CN115025131A - Application of lactobacillus reuteri E9 in preparing medicine for relieving anxiety and improving sleep - Google Patents
Application of lactobacillus reuteri E9 in preparing medicine for relieving anxiety and improving sleep Download PDFInfo
- Publication number
- CN115025131A CN115025131A CN202210625111.6A CN202210625111A CN115025131A CN 115025131 A CN115025131 A CN 115025131A CN 202210625111 A CN202210625111 A CN 202210625111A CN 115025131 A CN115025131 A CN 115025131A
- Authority
- CN
- China
- Prior art keywords
- lactobacillus reuteri
- improving sleep
- zebra fish
- time
- relieving anxiety
- 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.)
- Granted
Links
- 241000186604 Lactobacillus reuteri Species 0.000 title claims abstract description 47
- 229940001882 lactobacillus reuteri Drugs 0.000 title claims abstract description 47
- 208000019901 Anxiety disease Diseases 0.000 title claims abstract description 22
- 230000036506 anxiety Effects 0.000 title claims abstract description 22
- 239000003814 drug Substances 0.000 title claims abstract description 15
- 230000001580 bacterial effect Effects 0.000 claims abstract description 26
- 239000006228 supernatant Substances 0.000 claims abstract description 26
- 238000000855 fermentation Methods 0.000 claims abstract description 25
- 230000004151 fermentation Effects 0.000 claims abstract description 25
- 239000000725 suspension Substances 0.000 claims abstract description 25
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000004321 preservation Methods 0.000 claims description 6
- 235000021001 fermented dairy product Nutrition 0.000 claims description 3
- 235000013305 food Nutrition 0.000 claims description 3
- 241000252212 Danio rerio Species 0.000 abstract description 38
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 abstract description 20
- 206010022437 insomnia Diseases 0.000 abstract description 20
- 206010026749 Mania Diseases 0.000 abstract description 14
- 230000033001 locomotion Effects 0.000 abstract description 11
- 238000001727 in vivo Methods 0.000 abstract description 6
- 244000005700 microbiome Species 0.000 abstract description 4
- 230000004913 activation Effects 0.000 abstract description 3
- 230000000694 effects Effects 0.000 description 18
- 239000006041 probiotic Substances 0.000 description 13
- 235000018291 probiotics Nutrition 0.000 description 13
- CWRVKFFCRWGWCS-UHFFFAOYSA-N Pentrazole Chemical compound C1CCCCC2=NN=NN21 CWRVKFFCRWGWCS-UHFFFAOYSA-N 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 6
- 229960004535 oxazepam Drugs 0.000 description 6
- ADIMAYPTOBDMTL-UHFFFAOYSA-N oxazepam Chemical compound C12=CC(Cl)=CC=C2NC(=O)C(O)N=C1C1=CC=CC=C1 ADIMAYPTOBDMTL-UHFFFAOYSA-N 0.000 description 6
- 239000013641 positive control Substances 0.000 description 6
- 230000000529 probiotic effect Effects 0.000 description 6
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 230000006399 behavior Effects 0.000 description 4
- 230000007150 microbiota gut brain axis Effects 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000000147 hypnotic effect Effects 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000000284 resting effect Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 208000019116 sleep disease Diseases 0.000 description 3
- 208000000044 Amnesia Diseases 0.000 description 2
- 230000000949 anxiolytic effect Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 244000005709 gut microbiome Species 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000003860 sleep quality Effects 0.000 description 2
- 208000011117 substance-related disease Diseases 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 208000031091 Amnestic disease Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 208000019888 Circadian rhythm sleep disease Diseases 0.000 description 1
- 208000028698 Cognitive impairment Diseases 0.000 description 1
- 206010013654 Drug abuse Diseases 0.000 description 1
- 102000004300 GABA-A Receptors Human genes 0.000 description 1
- 108090000839 GABA-A Receptors Proteins 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010022998 Irritability Diseases 0.000 description 1
- 208000001456 Jet Lag Syndrome Diseases 0.000 description 1
- 208000026139 Memory disease Diseases 0.000 description 1
- 208000001145 Metabolic Syndrome Diseases 0.000 description 1
- 241000736262 Microbiota Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 208000010340 Sleep Deprivation Diseases 0.000 description 1
- 206010041349 Somnolence Diseases 0.000 description 1
- 201000000690 abdominal obesity-metabolic syndrome Diseases 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000006986 amnesia Effects 0.000 description 1
- 230000009604 anaerobic growth Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000000935 antidepressant agent Substances 0.000 description 1
- 229940005513 antidepressants Drugs 0.000 description 1
- 229940125715 antihistaminic agent Drugs 0.000 description 1
- 239000000739 antihistaminic agent Substances 0.000 description 1
- 239000000164 antipsychotic agent Substances 0.000 description 1
- 229940005529 antipsychotics Drugs 0.000 description 1
- 239000002249 anxiolytic agent Substances 0.000 description 1
- 229940005530 anxiolytics Drugs 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000002457 bidirectional effect Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 208000010877 cognitive disease Diseases 0.000 description 1
- 230000003920 cognitive function Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 206010013663 drug dependence Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007366 host health Effects 0.000 description 1
- 229940053999 hypnotics and sedatives melatonin receptor agonists Drugs 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- -1 irregular working hours Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 208000033915 jet lag type circadian rhythm sleep disease Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000006984 memory degeneration Effects 0.000 description 1
- 208000023060 memory loss Diseases 0.000 description 1
- 230000004630 mental health Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000000955 neuroendocrine Effects 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000000270 postfertilization Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108010008359 protein kinase C lambda Proteins 0.000 description 1
- 229940044601 receptor agonist Drugs 0.000 description 1
- 239000000018 receptor agonist Substances 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 208000020685 sleep-wake disease Diseases 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 230000001515 vagal effect Effects 0.000 description 1
- 108700024526 zebrafish sox32 Proteins 0.000 description 1
Images
Classifications
-
- 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
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/20—Hypnotics; Sedatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/22—Anxiolytics
-
- 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)
- Public Health (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Anesthesiology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an application of lactobacillus reuteri E9 in preparing a medicine for relieving anxiety and improving sleep, and belongs to the technical field of microorganisms. The invention discloses application of lactobacillus reuteri E9 in preparing a medicine for relieving anxiety and improving sleep, wherein the fermentation supernatant and bacterial suspension of lactobacillus reuteri E9 can obviously reduce the movement distance, mania time and activation time of zebra fish in an in-vivo insomnia model, obviously increase the rest time, and have the potential of relieving anxiety and improving sleep in vivo. The lactobacillus reuteri E9 disclosed by the invention has great potential application prospects in the aspects of relieving anxiety and improving sleep.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to application of lactobacillus reuteri E9 in preparing a medicine for relieving anxiety and improving sleep.
Background
Insomnia, including difficulty in initiating or maintaining sleep, is the most common sleep disorder in the population. Over 20% of adults suffer from chronic insomnia. Many factors that lead to chronic insomnia, such as shift work, irregular working hours, jet lag, and stress, are associated with modern lifestyles. Insufficient sleep can lead to memory loss, irritability, depression, inattention and fatigue. In addition to cognitive function, sleep disorders are also associated with metabolic syndrome, such as obesity, inflammation, diabetes and cardiovascular disease. Although there are many drugs available for the treatment of insomnia, including benzodiazepine receptor agonists, antihistamines, melatonin receptor agonists, anxiolytics, antidepressants, and antipsychotics, the potential problems of drug dependence and abuse are alarming. In addition, these drugs are often associated with side effects such as dizziness, headache, drowsiness, amnesia and cognitive impairment, even leading to an increased risk of death. Therefore, it is important to find a potential hypnotic adjunct to replace or reduce the use of these hypnotic drugs in order to have a safer choice to improve sleep quality and efficiency without significant adverse effects.
Probiotics were defined as living microorganisms that promote host health, as proposed by FAO/WHO in 2002. There is increasing evidence that long-term intake of probiotics can improve health without significant side effects. Thus, many potential functions of probiotics are proposed, even in the area of mental health. Microbiota-gut-brain axis (MGBA) specifically refers to the bidirectional signaling between the gastrointestinal tract and the central nervous system by gut microbes, which may be mediated by immunomodulation, neuroendocrine or vagal pathways. Probiotics that are beneficial to the mind or behavior through MGBA are called psychobiologies, and have a wide potential for future applications.
Studies have shown that sleep disorders lead to metabolic disturbances with slight changes in the intestinal microbiota. Recently, several studies have reported preliminary evidence that the gut microbiota affects sleep. Mice taken chronically with antibiotics exhibit sleep disordered architecture with changes in intestinal neurotransmitters. In addition, dietary probiotics have been found to be effective in improving sleep conditions affected by stress. These results indicate that remodeling microbiota may improve sleep quality in the host through MGBA.
However, probiotics are currently less studied and used in relieving anxiety and improving sleep. Probiotic strains used by domestic production enterprises are imported for a long time, and foreign strains are not necessarily suitable for the gastrointestinal physiological conditions of residents in China. In addition, the function of the probiotics lacks strong scientific research evidence, and the popularization of the probiotics and the products thereof is seriously influenced. Based on the method, aiming at the deep excavation of the functions of the strain resources, the novel probiotic strain which has independent intellectual property rights, has specific functional properties and is suitable for the physiological characteristics of Chinese people is screened out, and the method is particularly important for improving the core competitiveness of probiotic production enterprises in China and promoting the development of probiotic products in China.
Therefore, the application of lactobacillus reuteri E9 in preparing a medicine for relieving anxiety and improving sleep is a problem to be solved urgently by the technical personnel in the field.
Disclosure of Invention
In view of this, the invention provides an application of lactobacillus reuteri E9 in preparing a medicine for relieving anxiety and improving sleep.
In order to achieve the purpose, the invention adopts the following technical scheme:
the Lactobacillus reuteri E9 is applied to the preparation of the medicine for relieving anxiety and improving sleep, the preservation number of the Lactobacillus reuteri E9 is CGMCC No.21768, the CGMCC is used for short and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the institute of microbiology of China academy of sciences No.3 of West Lu 1 institute of North City, the Korean district, Beijing, the preservation date is 2021 year 01 month 29 days, and the Lactobacillus reuteri is classified and named.
The lactobacillus reuteri E9 can remarkably reduce the movement distance, the mania time and the activity time of zebra fish and remarkably increase the rest time in a PTZ induced zebra fish insomnia model, and shows good probiotic effects of relieving anxiety and improving sleep.
Further, the lactobacillus reuteri E9 is applied to preparing fermented dairy products and other food.
Further, the lactobacillus reuteri E9 is a bacterial suspension or a fermentation supernatant.
The strain capable of remarkably reducing the movement distance, the mania time and the activation time of the zebra fish and remarkably increasing the rest time in the in-vivo insomnia model comprises fermentation supernatant (extracellular secretion) and bacterial suspension (thallus) of the strain E9.
According to the technical scheme, compared with the prior art, the invention discloses the application of lactobacillus reuteri E9 in preparing the medicine for relieving anxiety and improving sleep, the lactobacillus reuteri E9 is obtained by separating and screening excrements of the elderly with long lives in Mei Ling county of Meizhou city, Guangdong province, the movement distance, the mania time and the activation time of zebra fish can be obviously reduced in a zebra fish insomnia model, and the rest time is obviously increased; has the potential of being applied to the in vivo anxiety relieving and sleep improving, and provides theoretical reference and guidance basis for developing the probiotic preparation for relieving the anxiety and improving the sleep by utilizing the lactobacillus reuteri E9.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a drawing showing the colony morphology of Lactobacillus reuteri E9 on MRS agar plates;
FIG. 2 is a graph showing the influence of fermentation supernatant and bacterial suspension of Lactobacillus reuteri E9 on the motion trail of a PTZ-induced zebra fish insomnia model;
FIG. 3 is a graph showing the effect of fermentation supernatant and bacterial suspension of Lactobacillus reuteri E9 on the movement distance of PTZ-induced zebrafish insomnia model;
FIG. 4 is a graph showing the effect of fermentation supernatant and bacterial suspension of Lactobacillus reuteri E9 on manic time in a PTZ-induced insomnia model of zebrafish;
FIG. 5 is a graph showing the effect of fermentation supernatant and bacterial suspension of Lactobacillus reuteri E9 on the activity time of PTZ-induced insomnia model of zebrafish;
FIG. 6 is a graph showing the effect of fermentation supernatant and bacterial suspension of Lactobacillus reuteri E9 on the resting time of PTZ-induced insomnia model of zebrafish.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 isolation, identification and preservation of Lactobacillus reuteri E9
(1) Separation: the method comprises the following steps of diluting the feces of the elderly with a gradient, respectively inoculating the feces into an MRS solid culture medium, a BS solid culture medium and a BHI solid culture medium, carrying out anaerobic culture at 37 ℃ for 48h, and selecting a single colony on a flat plate to carry out streaking separation to obtain a pure colony. Inoculating pure bacterial colonies on the plate into an MRS liquid culture medium, carrying out anaerobic culture at 37 ℃ for 12-16 h, adding 20% glycerol, and storing in a refrigerator at-80 ℃.
(2) Molecular biological identification of the strains: extracting genome DNA of the obtained strain, amplifying a 16S rDNA full-length fragment by utilizing 16S rDNA universal primers 27F and 1492R through a PCR technology, and then sequencing to identify the strain species.
Wherein, the primer sequences of the universal primers 27F and 1492R are as follows:
27F：5’-AGAGTTTGATCCTGGCTCAG-3’；SEQ ID NO.1；
1492R：5’-GGTTACCTTGTTACGACTT-3’；SEQ ID NO.2。
the experimental results are as follows: the strain screened from the excrement of the elderly people in Canine county of Meizhou city, Guangdong province is identified as lactobacillus reuteri through morphological observation and 16S rDNA identification, wherein the strain E9 is identified as lactobacillus reuteri, and the 16S rDNA sequence of the strain is shown as SEQ ID NO. 3.
GGCTCCCTCCATAAAGGTTAGGCCACCGACTTTGGGCGTTACAAAC TCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCAC CGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCG AGTTGCAGCCTACAGTCCGAACTGAGAACGGCTTTAAGAGATTAGCTT ACTCTCGCGAGCTTGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGT AGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCT CCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCA ACTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCAC GACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTCCCCGAA GGGAACGCCTTATCTCTAAGGTTAGCGCAAGATGTCAAGACCTGGTAA GGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCG GGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAG GCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTC CAACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAAT CCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGA CAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCG CTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCGCCCGGTTT CCGATGCACTTCTTCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGC AACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCA CCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGG TTGGATACCGTCACTGCGTGAACAGTTACTCTCACGCACGTTCTTCTCC AACAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGTGTTG CTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCC GTAGGAGTATGGACCGTGTCTCAGTTCCATTGTGGCCGATCAGTCTCTC AACTCGGCTATGCATCATCGCCTTGGTAAGCCGTTACCTTACCAACTAG CTAATGCACCGCAGGTCCATCCCAGAGTGATAGCCAAAGCCATCTTTCA AACAAAAGCCATGTGGCTTTTGTTGTTATGCGGTATTAGCATCTGTTTC CAAATGTTATCCCCCGCTCCGGGGCAGGTTACCTACGTGTTACTCACCC GTCCGCCACTCACTGGTGATCCATCGTCAATCAGGTGCAAGCACCATCA ATCAGTTGGGCCAGTGCGTACGACTGCA；SEQ ID NO.3。
A single colony of the strain E9 is inoculated on an MRS solid culture medium, the anaerobic growth is good at 37 ℃, and the colony is white, spherical, smooth in surface and regular in edge (figure 1). The strain E9 has been preserved in China general microbiological culture Collection center (CGMCC), CGMCC for short, the microbiological research institute of China academy of sciences No.3, Xilu No. 1, North Cheng, the area of Chaozhou, the date of preservation is 29 months at 2021 years, 01, and is classified and named as Lactobacillus reuteri, the number of preservation is CGMCC number 21768.
Example 2 preparation of fermentation supernatant (extracellular secretion) and bacterial suspension (thallus) of Lactobacillus reuteri E9
Activating and culturing Lactobacillus reuteri E9, inoculating in MRS liquid culture medium, culturing at 37 deg.C for 17 hr, and adjusting the concentration of fermentation bacteria to 1 × 10 7 Centrifuging at 4 deg.C and 6000r/min for 10min to obtain culture supernatant and thallus precipitate, and filtering the supernatant with 0.22 μm filter membrane to obtain fermentation supernatant (extracellular secretion); after the pellet was washed twice with PBS, the pellet was resuspended in PBS to adjust the cell concentration to 1X 10 7 CFU/mL to obtain bacterial suspension (thallus).
Example 3 sleep and anxiety improving effects of lactobacillus reuteri E9 on insomnia models of zebrafish oxazepam and Pentylenetetrazol (PTZ) were purchased from Sigma company, usa.
Healthy wild-type AB line zebrafish that developed to 6dpf (days post fertilization) were picked and placed in 96-well cell culture plates. The experiment is provided with a blank control group, a model group, a positive control group and a sample (bacterial suspension and fermentation supernatant) pretreatment group, wherein each group is provided with 20 multiple wells, and each well contains 1 fish. Adding PBS into a blank control group, adding PBS into a model group, adding oxazepam solution (30 mu g/mL) into a positive control group, adding lactobacillus reuteri E9 bacterial suspension into a bacterial suspension group, and adding lactobacillus reuteri E9 fermentation supernatant into a fermentation supernatant group, wherein 150 mu L of each hole is obtained; after incubation for 24h at 28 ℃, adding 50 μ L PBS into a blank control group, adding PTZ (60mM) into a model group, a positive control group, a bacterial suspension group and a fermentation supernatant group respectively, incubating for 2h at 28 ℃ with 20 μ L of each hole, placing a 96-hole cell culture plate in a zebra fish behavior analysis system (EthoVision XT 10.4, Nodasius information technology Limited liability company) for trajectory tracking, turning off a light source to enable the zebra fish to adapt to the environment for 5min, then turning on behavior trajectory analysis software, and collecting behavior trajectories of the zebra fish in a dark environment for 5 min. Counting the moving distance of the zebra fish; meanwhile, dividing the zebra fish into 3 motion states according to the swimming speed of the zebra fish: rest (<4mm/s), active (4-20mm/s), manic (>20mm/s), and count manic, active, and rest times of zebrafish.
The data were statistically processed using SPSS 19.0 software, and the experimental data were expressed as x + -SEM data and analyzed by one-way anova. Compared to the blank control group: ### p<0.005; compared to the model set: p<0.05， **P<0.01，***P<0.005。
The results are shown in FIGS. 2-6; as can be seen from fig. 2 and 3, the red area is the area covered by the moving path of the zebra fish; compared with a blank control group, the movement track of the zebra fish of the model group is increased, which indicates that the activity degree of the zebra fish of the model group is increased; meanwhile, compared with a blank control group (51.52 +/-5.39 cm), the moving distance (95.39 +/-4.60 cm) of the zebra fish in the model group is remarkably increased (p is less than 0.005), and the establishment of the zebra fish insomnia model is successful.
Compared with a model group, the motion trail of the positive control group (oxazepam) zebra fish is reduced, which indicates that the oxazepam can reduce the activity degree of the zebra fish in a PTZ induced zebra fish insomnia model; meanwhile, the moving distance of the zebra fish in the positive control group is 64.32 +/-2.30 cm, and the zebra fish is remarkably different from the model group (95.39 +/-4.60 cm) (P < 0.005). Therefore, oxazepam has an anxiolytic effect, consistent with clinical outcomes. Compared with the model group, the movement tracks of the zebra fish in the fermentation supernatant group and the bacterial suspension group of the lactobacillus reuteri E9 are reduced, which shows that the activity degree of the zebra fish in the PTZ induced zebra fish insomnia model can be reduced in the fermentation supernatant group and the bacterial suspension group of the lactobacillus reuteri E9; meanwhile, the moving distances of the fermented supernatant group of the lactobacillus reuteri E9 and the zebra fish in the bacterial suspension group are 72.66 +/-3.23 cm and 78.72 +/-3.68 cm respectively, and the difference is obvious (P is less than 0.05) compared with that of a model group (95.39 +/-4.60 cm). Therefore, the results show that the fermentation supernatant and the bacterial suspension of the lactobacillus reuteri E9 can obviously reduce the movement distance of the zebra fish in an in-vivo insomnia model, and show the effect of relieving anxiety.
As can be seen from FIG. 4, FIG. 5 and FIG. 6, compared with the blank control group (manic time: 15.65 + -0.85 s, active time: 27.32 + -1.79 s and resting time: 257.03 + -2.19 s), the manic time (62.84 + -2.85 s) and the active time (72.70 + -4.58 s) of the zebra fish in the model group are both significantly increased (p <0.005) and the resting time (164.46 + -4.90 s) is significantly decreased (p <0.005), which indicates that the establishment of the zebra fish insomnia model is successful.
Compared with the model group (mania time: 62.84 +/-2.85 s, active time: 72.70 +/-4.58 s and rest time: 164.46 +/-4.90 s), the positive control group zebra fish has the advantages that the mania time (25.64 +/-0.73 s) and the active time (36.08 +/-3.19 s) are both obviously reduced (p is less than 0.005) and the rest time (238.28 +/-3.44 s) is obviously increased (p is less than 0.005), which shows that oxazepam has the effects of relieving anxiety and hypnosis, and is consistent with clinical results. Compared with the model group (mania time: 62.84 +/-2.85 s, activity time: 72.70 +/-4.58 s and rest time: 164.46 +/-4.90 s), the fermentation supernatant group of the lactobacillus reuteri E9 and the mania time of the zebra fish in the bacterial suspension group (fermentation supernatant: 31.99 +/-2.48 s and bacterial suspension: 42.64 +/-2.36 s) are obviously reduced, the activity time (fermentation supernatant: 36.50 +/-3.67 s and bacterial suspension: 50.66 +/-3.78 s) are obviously reduced (p is less than 0.005), and the rest time (fermentation supernatant: 231.51 +/-4.09 s and bacterial suspension: 206.70 +/-4.03 s) is obviously increased (p is less than 0.005). Therefore, the results show that the bacterial suspension and the fermentation supernatant of the lactobacillus reuteri E9 can obviously reduce the manic time and the active time of the zebra fish and obviously increase the rest time in an in-vivo insomnia model, and show the effects of relieving anxiety and improving sleep.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> Guangdong Yinlanxin Biotech Co., Ltd, south China core medical science & technology Co., Ltd
Application of <120> lactobacillus reuteri E9 in preparing medicine for relieving anxiety and improving sleep
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 1
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 2
ggttaccttg ttacgactt 19
<210> 3
<211> 1441
<212> DNA
<213> Artificial Sequence
<400> 3
ggctccctcc ataaaggtta ggccaccgac tttgggcgtt acaaactccc atggtgtgac 60
gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc atgctgatcc gcgattacta 120
gcgattccga cttcgtgtag gcgagttgca gcctacagtc cgaactgaga acggctttaa 180
gagattagct tactctcgcg agcttgcgac tcgttgtacc gtccattgta gcacgtgtgt 240
agcccaggtc ataaggggca tgatgatctg acgtcgtccc caccttcctc cggtttgtca 300
ccggcagtct cactagagtg cccaacttaa tgctggcaac tagtaacaag ggttgcgctc 360
gttgcgggac ttaacccaac atctcacgac acgagctgac gacgaccatg caccacctgt 420
cattgcgtcc ccgaagggaa cgccttatct ctaaggttag cgcaagatgt caagacctgg 480
taaggttctt cgcgtagctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 540
tcaattcctt tgagtttcaa ccttgcggtc gtactcccca ggcggagtgc ttaatgcgtt 600
agctccggca ctgaagggcg gaaaccctcc aacacctagc actcatcgtt tacggcatgg 660
actaccaggg tatctaatcc tgttcgctac ccatgctttc gagcctcagc gtcagttgca 720
gaccagacag ccgccttcgc cactggtgtt cttccatata tctacgcatt ccaccgctac 780
acatggagtt ccactgtcct cttctgcact caagtcgccc ggtttccgat gcacttcttc 840
ggttaagccg aaggctttca catcagacct aagcaaccgc ctgcgctcgc tttacgccca 900
ataaatccgg ataacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc 960
gtgactttct ggttggatac cgtcactgcg tgaacagtta ctctcacgca cgttcttctc 1020
caacaacaga gctttacgag ccgaaaccct tcttcactca cgcggtgttg ctccatcagg 1080
cttgcgccca ttgtggaaga ttccctactg ctgcctcccg taggagtatg gaccgtgtct 1140
cagttccatt gtggccgatc agtctctcaa ctcggctatg catcatcgcc ttggtaagcc 1200
gttaccttac caactagcta atgcaccgca ggtccatccc agagtgatag ccaaagccat 1260
ctttcaaaca aaagccatgt ggcttttgtt gttatgcggt attagcatct gtttccaaat 1320
gttatccccc gctccggggc aggttaccta cgtgttactc acccgtccgc cactcactgg 1380
tgatccatcg tcaatcaggt gcaagcacca tcaatcagtt gggccagtgc gtacgactgc 1440
a 1441
Claims (4)
1. The application of lactobacillus reuteri E9 in preparing the medicine for relieving anxiety and improving sleep is characterized in that the preservation number of the lactobacillus reuteri E9 is CGMCC No. 21768.
2. The use of Lactobacillus reuteri E9 for the preparation of a medicament for the relief of anxiety and improvement of sleep as claimed in claim 1, wherein said Lactobacillus reuteri E9 is a bacterial suspension or fermentation supernatant.
3. Use of lactobacillus reuteri E9 according to claim 1 for the preparation of fermented dairy products and other food products.
4. Use of Lactobacillus reuteri E9 in the preparation of fermented dairy products and other food products according to claim 3, wherein the Lactobacillus reuteri E9 is a bacterial suspension or a fermentation supernatant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210625111.6A CN115025131B (en) | 2022-06-02 | 2022-06-02 | Application of lactobacillus reuteri E9 in preparation of anxiety-relieving and sleep-improving drugs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210625111.6A CN115025131B (en) | 2022-06-02 | 2022-06-02 | Application of lactobacillus reuteri E9 in preparation of anxiety-relieving and sleep-improving drugs |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115025131A true CN115025131A (en) | 2022-09-09 |
| CN115025131B CN115025131B (en) | 2023-06-30 |
Family
ID=83122114
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210625111.6A Active CN115025131B (en) | 2022-06-02 | 2022-06-02 | Application of lactobacillus reuteri E9 in preparation of anxiety-relieving and sleep-improving drugs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115025131B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110206649A1 (en) * | 2008-11-03 | 2011-08-25 | Nestec S.A. | Nutritional composition comprising probiotics and improving sleep patterns |
| CN107523526A (en) * | 2017-10-17 | 2017-12-29 | 无限极（中国）有限公司 | A kind of lactobacillus reuteri and application thereof |
-
2022
- 2022-06-02 CN CN202210625111.6A patent/CN115025131B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110206649A1 (en) * | 2008-11-03 | 2011-08-25 | Nestec S.A. | Nutritional composition comprising probiotics and improving sleep patterns |
| CN107523526A (en) * | 2017-10-17 | 2017-12-29 | 无限极（中国）有限公司 | A kind of lactobacillus reuteri and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| YOSHIKI MATSUDA 等: ""Ergothioneine, a metabolite of the gut bacterium Lactobacillus reuteri, protects against stress-induced sleep disturbances"", 《TRANSLATIONAL PSYCHIATRY》, vol. 10, no. 170, pages 1 - 11 * |
| 骆鹏飞 等: ""乳酸菌对精神性疾病作用的研究进展"", 《食品工业科技》, vol. 38, no. 16, pages 347 - 351 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115025131B (en) | 2023-06-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105132318B (en) | Lactobacillus plantarum Grx16 and its application | |
| CN111826315B (en) | Lactobacillus rhamnosus NX-2 and application thereof in preparation of uric acid reducing medicines | |
| CN109182165B (en) | Lactobacillus helveticus strain and application thereof in bee breeding process | |
| CN113564078B (en) | Bifidobacterium lactis BLa80 for reducing cholesterol and application thereof | |
| CN113846035B (en) | Lactobacillus salivarius capable of relieving enteritis and meningitis and promoting intestinal development and application thereof | |
| CN111676175B (en) | Bifidobacterium animalis NX-6 and application thereof in preparation of lipid-lowering and weight-losing medicines | |
| CN113862188B (en) | Lactobacillus gasseri LS03 and application thereof | |
| CN111733111A (en) | Lactobacillus plantarum NX-1 and application thereof in preparation of hypoglycemic drugs | |
| CN115786207A (en) | Lactobacillus salivarius and application thereof in preparation of medicines for treating and/or preventing diabetes | |
| CN109576175A (en) | Streptococcus thermophilus JMCC0022, its isolation and purification method and application | |
| CN109182164B (en) | Lactobacillus reuteri strain and application thereof in bee breeding process | |
| CN114480213A (en) | Lactobacillus fermentum E15 producing gamma-aminobutyric acid and application thereof in preparation of sleep improvement medicines | |
| CN112159784A (en) | Bifidobacterium longum NX-4 and application thereof in preparing medicament for treating and/or preventing allergic diseases | |
| CN116254190A (en) | Lactobacillus paracasei subspecies and application thereof | |
| CN113337428A (en) | Lactobacillus plantarum HNU082 and application thereof | |
| CN115772487A (en) | Lactobacillus reuteri C2 and application thereof in preparation of monoamine oxidase inhibiting medicines | |
| CN115025131A (en) | Application of lactobacillus reuteri E9 in preparing medicine for relieving anxiety and improving sleep | |
| CN113151070A (en) | Lactobacillus fermentum capable of improving relative abundance of Guttiferae in intestinal tract | |
| CN115992079A (en) | Lactobacillus fermentum E1 and application thereof in preparation of uric acid reducing drugs | |
| CN115025130A (en) | Application of lactobacillus reuteri E9 in preparing medicine for treating or preventing allergic diseases | |
| CN113980843B (en) | Application of lactobacillus acidophilus LA-03 in preparation of helicobacter pylori resistant medicines | |
| CN115838673A (en) | Lactobacillus salivarius LS08 and application thereof in preparation of hypoglycemic products | |
| CN115895971A (en) | Lactobacillus johnsonii LS04 and application thereof in preparation of lipid-lowering and weight-losing products | |
| CN116162569A (en) | Bifidobacterium animalis subspecies BL03 and application thereof | |
| CN113215045B (en) | Lactobacillus gasseri LGV03 and application thereof |
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 |