过与宿主长期的共同进化,已经形成了密不可分的结构、功能联系,它通过消化营养成分、提供宿主维生素及能量、参与正常免疫构建等一系列功能参与宿主稳态平衡。肠道菌群的失调已经被认为与超过50种疾病有关。近年来研究发现代谢性疾病,尤其是肥胖和糖尿病与肠道菌群密切相关,肠道菌群可以调节宿主的脂肪积累和胰岛素敏感性。临床FMT的研究表明,肥胖患者接受来自正常个体的肠道FMT6周后,胰岛素抵抗得到了显著改善。另一项研究在对171名中国成人2型糖尿病患者和174名健康志愿者的肠道菌群研究中,共识别出52484个与2型糖尿病相关的肠道细菌基因。因此益生菌在治疗肥胖、糖尿病中具有巨大的潜力。
GPR120是长链不饱和游离脂肪酸受体,具有调节胃肠道激素分泌、调节脂肪细胞发育和分化等多种生理功能。动物实验显示,高脂饮食诱导GPR120缺失小鼠会产生更为严重的肥胖、胰岛素抵抗和肝脏脂肪变性。一系列临床前研究显示GPR120激动剂能够调节葡萄糖和能量稳态,包括改善肥胖引起的慢性炎症及胰岛素抵抗、调节脂肪细胞生热和调节食欲等。人的队列研究也显示,GPR120L氨基酸序列中R270H突变与肥胖显著相关。因此,GPR120是治疗肥胖、糖尿病等代谢综合症的潜在重要靶点。
脂肪组织中有两种甘油三酯脂肪酶,脂肪组织甘油三酯脂肪酶(adipose triglyceride lipase,ATGL)和激素敏感脂肪酶(hormone-sensitive lipase,HSL)。前者不需要激素激活即有活性,所以在基础脂解中非常重要,也是最主要的脂解酶。后者则需由脂解激素激活才有活性。二者水解的甘油三酯(TG)约占水解总量的95%。
HSL是1962年发现的,因其脂肪酶活性受激素影响很大而得名。研究表明,HSL激活剂可以通过PKA将HSL磷酸化,使其易位到脂滴中,从而促进脂解过程。胰岛素是其最重要的抑制剂。
ATGL是2004年发现的。其C端含有疏水性脂滴结合区,所以主要定位于脂滴表面。ATGL能特异性地水解TG的第一酯键,被认为是TG水解过程的限速酶,其表达减少会导致脂肪细胞和其他组织的TG大量积累,造成肥胖和其他代谢并发症。
本发明的目的是提供治疗糖尿病及相关病症的两歧双歧杆菌(
Bifidobacterium bifidum)。
本发明是通过以下技术方案实现的:
两歧双歧杆菌(
Bifidobacterium bifidum)ibiome001,所述的两歧双歧杆菌菌株在广东省微生物菌种保藏中心保藏,地址为广东省广州市先烈中路100号大院59号楼5楼,广东省科学院微生物研究所,保藏日期2022年5月17日,保藏号为GDMCC No. 62473,全基因组序列如SEQ ID NO.2所示。
两歧双歧杆菌(
Bifidobacterium bifidum)ibiome001,所述的两歧双歧杆菌菌株含有SEQ ID NO.2-5中至少一条特异性基因片段或其互补片段。
本发明还保护所述的两歧双歧杆菌(
Bifidobacterium bifidum)ibiome001及其代谢物,或含有该菌和/或其代谢物的混合物在制备功能性菌剂或药物中的用途,其中所述功能性菌剂或药物用于预防或治疗哺乳动物的一种或两种以上下列疾病和病症(a)至(j):
(a)糖尿病;
(b)代谢综合症;
(c)糖化血红蛋白含量异常;
(d)胰岛素敏感度异常;
(e)空腹血糖值异常;
(f)口服糖耐量异常;
(g)空腹胰岛素含量异常;
(h)肥胖;
(i)体重增长;
(j)脂肪组织重量增长。
本发明还保护所述的两歧双歧杆菌(
Bifidobacterium bifidum)ibiome001及其代谢物,或含有该菌和/或其代谢物的混合物在制备激活GPR120的功能性菌剂或药物中的应用。
本发明还保护所述的两歧双歧杆菌(
Bifidobacterium bifidum)ibiome001及其代谢物,或含有该菌和/或其代谢物的混合物在制备提高脂解基因ATGL和/或HSL表达的功能性菌剂或药物中的应用。
进一步的,所述糖尿病为2型糖尿病。
进一步的,所述哺乳动物为高脂饮食哺乳动物。
本发明还保护所述的两歧双歧杆菌(
Bifidobacterium bifidum)ibiome001及其代谢物,或含有该菌和/或其代谢物的混合物在制备食品或膳食补充剂中的用途。
本发明还保护一种组合物,所述组合物包括所述的两歧双歧杆菌(
Bifidobacterium bifidum)ibiome001或其代谢物,以及在药学上可接受的载体。
进一步的,所述在药学上可接受的载体包括医学上通常使用的填充剂、粘合剂、润湿剂、崩解剂、润滑剂、矫味剂、稀释剂、促吸收剂中的一种或两种以上。
本发明的有益效果在于:
灌胃本发明的两歧双歧杆菌(
Bifidobacterium bifidum)ibiome001能够显著降低高脂饲料诱导的肥胖、糖尿病小鼠的体重、白色脂肪重量,提高脂解基因ATGL和HSL的表达;并且显著降低小鼠禁食血糖、口服葡萄糖耐量曲线下面积、血浆糖化血红蛋白和胰岛素含量。两歧双歧杆菌(
Bifidobacterium bifidum)ibiome001是潜在的治疗肥胖、糖尿病等代谢综合症的功能菌株,效果优于与其他两歧双歧杆菌的组合。
两歧双歧杆菌(
Bifidobacterium bifidum)ibiome001,保藏日期2022年5月17日,保藏地点为广东省微生物菌种保藏中心,地址为广东省广州市先烈中路100号大院59号楼5楼,广东省科学院微生物研究所,保藏号为GDMCC No. 62473。
图1为本发明ibiome001的涂片镜检图(40X)。
图2为本发明ibiome001在固体培养基上的菌落形态。
图3为本发明ibiome001与其他两歧双歧杆菌的基因比对图。
图4-7为本发明ibiome001与其他两歧双歧杆菌的基因比对在SEQ ID NO.2-5处的放大图。
图8为不同株的两歧双歧杆菌激活GPR120的结果。
图9为本发明ibiome001及其他3组对照对肥胖、糖尿病小鼠体重的影响,其中,a为体重变化率,b为体重值;*表示
P<0.05;**表示
P<0.01。
图10为本发明ibiome001及其他3组对照对肥胖、糖尿病小鼠血糖值的影响,其中,*表示
P<0.05。
图11为本发明ibiome001及其他3组对照对肥胖、糖尿病小鼠口服葡萄糖耐量(OGTT)及曲线下面积(AUC of OGTT)的影响,其中,*表示
P<0.05。
图12为本发明ibiome001及其他3组对照对肥胖、糖尿病小鼠脂肪含量的影响,其中,*表示
P<0.05;**表示
P<0.01。
图13为本发明ibiome001及其他3组对照对肥胖、糖尿病小鼠白色脂肪中脂解基因表达的影响,其中,*表示
P<0.05。
图14为本发明ibiome001及其他3组对照对肥胖、糖尿病小鼠糖化血红蛋白含量的影响,其中,**表示
P<0.01。
图15为本发明ibiome001及其他3组对照对肥胖、糖尿病小鼠血浆胰岛素含量的影响,其中,*表示
P<0.05,**表示
P<0.01。
为更好理解本发明,下面结合实施例及附图对本发明作进一步描述,以下实施例仅是对本发明进行说明而非对其加以限定。另外,如无特别说明,未具体记载条件或者步骤的方法均为常规方法,所采用的试剂和材料均可从商业途径获得。
1 分离
取10名健康志愿者粪便样品,用20%体积分数的甘油磷酸盐缓冲液保存,对粪便样品分别梯度稀释至10
-5、10
-6、10
-7。
将各浓度涂布于MRS肉汤培养基上(购自Solarbio,货号M8540),37℃,厌氧培养48小时。
挑取单克隆至MRS肉汤液体培养基中进行培养,通过16s rRNA通用引物进行PCR扩增(上游引物27F:AGAGTTTG ATCCTGGCTCAG,下游引物1492R:GGTTA CCTTGTTACGACTT)。
2 鉴定
2.1 16s rRNA测序
扩增产物送测序,通过16s rRNA基因序列比对选取两歧双歧杆菌进行体外筛选,编为两歧双歧杆菌(
Bifidobacterium bifidum)ibiome001。
实验方法:
取100μL菌液,12000rpm离心2min,弃培养基,加灭菌ddH
2O重悬菌体用于PCR。
PCR体系(20μL): 2 × Taq Master Mix:10μL; 引物1(341F):1μL; 引物2(1492R):1μL;ddH
2O:6μL;菌液:2μL。
PCR反应程序:95℃ 3min;95℃ 15s,58℃ 15s,72℃ 30s,step2-4 35×;72℃ 5min。
将16S rRNA基因的PCR产物进行测序,结果如SEQ ID NO.1所示。
2.2 涂片镜检
使用显微镜将ibiome001在40X下进行涂片镜检,得到如图1所示镜检图。由图中可看出,ibiome001革兰氏染色阳性,呈短杆状、纤细杆状或球形,可形成各种分支或分叉等多形态,无芽孢,无动力。
2.3 单菌落照片
将ibiome001在MRS培养基上培养48h后拍照,单菌落照片如图2所示,菌落呈白色,圆形,边缘整齐,表面湿润。
2.4 过氧化氢酶试验和糖醇发酵生化反应检测
取300μL冻存的ibiome001到1mL MRS培养基中复苏菌株,取液体培养的ibiome001在MRS固体培养基划线中纯化,挑取单菌落接种于1mL MRS液体培养基中培养24h,再取液体培养的菌株,经过梯度稀释,涂布于MRS固体培养基中,培养72h。再分别进行过氧化氢酶试验和糖醇发酵生化反应检测。
过氧化氢酶试验:滴加2-3滴过氧化氢酶反应试剂(购自青岛海博生物,货号HB8650)至ibiome001的菌落上,结果无气泡,即阴性。
糖醇发酵生化反应检测:用灭菌的枪头挑取单个菌落到商品化的细菌生化检测的安瓿瓶(购自青岛海博生物,货号GB057、GB102-1、GB104-1、GB176、GB178、GB188、GB189、GB193、GB195、GB196、GB197、GB199、GB200、GB201、GB202、GB203、GB204、GB206、GB207)中,接种后置于37℃厌氧培养48h,根据试剂盒说明书判断检测结果,如表1所示:
表1. 两歧双歧杆菌生化反应鉴定结果
检测项目 | 检测结果 | 检测项目 | 检测结果 |
纤维二糖 | - | 卫矛醇 | - |
海藻糖 | - | D-葡萄糖 | + |
蜜二糖 | - | D-半乳糖 | + |
肌醇 | - | 阿东醇 | - |
D-麦芽糖 | - | 苦杏仁甙 | - |
L-阿拉伯糖 | - | D-果糖 | + |
水杨素 | - | 肝糖 | - |
D-木糖 | - | 菊糖 | + |
D-蔗糖 | - | N-乙酰-葡萄糖胺 | + |
七叶灵 | - |
注:+表示阳性;-表示阴性。
2.5 全基因组测序
将ibiome001送至基因检测公司进行全基因组测序,将得到的全基因组序列与ATCC 29521(=JCM 1255,GenBank Assembly Accession:GCA_001025135.1)、YIT 10347(GenBank Assembly Accession:GCA_020892075.1)、TMC 3115(GenBank Assembly Accession:GCA_003573895.1)、NCTC13001(GenBank Assembly Accession:GCA_900637095.1)、JCM 7004(GenBank Assembly Accession:GCA_003573955.1)、PRL2010(GenBank Assembly Accession:GCA_000165905.1)、HN002(GenBank Assembly Accession:GCA_016838705.1)、BGN4(GenBank Assembly Accession:GCA_000265095.1)、BF3(GenBank Assembly Accession:GCA_001281345.1)、S17(GenBank Assembly Accession:GCA_000164965.1)、S6(GenBank Assembly Accession:GCA_003390735.1)等两歧双歧杆菌菌株序列比对,得到如SEQ ID NO.2-5所示特异性序列片段,如图3-7所示。
(1)将稳定表达β-arrestin-TEV和tTA-luciferase的HEK293细胞株放到含有10%胎牛血清和1%青霉素/链霉素的DMEM培养基中培养;
(2)转染混合物制备:200ng/孔GPR120-tango质粒在20μL DMEM中与400ng polyethylenimine(溶于20μL的DMEM中)混合,室温孵育20分钟;
(3)将步骤(1)的HEK293细胞培养两天(达到大约90%融合)后,将步骤(2)的转染混合物加入步骤(1)的HEK293细胞中;
(4)转染24小时后,用180mL含1%青霉素/链霉素和10mM HEPES的DMEM培养基和20μL细菌培养上清液(Bb-1、Bb-2、Bb-3或ibiome001,其中Bb-1、Bb-2、Bb-3均为实验室筛选的其他两歧双歧杆菌,16s rDNA序列与SEQ ID NO.1一致)替换培养基;
(5)细菌培养上清液刺激24小时后弃上清,每孔加入50μL 用含20mM HEPES的PBS稀释20倍的Bright-Glo溶液(Promega公司产品);
(6)室温孵育20分钟后,使用 Spectramax i3进行荧光定量。
实验结果如图8所示,与培养基相比,ibiome001对GPR120的激活作用最强,是培养基对照的4倍,其他的两歧双歧杆菌菌株激活作用均小于2倍,证明ibiome001对GPR120的激活作用明显优于其他两歧双歧杆菌菌株。
选取C57BL/6J(10周,雄性)小鼠,SPF级,购自江苏集萃药康生物科技股份有限公司。小鼠适应一周后,给予小鼠60%高脂饲料(购自美迪森,货号MD12033),并同时给药,实验共分为4组:
(1)control组:对照组,灌胃0.2mL PBS溶液;
(2)Bb组:灌胃10
9CFU两歧双歧杆菌ibiome001;
(3)Bb+BI组:灌胃两歧双歧杆菌ibiome001+长双歧杆菌(菌株按照1:1混合,菌落数共10
9CFU);
(4)5mix(Ba+Bf+BI+Bb+Bp)组:五种双歧杆菌混合,分别为两歧双歧杆菌ibiome001(Bb),长双歧杆菌(BI),青春双歧杆菌(Ba),粪双歧杆菌(Bf)和假链双歧杆菌(Bp)(菌株按照1:1:1:1:1混合,菌落数共10
9CFU)。
实验开始前将上述小鼠按体重分组,实验开始后每周记录小鼠体重,共记录13周。小鼠的体重变化率和体重绝对值如图9所示。
从图9a中可看出,灌胃两歧双歧杆菌ibiome001(Bb)从第四周开始就可以显著抑制小鼠体重增长,并且这种效果一直持续到实验结束;而与其他双歧杆菌组合后,抑制体重增长的效果不显著。从图9b可以看到在灌胃两歧双歧杆菌ibiome001 13周后,小鼠的绝对体重与对照组(control)相比降低了5.53g。
实验第10周时检测小鼠的禁食血糖值并进行口服葡萄糖耐量试验(OGTT)。
OGTT实验方法:动物禁食12小时,小鼠尾尖取血用血糖试纸检测小鼠空腹血糖值(0时)同时按照2g/kg的剂量给小鼠灌胃葡萄糖溶液,然后分别在灌胃葡萄糖后的30、60、120min尾尖取血测量血糖值,绘制血糖随时间变化曲线,计算曲线下面积。
禁食血糖值结果如图10所示:灌胃两歧双歧杆菌ibiome001(Bb)可以显著降低高脂饲料诱导的肥胖、糖尿病小鼠的禁食血糖值,而与其他双歧杆菌组合后没有改善作用。
口服葡萄糖耐量(OGTT)的结果如图11a所示:与对照组(control)相比,灌胃两歧双歧杆菌ibiome001(Bb)大幅降低了糖负荷后30 min的血糖水平(
P=0.051),并且显著降低了口服葡萄糖耐量曲线下面积(图11b)。而与其他双歧杆菌组合后没有显著改善作用。
给药13周后小鼠禁食12小时,牺牲小鼠,取血浆、脂肪组织(肠系膜白色脂肪,皮下白色脂肪,附睾白色脂肪和棕色脂肪),将脂肪组织分别称重,结果如图12所示:灌胃两歧双歧杆菌ibiome001(Bb)能够显著降低小鼠皮下白色脂肪和肠系膜白色脂肪的重量。灌胃两歧双歧杆菌和长双歧杆菌的组合(Bb+BI)仅能够显著降低小鼠的皮下白色脂肪重量。灌胃5种双歧杆菌混合物对小鼠各部位脂肪均没有影响。
取上述白色脂肪组织提取RNA,用qPCR方法检测脂解相关基因的表达量。具体方法如下:
组织RNA的提取:采用动物组织的总RNA提取试剂盒(购自天根生化科技(北京)有限公司,货号DP424)对组织中的RNA进行提取,具体操作参考试剂盒说明书,接着,利用NanoDrop及琼脂糖凝胶电泳测定RNA浓度和纯度。
逆转录:将2
μg组织中总RNA加入2
μL 5×g DNA Buffer,以RNase-Free ddH
2O补足至10
μL简短离心,置于42 ℃,孵育3 min后,冰上放置10 min,再加入2
μL 10×Fast RT Buffer 2
μL,RT Enzyme Mix 1
μL,FQ-RT Primer Mix 2
μL,以RNase-Free ddH
2O补足至20
μL,于42 ºC水浴15分钟后,于95 ºC反应3分钟,将产物保存于-80 ºC。
qPCR:2
μg cDNA、10
μL SYBR染液、0.8
μL引物,剩余体积用ddH
2O补足(总反应体系:20
μL)。反应条件:95 ºC变性10分钟,扩增(95 ℃ 15秒,60 ºC 1分钟,共40循环)。各基因分别设2个副孔,数据处理采用2
-ΔΔCT进行相对定量分析。
qPCR的结果如图13所示,结果表明:灌胃两歧双歧杆菌ibiome001(Bb)能够提高脂解基因ATGL和HSL的表达,其中ATGL的升高程度达到了显著性差异(
P<0.05)。
取上述血浆,用试剂盒(购自华美生物,货号CSB-E08141m和CSB-E05071m)检测血浆中糖化血红蛋白、胰岛素含量。
糖化血红蛋白水平是衡量血糖控制的金标准,如图14所示,灌胃两歧双歧杆菌ibiome001(Bb)显著降低高脂饲料诱导的肥胖、糖尿病小鼠血浆中糖化血红蛋白的含量,而与其他双歧杆菌组合后对血浆中糖化血红蛋白的含量没有影响。
如图15所示,灌胃两歧双歧杆菌ibiome001(Bb)显著降低高脂饲料诱导的肥胖、糖尿病小鼠血浆中胰岛素的含量,而灌胃两歧双歧杆菌和长双歧杆菌的组合(Bb+BI)对血浆胰岛素含量没有影响。
综上所述,灌胃两歧双歧杆菌ibiome001(Bb)能够显著降低高脂饲料诱导的肥胖、糖尿病小鼠的体重、白色脂肪重量,提高脂解基因ATGL和HSL的表达;并且显著降低小鼠禁食血糖、口服葡萄糖耐量曲线下面积、血浆糖化血红蛋白和胰岛素含量。两歧双歧杆菌ibiome001是潜在的治疗肥胖、糖尿病等代谢综合症的功能菌株,效果优于与其他两歧双歧杆菌的组合。
以上所述实施方式仅仅是对本发明的优选实施方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案作出的各种变形和改进,均应落入本发明的权利要求书确定的保护范围内。
灌胃本发明的两歧双歧杆菌(
Bifidobacterium bifidum)ibiome001能够显著降低高脂饲料诱导的肥胖、糖尿病小鼠的体重、白色脂肪重量,提高脂解基因ATGL和HSL的表达;并且显著降低小鼠禁食血糖、口服葡萄糖耐量曲线下面积、血浆糖化血红蛋白和胰岛素含量。两歧双歧杆菌(
Bifidobacterium bifidum)ibiome001是潜在的治疗肥胖、糖尿病等代谢综合症的功能菌株,效果优于与其他两歧双歧杆菌的组合,具有明显的工业实用性。
Claims (10)
- 两歧双歧杆菌( Bifidobacterium bifidum)ibiome001,其特征在于:所述的两歧双歧杆菌菌株在广东省微生物菌种保藏中心保藏,地址为广东省广州市先烈中路100号大院59号楼5楼,广东省科学院微生物研究所,保藏日期2022年5月17日,保藏号为GDMCC No. 62473。
- 一种检测权利要求1所述两歧双歧杆菌( Bifidobacterium bifidum)ibiome001的方法,其特征在于:所述的两歧双歧杆菌菌株含有SEQ ID NO.2-5中至少一条特异性基因片段或其互补片段。
- 一种组合物,其特征在于:所述组合物包括权利要求1所述的两歧双歧杆菌( Bifidobacterium bifidum)ibiome001,以及在药学上可接受的载体。
- 根据权利要求3所述的组合物,其特征在于:所述在药学上可接受的载体包括医学上通常使用的填充剂、粘合剂、润湿剂、崩解剂、润滑剂、矫味剂、稀释剂、促吸收剂中的一种或两种以上。
- 权利要求1所述的两歧双歧杆菌( Bifidobacterium bifidum)ibiome001、或权利要求3或4所述的组合物在制备功能性菌剂或药物中的用途,其中所述功能性菌剂或药物用于预防或治疗哺乳动物的一种或两种以上下列疾病和病症(a)至(j):(a)糖尿病;(b)代谢综合症;(c)糖化血红蛋白含量异常;(d)胰岛素敏感度异常;(e)空腹血糖值异常;(f)口服糖耐量异常;(g)空腹胰岛素含量异常;(h)肥胖;(i)体重增长;(j)脂肪组织重量增长。
- 权利要求1所述的两歧双歧杆菌( Bifidobacterium bifidum)ibiome001、或权利要求3或4所述的组合物在制备激活GPR120的功能性菌剂或药物中的应用。
- 权利要求1所述的两歧双歧杆菌( Bifidobacterium bifidum)ibiome001、或权利要求3或4所述的组合物在制备提高脂解基因ATGL和/或HSL表达的功能性菌剂或药物中的应用。
- 根据权利要求5所述的用途,其特征在于:所述糖尿病为2型糖尿病。
- 根据权利要求5或8所述的用途,其特征在于:所述哺乳动物为高脂饮食哺乳动物。
- 权利要求1所述的两歧双歧杆菌( Bifidobacterium bifidum)ibiome001、或权利要求3或4所述的组合物在制备食品或膳食补充剂中的用途。
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