CN114107088B - 一种罗伊氏乳杆菌lrsy523及其应用 - Google Patents
一种罗伊氏乳杆菌lrsy523及其应用 Download PDFInfo
- Publication number
- CN114107088B CN114107088B CN202111221173.2A CN202111221173A CN114107088B CN 114107088 B CN114107088 B CN 114107088B CN 202111221173 A CN202111221173 A CN 202111221173A CN 114107088 B CN114107088 B CN 114107088B
- Authority
- CN
- China
- Prior art keywords
- lactobacillus reuteri
- lrsy523
- mice
- strain
- lactobacillus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000186604 Lactobacillus reuteri Species 0.000 title claims abstract description 46
- 229940001882 lactobacillus reuteri Drugs 0.000 title claims abstract description 43
- 208000008589 Obesity Diseases 0.000 claims abstract description 13
- 235000020824 obesity Nutrition 0.000 claims abstract description 13
- 230000007413 intestinal health Effects 0.000 claims abstract description 8
- 238000002360 preparation method Methods 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 235000013376 functional food Nutrition 0.000 claims abstract description 5
- 230000000813 microbial effect Effects 0.000 claims abstract 2
- 230000001580 bacterial effect Effects 0.000 claims description 16
- 239000001963 growth medium Substances 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 3
- 238000009472 formulation Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 241000699670 Mus sp. Species 0.000 abstract description 12
- 238000013218 HFD mouse model Methods 0.000 abstract description 11
- 230000007358 intestinal barrier function Effects 0.000 abstract description 8
- 206010022489 Insulin Resistance Diseases 0.000 abstract description 6
- 208000001072 type 2 diabetes mellitus Diseases 0.000 abstract description 6
- 108020004465 16S ribosomal RNA Proteins 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 210000004534 cecum Anatomy 0.000 abstract description 3
- 238000009629 microbiological culture Methods 0.000 abstract description 3
- 206010009944 Colon cancer Diseases 0.000 abstract description 2
- 208000029742 colonic neoplasm Diseases 0.000 abstract description 2
- 244000005700 microbiome Species 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 14
- 241000186660 Lactobacillus Species 0.000 description 10
- 229940039696 lactobacillus Drugs 0.000 description 10
- 239000006041 probiotic Substances 0.000 description 10
- 230000000529 probiotic effect Effects 0.000 description 10
- 235000018291 probiotics Nutrition 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 9
- 238000012258 culturing Methods 0.000 description 7
- 210000005027 intestinal barrier Anatomy 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 210000001035 gastrointestinal tract Anatomy 0.000 description 6
- 230000000968 intestinal effect Effects 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 238000011529 RT qPCR Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000002757 inflammatory effect Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 235000009200 high fat diet Nutrition 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 230000003870 intestinal permeability Effects 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 102000002029 Claudin Human genes 0.000 description 3
- 108050009302 Claudin Proteins 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 230000002496 gastric effect Effects 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 102000000591 Tight Junction Proteins Human genes 0.000 description 2
- 108010002321 Tight Junction Proteins Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000002778 food additive Substances 0.000 description 2
- 235000013402 health food Nutrition 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 238000009631 Broth culture Methods 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 102000004162 Claudin-1 Human genes 0.000 description 1
- 108090000600 Claudin-1 Proteins 0.000 description 1
- 208000036649 Dysbacteriosis Diseases 0.000 description 1
- 208000027244 Dysbiosis Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000006833 Multifunctional Enzymes Human genes 0.000 description 1
- 108010047290 Multifunctional Enzymes Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000003940 Occludin Human genes 0.000 description 1
- 108090000304 Occludin Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 108700012920 TNF Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000007140 dysbiosis Effects 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 230000003871 intestinal function Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000006371 metabolic abnormality Effects 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 210000004279 orbit Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- C—CHEMISTRY; METALLURGY
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/173—Reuteri
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Genetics & Genomics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- Obesity (AREA)
- Tropical Medicine & Parasitology (AREA)
- Child & Adolescent Psychology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Diabetes (AREA)
- Epidemiology (AREA)
- General Engineering & Computer Science (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
本发明公开了一种罗伊氏乳杆菌LRSY523及其应用,属于微生物技术领域。本发明提供的罗伊氏乳杆菌(Lactobacillus reuteri)LRSY523分离自小鼠盲肠,该菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.22953。保藏日期:2021年07月26日。菌株的16S rDNA序列提交至Genbank数据库,序列登录号为:MW530426.2。研究结果证明,罗伊氏乳杆菌LRSY523对人结肠癌细胞Caco‑2和HT‑29黏附能力强,并且可以有效保护肠道屏障功能、改善高脂饮食小鼠的肥胖和胰岛素抵抗效应。因此,本发明所述的罗伊氏乳杆菌可在制备改善肠道健康、缓解肥胖的微生物制剂和/或功能性食品中应用。
Description
技术领域
本发明涉及微生物领域,具体涉及一种高黏附性能的罗伊氏乳杆菌LRSY523及其应用。
背景技术
近年来,随着我国经济水平的不断发展,居民饮食结构中高脂饮食比重日益增加,肠道问题及肥胖人群占比逐年增加。研究发现,高脂饮食的摄入会导致肠道菌群失调和肠道屏障功能紊乱,引发肠屏障受损及代谢异常,进而引发肥胖。所以,肠道健康问题与肥胖之间的联系愈发紧密。目前以益生菌为代表的新型膳食添加剂被证明其能在肠道中通过影响肠道菌群和肠道屏障发挥益生作用。上述新型膳食添加剂的可行性促使继续寻找应用更广泛、潜力更巨大,同时又具有改善肠道健康、缓解肥胖的益生菌膳食添加剂。
罗伊氏乳杆菌是一种在哺乳动物肠道内常见的乳杆菌,由于罗伊氏乳杆菌的益生能力和对胃肠道环境较好的耐受能力,2003年,其被卫生部批准可作为保健食品益生菌种并纳入《可用于保健食品的益生菌菌种名单》。研究显示:罗伊氏乳杆菌具有调节肠道菌群、增强免疫力、保护肠道黏膜、改善肠道功能、促进消化、抗肿瘤及抗氧化等功效,而乳杆菌在肠道的黏附定植是其发挥益生功能的前提因素。因此,挖掘高黏附能力的益生菌株对于开发新型的益生菌制剂和/或绿色安全的功能性食品尤为重要。
发明内容
有鉴于此,本发明的目的在于提供一种高黏附性能的罗伊氏乳杆菌及其在改善肠道健康、缓解肥胖中的应用。
本发明提供的一种罗伊氏乳杆菌(Lactobacillus reuteri),所述菌株分离于口服谷糠蛋白提取物的小鼠盲肠内容物,提取该菌株的16S rDNA,测序比对分析结果为罗伊氏乳杆菌,命名为罗伊氏乳杆菌LRSY523。其16S rDNA序列提交至Genbank数据库,序列登录号为:MW530426.2,GI:2035167899。该罗伊氏乳杆菌保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.22953,保藏日期:2021年07月26日。
所述罗伊氏乳杆菌LRSY523菌株在MRS固体培养基上的菌落形态突起,边缘光滑、形状不规则、乳白色、不透明至半透明、直径为1-3mm。
本发明提供的一种罗伊氏乳杆菌菌剂的制备方法,将权利要求1所述的罗伊氏乳杆菌LRSY523菌株在MRS培养基中,于37℃条件下驯化和扩大培养,培养至活菌数高于1×109CFU/mL,菌液分装后置于4℃待用。
通过体外菌胞共培养实验表明,罗伊氏乳杆菌LRSY523可以高效黏附于人结肠癌细胞Caco-2和HT-29上,显示罗伊氏乳杆菌LRSY523能够定植在肠道上皮细胞发挥益生作用。
经高脂饮食诱导构建小鼠肥胖和肠道屏障渗漏模型,培养罗伊氏乳杆菌LRSY523,使之菌液中活菌数达到为109-1010CFU/mL,每日灌胃给予小鼠,实验结果表明:罗伊氏乳杆菌LRSY523能够显著增加高脂饮食小鼠肠道紧密连接蛋白的表达改善肠道屏障功能;并缓解其胰岛素抵抗及肥胖效应。该罗伊氏乳杆菌LRSY523有潜力开发为改善肠道健康、缓解肥胖的功能性食品和/或益生菌制剂。
附图说明
图1罗伊氏乳杆菌LRSY523菌落形态照片和菌株16S rDNA核酸电泳扫描图;
图2罗伊氏乳杆菌LRSY523革兰氏染色显微照片图;
图3罗伊氏乳杆菌LRSY523在Caco-2和HT-29细胞黏附水平的统计柱状图;
图4LRSY523处理后高脂饮食小鼠体重变化曲线图;
图5LRSY523处理后高脂饮食小鼠胰岛素抵抗指数统计柱状图;
图6FITC-葡聚糖检测LRSY523处理后小鼠肠道通透性的统计柱状图;
图7LRSY523处理后小鼠血清炎症因子水平的统计柱状图;
图8qPCR检测LRSY523处理后肠道紧密连接蛋白表达的统计柱状图;
图1~8中,“*”、“**”表示显著性差异,误差以Mean±SEM形式展现。
具体实施方式
实施例1:罗伊氏乳杆菌LRSY523的分离、鉴定以及制备
1、材料准备
在厌氧条件下,取新鲜小鼠盲肠,分离内容物;
通用引物27F/1492R序列如下:
Forward AGAGTTTGATCCTGGCTCAG
Reverse GGTTACCTTGTTACGACTT
MRS肉汤培养基(加1.5%琼脂为固体培养基)
2、乳杆菌的分离筛选
(1)稀释涂布
称取100mg小鼠粪便,在厌氧箱中于1mL PBS中悬浮,震荡30min,使样品充分分散,纱布过滤去除残渣,10-4-10-6梯度涂布于MRS固体培养基上,37℃培养48h(如图1所示);
(2)取步骤(1)中的平板,观察挑取单菌落,在MRS固体培养基上划线,放至37℃厌氧条件下培养48h,重新得到单菌落;
(3)取步骤(2)中划线平板上单菌落接种至MRS液体培养基中,置于37℃摇床,200rpm培养14h,得到菌液。
3、乳杆菌的分子生物学鉴定
取步骤(3)中1mL菌液用于菌种鉴定,6000r/min离心3min,弃上清后无菌水重悬,再次弃上清6000g离心3min,重复3次,弃去上清得菌体。菌体使用QIAamp DNA Stool MiniKit(Qiagen)试剂盒提取细菌基因组DNA,并使用通用引物27F/1492R对16S rDNA进行PCR扩增。PCR条件:第一步:94℃,5min第二步:94℃,30s;第三步:55℃,30s;第四步:72℃,2min,第五步:72℃,10min,第二到四步进行30个循环。PCR产物经核酸电泳分析确认后,送华大基因测序(如图1所示);将测序返回的27F和1492R序列拼接后上传到NCBI的BLAST(http://www.ncbi.nlm.nih.gov/BLAST)进行种属确认;比对结果发现菌株为罗伊氏乳杆菌株,命名为罗伊氏乳杆菌Lactobacillus reuteri strain SY523(LRSY523)。
4、革兰氏染色
挑取菌液,涂片,进行革兰氏染色,对菌株的染色情况和形态进行观察(如图2所示)。
5、菌株活化和保藏
将该罗伊氏乳杆菌在MRS液体培养基中活化,使用斜面保藏法在4℃进行保存,冷链运输至中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.22953。
6.菌液的制备
将罗伊氏乳杆菌LRSY523菌株接种至MRS液体培养基中,置于37℃摇床,200rpm培养14h,得到初步菌液。通过1100rpm/min离心,调整初步菌液中乳杆菌密度为1×109CFU/mL,分装后4℃保存。
实施例2:检测罗伊氏乳杆菌LRSY523在Caco-2和HT-29细胞黏附水平
采用活菌计数法检测罗伊氏乳杆菌LRSY523对Caco-2和HT-29细胞的黏附能力。
1、在24孔板中每孔1×105加入Caco-2或HT-29细胞培养;
2、待细胞生长至融合,在厌氧条件下与乳杆菌共培养4h(MOI=100感染复数:每个细胞感染细菌的单位数);
3、共培养后,去除培养基,用PBS缓慢洗涤细胞两次;
4、加入200μL的无菌水,溶解细胞10min,再加入800μL MRS培养基使细胞均质化;
5、将上述悬液梯度稀释(10-1-10-8),在乳杆菌MRS平板上均匀涂布;
6、在厌氧条件下培养,黏附在细胞表面的乳杆菌在平板上形成单克隆,统计菌落数,计算黏附的乳杆菌活菌数,除以加入总乳杆菌活菌数,得到黏附率,以此衡量乳杆菌在细胞表面的黏附程度。
结果表明:罗伊氏乳杆菌LRSY523在Caco-2和HT-29细胞上都有较强的黏附能力(如图3所示)。
实施例3:罗伊氏乳杆菌LRSY523预防高脂饮食小鼠肥胖和胰岛素抵抗
1、动物来源:雄性SPF级C57BL/6小鼠,5周龄;
2、饲养条件和饲料:温度为23±2℃,相对湿度为50-55%,室内灯光模拟昼夜,12小时明暗循环。除正常对照组外均为高脂饲料喂养,正常对照组为普通维持饲料喂养,持续喂养8周。LRSY523治疗组采用灌胃方法,给小鼠每日灌胃的菌液中LRSY523活菌数为1×109CFU/mL,每只小鼠灌胃200μL,对照和模型组灌胃同等体积MRS培养基,直到实验结束。
3、实验动物分组(见表1):
表1实验分组
4、实验进行每周对小鼠进行称重并记录,绘制折线图,发现LRSY523治疗后,高脂饮食小鼠体重显著下降(如图4所示)。血糖检测前24h断饲料并停止给药,只供应水,然后尾静脉取血。使用One-Touch血糖仪检测血糖;将血液置于无菌干燥管,室温静置半小时,打开离心机,3500rpm室温离心10分钟,收集上层微黄色液体得到血清,利用ELISA试剂盒(江苏酶免实业有限公司)检测小鼠血清胰岛素;胰岛素抵抗指数用空腹血糖和空腹胰岛素的积比22.5计算,发现LRSY523治疗后,高脂饮食小鼠胰岛素抵抗指数明显降低(如图5所示)。
实施例4:罗伊氏乳杆菌LRSY523缓解高脂饮食引起的小鼠肠道屏障渗漏
在体内评估肠道屏障通透性水平利用口服荧光素FITC-dextran(MW:4000Da)来测定。实验取血:口服FITC后4h,麻醉并固定小鼠,用镊子快速摘取眼球,并使血液从眼眶内流入无菌EP管中,于室温(37℃)静置半小时以上,打开离心机,3500rpm室温离心10min,收集上层微黄色液体即为血清;使用多功能酶标仪检测肠道通透性,选择激发波长为493nm,吸收波长为518nm,检测血清荧光强度;血清炎症因子检测使用ELISA试剂盒(上海西唐生物科技有限公司)检测各组小鼠血清中炎症因子TNFα和IL-1β的水平。
结果表明:与对照组相比,高脂饮食小鼠肠道通透性显著增加,血清中炎症因子TNFα和IL-1β表达的水平明显上升,即肠道屏障被破坏且血液循环有炎症发生。LRSY523治疗后,高脂饮食小鼠肠道通透性显著恢复,血清中炎症因子表达的水平明显降低(如图6和7所示)。
实施例5:qPCR检测罗伊氏乳杆菌LRSY523治疗后肠道紧密连接蛋白表达
小鼠肠组织总RNA的提取:麻醉处死小鼠后,切取适量结肠组织10mm左右,将切好的组织用PBS清洗三遍,浸泡入1mL的Trizol中,匀浆后分装于进口EP管中(不立即提RNA的保存于-80℃);加入200μL氯仿,剧烈振荡15s,静置5min,于4℃ 13,000rpm下离心15min,至分层(无色水相、酚-氯仿相、浅红色相)吸取上清存放在灭菌的EP管中;加入等体积的异丙醇,上下颠倒离心管,充分混匀,在室温环境下,静置10min;于4℃ 13,000rpm下离心10min,见白色沉淀;小心倒掉上清,沿管壁缓慢加入1mL 75%的乙醇(可轻轻上下颠倒);于4℃13,000rpm下离心15min,丢弃上清,并用枪头吸干净;吹干沉淀,直至变为透明;加入适量DEPC水,溶解沉淀,在57℃金属浴助溶10min;
RNA浓度检测:使用Nanodrop 2000测定RNA浓度。
RNA模板反转录:将上述方法提取的总RNA,用HiScriptⅡReverse Transcriptase反转录试剂盒,进行反转录,反应条件为:37℃ 15min,85℃ 5s,反应结束后,cDNA存放于-20℃长期保存。
实时定量PCR:设计并合成小鼠源的紧密连接蛋白的引物,使用ChamQ SYBR qPCRMaster Mix试剂盒进行实时定量PCR。
结果表明:与对照组相比,高脂饮食小鼠紧密连接蛋白Claudin-1、Occludin以及ZO-1表达均显著降低,罗伊氏乳杆菌LRSY523治疗可以明显增加高脂饮食小鼠肠道中紧密连接蛋白的表达水平(如图8所示)。
Claims (5)
1.一种罗伊氏乳杆菌(Lactobacillus reuteri)LRSY523,其特征在于,保藏编号为CGMCC No.22953。
2.一种罗伊氏乳杆菌菌剂的制备方法,其特征在于,将权利要求1所述的罗伊氏乳杆菌LRSY523菌株在MRS培养基中,于37℃条件下驯化和扩大培养,培养至活菌数大于1×109CFU/mL,菌液分装后置于4℃待用。
3.如权利要求1所述的罗伊氏乳杆菌LRSY523在制备改善肠道健康、缓解肥胖的功能性食品中的应用。
4.如权利要求1所述的罗伊氏乳杆菌LRSY523在制备改善肠道健康、缓解肥胖的微生物制剂中的应用。
5.如权利要求2所述方法制备的罗伊氏乳杆菌菌剂在制备改善肠道健康、缓解肥胖的功能性食品中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111221173.2A CN114107088B (zh) | 2021-10-20 | 2021-10-20 | 一种罗伊氏乳杆菌lrsy523及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111221173.2A CN114107088B (zh) | 2021-10-20 | 2021-10-20 | 一种罗伊氏乳杆菌lrsy523及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114107088A CN114107088A (zh) | 2022-03-01 |
CN114107088B true CN114107088B (zh) | 2023-07-21 |
Family
ID=80376271
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111221173.2A Active CN114107088B (zh) | 2021-10-20 | 2021-10-20 | 一种罗伊氏乳杆菌lrsy523及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114107088B (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114344344B (zh) * | 2022-01-05 | 2023-06-13 | 东北农业大学 | 罗伊氏乳杆菌在缓解高脂饮食诱导小鼠肥胖功能中的应用及含有罗伊氏乳杆菌的复合物 |
CN116948858A (zh) * | 2022-12-13 | 2023-10-27 | 广西爱生生命科技有限公司 | 一株具有抗肿瘤作用的罗伊氏乳杆菌a21099及其应用 |
CN116077536B (zh) * | 2023-03-17 | 2023-11-28 | 微康益生菌(苏州)股份有限公司 | 一种用于改善肥胖相关代谢疾病的微生态活菌制剂及其制备方法和应用 |
Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20040014058A (ko) * | 2002-08-09 | 2004-02-14 | (주)바이오니아 | 비만 또는 당뇨 예방 및 치료용 미생물 |
WO2010077682A2 (en) * | 2008-12-08 | 2010-07-08 | James Allen Lemke | Treatment of hiv and aids using probiotic lactobacillus reuteri |
KR20110009626A (ko) * | 2009-07-22 | 2011-01-28 | 대한민국(관리부서 : 농림수산식품부 국립수의과학검역원) | 신규 락토바실러스 루테리 및 이를 포함하는 사료첨가제 조성물 |
EP2457575A1 (en) * | 2010-11-29 | 2012-05-30 | Eurochit Danuta Kruszewska | New method for obtaining a Lactobacillus reuteri strain useful in medical and veterinary prohylaxis and treatment |
WO2013098032A1 (en) * | 2011-12-30 | 2013-07-04 | Nestec S.A. | Lactobacillus reuteri dsm 17938 for the development of the enteric nervous system |
CN106794207A (zh) * | 2014-10-29 | 2017-05-31 | 雀巢产品技术援助有限公司 | 使用罗伊氏乳杆菌在生命早期恢复微生物群生态失调 |
CN110205261A (zh) * | 2019-05-10 | 2019-09-06 | 湖南菲勒生物技术有限公司 | 一种母乳来源罗伊氏乳杆菌降脂及调节脂代谢节律的应用 |
CN110540950A (zh) * | 2019-08-27 | 2019-12-06 | 南京农业大学 | 一株罗伊氏乳杆菌22及其应用 |
CN111686133A (zh) * | 2020-07-24 | 2020-09-22 | 江南大学 | 杜氏杆菌在预防和改善肥胖及其相关疾病中的应用 |
CN112322527A (zh) * | 2020-11-03 | 2021-02-05 | 江南大学 | 一株可干预代谢综合征的罗伊氏乳杆菌及应用 |
CN113243446A (zh) * | 2021-05-20 | 2021-08-13 | 山西大学 | 一种谷糠蛋白提取物及其制备方法和应用 |
CN113403231A (zh) * | 2021-07-08 | 2021-09-17 | 江南大学 | 一株可干预代谢综合征的罗伊氏乳杆菌ccfm1178及应用 |
-
2021
- 2021-10-20 CN CN202111221173.2A patent/CN114107088B/zh active Active
Patent Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20040014058A (ko) * | 2002-08-09 | 2004-02-14 | (주)바이오니아 | 비만 또는 당뇨 예방 및 치료용 미생물 |
WO2010077682A2 (en) * | 2008-12-08 | 2010-07-08 | James Allen Lemke | Treatment of hiv and aids using probiotic lactobacillus reuteri |
KR20110009626A (ko) * | 2009-07-22 | 2011-01-28 | 대한민국(관리부서 : 농림수산식품부 국립수의과학검역원) | 신규 락토바실러스 루테리 및 이를 포함하는 사료첨가제 조성물 |
EP2457575A1 (en) * | 2010-11-29 | 2012-05-30 | Eurochit Danuta Kruszewska | New method for obtaining a Lactobacillus reuteri strain useful in medical and veterinary prohylaxis and treatment |
WO2013098032A1 (en) * | 2011-12-30 | 2013-07-04 | Nestec S.A. | Lactobacillus reuteri dsm 17938 for the development of the enteric nervous system |
CN106794207A (zh) * | 2014-10-29 | 2017-05-31 | 雀巢产品技术援助有限公司 | 使用罗伊氏乳杆菌在生命早期恢复微生物群生态失调 |
CN110205261A (zh) * | 2019-05-10 | 2019-09-06 | 湖南菲勒生物技术有限公司 | 一种母乳来源罗伊氏乳杆菌降脂及调节脂代谢节律的应用 |
CN110540950A (zh) * | 2019-08-27 | 2019-12-06 | 南京农业大学 | 一株罗伊氏乳杆菌22及其应用 |
CN111686133A (zh) * | 2020-07-24 | 2020-09-22 | 江南大学 | 杜氏杆菌在预防和改善肥胖及其相关疾病中的应用 |
CN112322527A (zh) * | 2020-11-03 | 2021-02-05 | 江南大学 | 一株可干预代谢综合征的罗伊氏乳杆菌及应用 |
CN113243446A (zh) * | 2021-05-20 | 2021-08-13 | 山西大学 | 一种谷糠蛋白提取物及其制备方法和应用 |
CN113403231A (zh) * | 2021-07-08 | 2021-09-17 | 江南大学 | 一株可干预代谢综合征的罗伊氏乳杆菌ccfm1178及应用 |
Non-Patent Citations (2)
Title |
---|
Lactobacillus reuteri prevents obesity by altering the gut microbiota and regulating bile acid metabolism in obese mice;Chenxi Zhang等;Food Funct;全文 * |
具有潜在改善炎症反应益生菌的筛选;崔鹏月等;食品工业科技;全文 * |
Also Published As
Publication number | Publication date |
---|---|
CN114107088A (zh) | 2022-03-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN114107088B (zh) | 一种罗伊氏乳杆菌lrsy523及其应用 | |
CN111235070B (zh) | 一株母乳婴儿源植物乳杆菌bf_15及其应用 | |
CN112458027B (zh) | 一株格氏乳杆菌及其缓解和治疗高尿酸血症的用途 | |
CN113604384B (zh) | 一种鼠李糖乳杆菌及其应用 | |
CN112813003B (zh) | 一株植物乳杆菌及其在制备缓解高血脂症引发的疾病的药物或食品中的应用 | |
CN114574390B (zh) | 一株缓解结肠炎的长双歧杆菌婴儿亚种及应用 | |
CN112574917B (zh) | 一株缓解高尿酸血症的鼠李糖乳杆菌ccfm1131 | |
CN114350577B (zh) | 一种改善便秘的动物双歧杆菌乳亚种BLa36及其培养方法与应用 | |
CN114181864B (zh) | 一种鼠李糖乳杆菌hf01及其应用 | |
CN109593678B (zh) | 长双歧杆菌yh295及其在制备降低腹型肥胖风险产品中的应用 | |
CN110023484B (zh) | 一种假小链状双歧杆菌及其培养方法和应用 | |
CN110023486B (zh) | 一种嗜酸乳杆菌及其培养方法和应用 | |
CN116769658A (zh) | 一种凝结芽孢杆菌ap15及其在肠道调节与尿酸、胆固醇代谢的应用 | |
CN113797232B (zh) | 具有缓解胰岛素抵抗功能的组合物及其应用 | |
CN114854638A (zh) | 一株高效表达腺苷脱氨酶缓解结肠炎副干酪乳杆菌 | |
CN116445356B (zh) | 一种调节肠道菌群及增强免疫力的动物双歧杆菌乳亚种ba67及其应用 | |
CN117264839A (zh) | 唾液联合乳杆菌mb1在制备美白和缓解痛风食品药品中的应用 | |
CN116218735A (zh) | 一种单形拟杆菌菌株及其培养方法和应用 | |
CN112980737B (zh) | 一株促进动物双歧杆菌增殖的青春双歧杆菌及其应用 | |
CN111700918B (zh) | 一种用于缓解酒精性肠道损伤的药品 | |
CN113249256A (zh) | 一株缓解雌激素相关代谢紊乱及肥胖的植物乳杆菌及应用 | |
CN113615776A (zh) | 一种改善家禽腺胃发育的乳酸菌复合微生态制剂及其应用 | |
CN112481172B (zh) | 鼠李糖乳杆菌ccfm1130及其缓解治疗痛风的应用 | |
CN115109718B (zh) | 一株屎肠球菌菌株及其用途 | |
CN114392281A (zh) | 抑制幽门螺杆菌的嗜酸乳杆菌La28在作为胃黏膜保护产品中的新应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |