CN112980712A - 一种调节高原人群情绪及肠道菌群稳定的微生物组合物及其应用 - Google Patents
一种调节高原人群情绪及肠道菌群稳定的微生物组合物及其应用 Download PDFInfo
- Publication number
- CN112980712A CN112980712A CN201911286718.0A CN201911286718A CN112980712A CN 112980712 A CN112980712 A CN 112980712A CN 201911286718 A CN201911286718 A CN 201911286718A CN 112980712 A CN112980712 A CN 112980712A
- Authority
- CN
- China
- Prior art keywords
- plateau
- population
- microbial composition
- han
- han population
- 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.)
- Pending
Links
- 230000000813 microbial effect Effects 0.000 title claims abstract description 25
- 239000000203 mixture Substances 0.000 title claims abstract description 25
- 230000000968 intestinal effect Effects 0.000 title claims abstract description 15
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 9
- 230000008451 emotion Effects 0.000 title abstract description 10
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 16
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 16
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 16
- 241000588720 Escherichia fergusonii Species 0.000 claims abstract description 15
- 241000894007 species Species 0.000 claims description 11
- 241001134770 Bifidobacterium animalis Species 0.000 claims description 5
- 229940118852 bifidobacterium animalis Drugs 0.000 claims description 5
- 230000036651 mood Effects 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 4
- 239000002775 capsule Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 239000006190 sub-lingual tablet Substances 0.000 claims description 2
- 229940098466 sublingual tablet Drugs 0.000 claims description 2
- 239000000725 suspension Substances 0.000 claims description 2
- 239000006188 syrup Substances 0.000 claims description 2
- 235000020357 syrup Nutrition 0.000 claims description 2
- 239000003826 tablet Substances 0.000 claims description 2
- 241000901050 Bifidobacterium animalis subsp. lactis Species 0.000 abstract description 11
- 241000194032 Enterococcus faecalis Species 0.000 abstract description 11
- 229940032049 enterococcus faecalis Drugs 0.000 abstract description 10
- 238000011160 research Methods 0.000 abstract description 4
- 150000001875 compounds Chemical class 0.000 abstract description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 25
- 239000008103 glucose Substances 0.000 description 17
- 102000004420 Creatine Kinase Human genes 0.000 description 16
- 108010042126 Creatine kinase Proteins 0.000 description 16
- 239000008280 blood Substances 0.000 description 10
- 210000004369 blood Anatomy 0.000 description 10
- 210000001035 gastrointestinal tract Anatomy 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 4
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 238000012352 Spearman correlation analysis Methods 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 229940076279 serotonin Drugs 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 108010073254 Colicins Proteins 0.000 description 1
- 206010012374 Depressed mood Diseases 0.000 description 1
- 241000194033 Enterococcus Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 238000012313 Kruskal-Wallis test Methods 0.000 description 1
- 241000866650 Lactobacillus paraplantarum Species 0.000 description 1
- 108700005443 Microbial Genes Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 241000566145 Otus Species 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000006196 drop Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 208000018685 gastrointestinal system disease Diseases 0.000 description 1
- -1 glaze Substances 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 230000006996 mental state Effects 0.000 description 1
- 229940127249 oral antibiotic Drugs 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- 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
- 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
-
- 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
-
- 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
- A61K35/745—Bifidobacteria
-
- 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
- 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
- A23V2400/169—Plantarum
-
- 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
- A23V2400/51—Bifidobacterium
- A23V2400/515—Animalis
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明涉及一种调节高原人群情绪及肠道菌群稳定的微生物组合物及其应用。本发明研究了高原汉族人群和平原汉族人群肠道微生物菌群的差异，发现Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacterium animalis subsp.lactis和Enterococcus faecalis四种菌种在高原汉族人群和平原汉族人群之间差异显著，并且这四种菌种均与和人群情绪有关的临床指标密切相关，因此本发明提供了一种包含上述四种菌种的微生物复合物，通过施用该微生物复合物，可针对性的稳定高原汉族人群的情绪和肠道微生态环境。
Description
技术领域：
本发明涉及一种调节高原人群情绪及肠道菌群稳定的微生物组合物及其应用，属于生物医药技术领域。
背景技术：
人体肠道中存在大量的共生菌群，其数量超过1000万亿个，大约是人体细胞总数的10倍。同时肠道内微生物基因的数量约为300万个，大约是人类基因组基因数量的100多倍，如此海量的微生物形成了与人体密不可分的共生关系，能够帮助人群适应多变的环境。人体内微生态环境与人体健康密切相关，微生态环境的改变会影响人体的临床指标。维持人体内微生态环境的稳定有利于维护人体的身体健康。可以通过补充益生菌来帮助维护肠道微生态环境的稳定，预防和治疗疾病，提高人体机能。
文献“Colicins produced by the Escherichia fergusonii strains closelyresemble colicins encoded by Escherichia coli”报道Escherichia fergusonii菌种有助于分泌大肠杆菌素，而大肠杆菌素有助于调节人体肠道菌群数量。
迄今为止，尚未见肠道中同时补充Escherichia fergusonii,LactobacillusPlantarum,Bifidobacterium animalis subsp.lactis和Enterococcus faecalis四种菌种稳定高原人群情绪及肠道菌群的报道。
发明内容：
针对现有技术的不足，本发明提供了一种调节高原人群情绪及肠道菌群稳定的微生物组合物及其应用，本发明中的微生物组合物用于帮助高原人群稳定情绪和肠道的微生态环境。
术语说明：
Escherichia fergusonii：费格森埃希菌，
Lactobacillus plantarum：植物乳杆菌，
Bifidobacterium animalis subsp.Lactis：动物双歧杆菌乳亚种，
Enterococcus faecalis：粪肠球菌。
本发明的技术方案如下：
一种调节高原人群情绪及肠道菌群稳定的微生物组合物，所述微生物组合物包括安全有效剂量的Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacteriumanimalis subsp.lactis和Enterococcus faecalis四种菌种。
根据本发明优选的，所述微生物组合物还包括食品上或药学上可接受的载体。
根据本发明优选的，所述高原人群为汉族人群。
根据本发明优选的，所述微生物组合物为口服制剂。
根据本发明优选的，所述微生物组合物为粉末剂、散剂、片剂、糖衣剂、胶囊剂、颗粒剂、悬浮剂、溶液剂、糖浆剂、滴剂、舌下含片、或其组合。
上述微生物组合物在调节高原人群情绪及肠道菌群稳定中的应用。
根据本发明优选的，所述高原人群为汉族人群。
一种调节高原人群情绪及肠道菌群稳定的方法，所述方法为给高原人群施用上述微生物组合物。
根据本发明优选的，所述高原人群为汉族人群。
有益效果：
本发明研究了高原汉族人群和平原汉族人群肠道微生物菌群的差异，发现Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacterium animalissubsp.lactis和Enterococcus faecalis四种菌种在高原汉族人群和平原汉族人群之间差异显著，并且这四种菌种均与和人群情绪有关的临床指标密切相关，因此本发明提供了一种包含Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacterium animalissubsp.lactis和Enterococcus faecalis四种菌种的微生物复合物，通过施用该微生物复合物，可针对性的稳定高原汉族人群的情绪和肠道微生态环境。
附图说明
图1为三组人群肠道中Escherichia fergusonii菌种丰度的箱式图；图中，Han1000为平原汉族人群，H4A4为一周内的高原汉族人群，H4LB为两个月以上的高原汉族人群；
图2为三组人群肠道中Lactobacillus Paraplantarum菌种丰度的箱式图；图中，Han1000为平原汉族人群，H4A4为一周内的高原汉族人群，H4LB为两个月以上的高原汉族人群；
图3为三组人群肠道中Bifidobacterium animalis subsp.lactis菌种丰度的箱式图；图中，Han1000为平原汉族人群，H4A4为一周内的高原汉族人群，H4LB为两个月以上的高原汉族人群；
图4为三组人群肠道中Enterococcus faecalis菌种丰度的箱式图；图中，Han1000为平原汉族人群，H4A4为一周内的高原汉族人群，H4LB为两个月以上的高原汉族人群；
图5为三组人群临床指标CK(肌酸激酶)的箱式图；图中，Han1000为平原汉族人群，H4A4为一周内的高原汉族人群，H4LB为两个月以上的高原汉族人群；
图6为三组人群临床指标GLU(血糖)的箱式图；图中，Han1000为平原汉族人群，H4A4为一周内的高原汉族人群，H4LB为两个月以上的高原汉族人群。
具体实施方式：
下面结合实施例对本发明作进一步说明，但本发明的保护范围并不仅限于此。
本发明中涉及的微生物均为现有菌种，可从市场购得，不涉及微生物保藏。
研究对象：根据STROBE声明设计病例对照研究，纳入某年某月至某年某月在中国某部队的45个在海拔3500米高原以上服役一周以内的汉族男性士兵为实验组1，22个在海拔3500米高原以上服役两个月以上的汉族男性士兵为实验组2，根据实验组士兵年龄匹配96个平原汉族士兵进行对照。严格控制三组人群进行相同的中国部队混合饮食，保持相同的训练环境和训练强度，戒烟、酒，持续3个月。排除有慢性炎性疾病、口服抗生素、急性感染和胃肠道疾病的士兵。
每一个研究对象在纳入后均收到一个马桶粪便收集器，在排出大便后将大便装入粪便收集管，即刻保存在-20℃冰箱，次日将粪便标本转运至-80℃冰箱保存，统一进行检测。
实施例1平原汉族人群和高原汉族人群差异菌群的确定
1.1粪便样品DNA提取
吸取1000μL CTAB裂解液至2.0mL EP管中，加入溶菌酶，将500μL的粪便样品加入裂解液中，65℃水浴20min，期间颠倒混匀数次，以使样品充分裂解。离心取上清，加等体积苯酚(pH8.0)：氯仿：异戊醇(25：24：1体积比)混合液，颠倒混匀，12000rpm离心10min，取上清，加等体积氯仿：异戊醇(24:1体积比)混合液，颠倒混匀，12000rpm离心10min，吸取上清至1.5mL离心管中，加入0.8倍体积的异丙醇，上下摇晃，-20℃沉淀过夜。12000rpm离心10分钟，倒出液体，注意不要倒出沉淀。用1mL 75％乙醇洗涤沉淀2次，超净工作台吹干或者室温晾干。加入ddH2O溶解DNA样品，加RNase A 1μL消化RNA，37℃放置15min。之后利用琼脂糖凝胶电泳检测DNA的纯度和浓度，取适量样本DNA于离心管中，使用无菌水稀释样本至1ng/μL。
1.2PCR扩增、混样和纯化
以稀释至1ng/μL的基因组DNA为模板，根据测序区域16S V3-V4，选择使用带Barcode的特异引物515F和806R，New England Biolabs公司的
High-FidelityPCR Master Mix with GC Buffer，和高效高保真酶进行PCR，确保扩增效率和准确性。
PCR产物使用2％的琼脂糖凝胶进行电泳检测；根据PCR产物浓度进行等量混样，充分混匀后使用1×TAE、2％的琼脂糖凝胶电泳纯化PCR产物，切胶回收目标条带。产物纯化试剂盒使用的是Thermo Scientific公司GeneJET胶回收试剂盒回收产物。
1.3文库构建和上机测序
使用Thermo fisher公司的Ion Plus Fragment Library Kit 48rxns建库试剂盒进行文库的构建，构建好的文库经过Qubit定量和文库检测合格后，使用Thermo fisher的Ion S5TMXL进行上机测序。
1.4数据分析
1.4.1测序数据处理
使用Cutadapt(V1.9.1,http://cutadapt.readthedocs.io/en/stable/)先对reads进行低质量部分剪切，再根据Barcode从得到的reads中拆分出各样品数据，截去Barcode和引物序列初步质控得到原始数据，经过以上处理后得到的Reads需要进行去除嵌合体序列的处理，Reads序列通过与物种注释数据库进行比对，检测嵌合体序列，并最终去除其中的嵌合体序列，得到最终的有效数据。
1.4.2OTU聚类和物种注释
利用Uparse软件(Uparse v7.0.1001，http://www.drive5.com/uparse/)对所有样品的全部有效数据进行聚类，默认以97％的一致性将序列聚类成为OTUs(OperationalTaxonomic Units)，同时会选取OTUs的代表性序列，依据其算法原则，筛选的是OTUs中出现频数最高的序列作为OTUs的代表序列。对OTUs序列进行物种注释，用Usearch方法与rdp_16s_sp.udb数据库进行物种注释分析(设定阈值为0.8～1)，获得分类学信息并分别在属水平统计各样本的群落组成。
1.4.3菌属丰度计算和差异比较
用the Chao1估计量表示菌属丰度，绘制箱图对比三组人群中相关菌属/菌种丰度的差异，差异比较因为样本非正态分布采用Kruskal-Wallis检验，P＜0.05为差异有统计学意义。
1.5测序结果
平原汉族人群和高原汉族人群粪便样本中Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacterium animalis subsp.lactis和Enterococcusfaecalis 4种菌种丰度差异较大。
三组人群中Escherichia fergusonii和Bifidobacterium animalissubsp.lactis菌种的五分位数结果如图1和图2所示，比较中位数发现，Escherichiafergusonii和Bifidobacterium animalis subsp.lactis菌种都是在平原汉族人群中含量最高，上高原后降低，Kruskal-Wallis数值显示三组数据有明显差异；
三组人群中Lactobacillus Plantarum和Enterococcus faecalis菌种的五分位数结果如图3和图4所示，Lactobacillus Plantarum和Enterococcus faecalis菌种相对丰度的中位数在三组人群中没有明显差别，但是均值是在汉族平原人群中最高，上高原后显著降低，Kruskal-Wallis数值显示三组数据有明显差异。
实施例2平原汉族人群和高原汉族人群临床指标的确定
临床指标是由血液样本测得。血液样本是用EDTA-K2采集晨空腹静脉血(8mL)。血样以4000r/min离心10min，分离血浆，-80℃保存至301医院(北京，海拔约40米)测定。使用血液学分析仪(Roche-cobas6000，美国)测定76项临床试验项目，使用2毫升血浆。此外，1.5mL血浆用于11种酶联免疫吸附试验(ELISA)(Expand Bio,China)。临床数据共涵盖76项指标。
图5-图6显示了临床指标中CK(肌酸激酶)和GLU(血糖)的五分位数结果。根据临床研究，CK(肌酸激酶)与人体中的血清素的水平有关，而血清素的异常会导致抑郁症等精神疾病，而GLU(血糖)水平降低也会导致情绪低落。Kruskal-Wallis数值显示，CK(肌酸激酶)和GLU(血糖)水平在三组人群中均有明显差异。CK(肌酸激酶)在刚上高原的人群中出现降低，在上高原一段时间后的人群体内出现回升，可能与高原适应有关；上高原后的汉族人群体内的GLU(血糖)水平会下降，相对平原汉族人体中的血糖水平表现出不稳定。
实施例3菌群与临床指标之间的关系
采用R软件进行spearman关联分析，研究Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacterium animalis subsp.lactis和Enterococcusfaecalis4种菌种与临床指标之间的关联关系，研究结果如表1所示。
表1. 4种菌种与临床指标的spearman关联分析结果
| 菌种 | CK(肌酸激酶) | GLU(葡萄糖) |
| Escherichia fergusonii | 正相关 | 正相关 |
| Lactobacillus Plantarum | 正相关 | 正相关 |
| Bifidobacterium animalis subsp.lactis | 正相关 | 正相关 |
| Enterococcus faecalis | 正相关 | 正相关 |
以上结果显示，Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacterium animalis subsp.lactis和Enterococcus faecalis 4种菌种与CK(肌酸激酶)和GLU(葡萄糖)之间有较强关联关系，并且都呈正相关。因此补充这四种菌种可以稳定上高原后CK(肌酸激酶)和GLU(葡萄糖)水平，并帮助调节人体的精神状态。
结合实施例2和实施例3可知，Escherichia fergusonii,LactobacillusPlantarum,Bifidobacterium animalis subsp.lactis和Enterococcus faecalis四种菌种的组合物有助于稳定人体内肠道微生态环境稳定，并进一步帮助稳定临床指标，帮助高原汉族人群稳定情绪。
Claims (9)
1.一种调节高原人群情绪及肠道菌群稳定的微生物组合物，其特征在于，所述微生物组合物包括安全有效剂量的Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacterium animalis subsp.lactis和Enterococcus faecalis四种菌种。
2.如权利要求1所述的微生物组合物，其特征在于，所述微生物组合物还包括食品上或药学上可接受的载体。
3.如权利要求1所述的微生物组合物，其特征在于，所述高原人群为汉族人群。
4.如权利要求1所述的微生物组合物，其特征在于，所述微生物组合物为口服制剂。
5.如权利要求1所述的微生物组合物，其特征在于，所述微生物组合物为粉末剂、散剂、片剂、糖衣剂、胶囊剂、颗粒剂、悬浮剂、溶液剂、糖浆剂、滴剂、舌下含片、或其组合。
6.权利要求1所述的微生物组合物在调节高原人群情绪及肠道菌群稳定中的应用。
7.如权利要求6所述的应用，其特征在于，所述高原人群为汉族人群。
8.一种调节高原人群情绪及肠道菌群稳定的方法，其特征在于，所述方法为给高原人群施用权利要求1所述的微生物组合物。
9.如权利要求8所述的方法，其特征在于，所述高原人群为汉族人群。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911286718.0A CN112980712A (zh) | 2019-12-14 | 2019-12-14 | 一种调节高原人群情绪及肠道菌群稳定的微生物组合物及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911286718.0A CN112980712A (zh) | 2019-12-14 | 2019-12-14 | 一种调节高原人群情绪及肠道菌群稳定的微生物组合物及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112980712A true CN112980712A (zh) | 2021-06-18 |
Family
ID=76342610
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911286718.0A Pending CN112980712A (zh) | 2019-12-14 | 2019-12-14 | 一种调节高原人群情绪及肠道菌群稳定的微生物组合物及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112980712A (zh) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160348155A1 (en) * | 2013-09-06 | 2016-12-01 | Sofar S.P.A. | Method for evaluating the effects of a composition comprising microorganisms on intestinal microbiota |
| WO2018165764A1 (en) * | 2017-03-16 | 2018-09-20 | Microsintesis Inc. | Compositions and methods involving probiotic molecules |
| CN109652570A (zh) * | 2019-01-16 | 2019-04-19 | 中国人民解放军总医院 | 微生物在鉴别和/或区分不同种族个体中的应用 |
| CN109913525A (zh) * | 2019-02-13 | 2019-06-21 | 中国人民解放军总医院 | 丁酸弧菌属在鉴别和/或区分高原地区汉族人群和藏族人群中的应用 |
| CN110461337A (zh) * | 2017-02-23 | 2019-11-15 | 英特塞普特医药品公司 | 具有胆汁酸衍生物和微生物组的药物组合物及其用途 |
-
2019
- 2019-12-14 CN CN201911286718.0A patent/CN112980712A/zh active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160348155A1 (en) * | 2013-09-06 | 2016-12-01 | Sofar S.P.A. | Method for evaluating the effects of a composition comprising microorganisms on intestinal microbiota |
| CN110461337A (zh) * | 2017-02-23 | 2019-11-15 | 英特塞普特医药品公司 | 具有胆汁酸衍生物和微生物组的药物组合物及其用途 |
| WO2018165764A1 (en) * | 2017-03-16 | 2018-09-20 | Microsintesis Inc. | Compositions and methods involving probiotic molecules |
| CN110621689A (zh) * | 2017-03-16 | 2019-12-27 | 微合成有限公司 | 涉及益生菌分子的组合物和方法 |
| CN109652570A (zh) * | 2019-01-16 | 2019-04-19 | 中国人民解放军总医院 | 微生物在鉴别和/或区分不同种族个体中的应用 |
| CN109913525A (zh) * | 2019-02-13 | 2019-06-21 | 中国人民解放军总医院 | 丁酸弧菌属在鉴别和/或区分高原地区汉族人群和藏族人群中的应用 |
Non-Patent Citations (4)
| Title |
|---|
| DAVID J. NISBET等: "Effect of a Defined Continuous-Flow Derived Bacterial Culture and Dietary Lactose on Salmonella typhimurium Colonization in Broiler Chickens", 《AVIAN DISEASES》 * |
| W.GAASTRA等: "Escherichia fergusonii", 《VETERINARY MICROBIOLOGY》 * |
| 刘志鹏等: "肠道微生态与疾病", 《营养学报》 * |
| 张顺三等: "《临床细菌学检验》", 31 March 2009, 军事医学科学出版社 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhai et al. | Strain-specific anti-inflammatory properties of two Akkermansia muciniphila strains on chronic colitis in mice | |
| Wang et al. | High-salt diet has a certain impact on protein digestion and gut microbiota: a sequencing and proteome combined study | |
| Serafini et al. | Kefir fermented milk and kefiran promote growth of Bifidobacterium bifidum PRL2010 and modulate its gene expression | |
| Ding et al. | Geographical location specific composition of cultured microbiota and Lactobacillus occurrence in human breast milk in China | |
| CN113038957A (zh) | 微生物群 | |
| CN109913525B (zh) | 丁酸弧菌属在鉴别和/或区分高原地区汉族人群和藏族人群中的应用 | |
| US20230172999A1 (en) | Microbial compositions, strains and methods | |
| CN107937581B (zh) | 用于乳酸菌测序的扩增引物对、乳酸菌种属鉴定方法及应用 | |
| Gaspardo et al. | Influence of Lactobacillus kefiri on intestinal microbiota and fecal IgA content of healthy dogs | |
| Dang et al. | Interaction between Fumonisin B and the Intestinal Microflora of Pigs | |
| Zielińska et al. | Enumeration and identification of probiotic bacteria in food matrices | |
| Wu et al. | Functional genomic analyses of exopolysaccharide-producing Streptococcus thermophilus ASCC 1275 in response to milk fermentation conditions | |
| CN112980712A (zh) | 一种调节高原人群情绪及肠道菌群稳定的微生物组合物及其应用 | |
| WO2020150717A1 (en) | Monitoring tools and diagnostic methods for determining a canid's microbiome age status | |
| CN115029273B (zh) | 发酵乳杆菌ty-g03及其应用 | |
| CN113699088B (zh) | 一株溶藻弧菌pstS基因稳定沉默菌株及其应用 | |
| CN112957376A (zh) | 一种调节高原人群血糖的微生物组合物及其应用 | |
| CN109652493B (zh) | 颤杆菌克属在鉴别和/或区分不同民族个体中的应用 | |
| CN112029884B (zh) | 用于鉴定干酪乳杆菌类群的分子标记、检测引物和检测方法 | |
| CN112972500A (zh) | 一种提高高原人群运动潜能的微生物组合物及其应用 | |
| CN109825561B (zh) | 拟普雷沃菌属在鉴别和/或区分不同种族个体中的应用 | |
| Yi et al. | Compositional and functional features of the intestinal lactobacilli associated with different long-term diet types | |
| CN107446988B (zh) | 一种检测胃癌的生物标志物及其应用 | |
| Al Bayatee et al. | Phenetic and phylogenetic analysis of Kocuria palustris and Kocuria rhizophila strains isolated from healthy and thalassemia persons | |
| US12012589B2 (en) | Lactobacillus composition and use thereof for improving anxiety caused by antibiotics |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20210618 |
|
| WD01 | Invention patent application deemed withdrawn after publication |