CN106883999A - Bacillus acidi lactici NK318.1 and application thereof - Google Patents
Bacillus acidi lactici NK318.1 and application thereof Download PDFInfo
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- CN106883999A CN106883999A CN201710269312.6A CN201710269312A CN106883999A CN 106883999 A CN106883999 A CN 106883999A CN 201710269312 A CN201710269312 A CN 201710269312A CN 106883999 A CN106883999 A CN 106883999A
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- 239000003814 drug Substances 0.000 claims abstract description 7
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- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 5
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- 235000005911 diet Nutrition 0.000 claims description 3
- 230000036541 health Effects 0.000 claims description 3
- 206010022498 insulinoma Diseases 0.000 claims description 3
- 208000021255 pancreatic insulinoma Diseases 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 abstract description 22
- 241000186660 Lactobacillus Species 0.000 abstract description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 abstract description 4
- 229940039696 lactobacillus Drugs 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000007622 bioinformatic analysis Methods 0.000 abstract description 2
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- 239000004310 lactic acid Substances 0.000 abstract description 2
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- 241000192125 Firmicutes Species 0.000 abstract 1
- 101001096073 Mus musculus Regenerating islet-derived protein 3-gamma Proteins 0.000 abstract 1
- 238000012300 Sequence Analysis Methods 0.000 abstract 1
- 239000008272 agar Substances 0.000 abstract 1
- 238000012216 screening Methods 0.000 abstract 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 20
- 241000699660 Mus musculus Species 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- 210000002540 macrophage Anatomy 0.000 description 5
- 238000011830 transgenic mouse model Methods 0.000 description 5
- 206010061218 Inflammation Diseases 0.000 description 4
- 206010022489 Insulin Resistance Diseases 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 208000008589 Obesity Diseases 0.000 description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 description 4
- 230000004054 inflammatory process Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
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- 230000001580 bacterial effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000003304 gavage Methods 0.000 description 3
- 238000007446 glucose tolerance test Methods 0.000 description 3
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- 235000020824 obesity Nutrition 0.000 description 3
- 230000010412 perfusion Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 2
- 210000001789 adipocyte Anatomy 0.000 description 2
- 210000004982 adipose tissue macrophage Anatomy 0.000 description 2
- 208000016097 disease of metabolism Diseases 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 2
- 241000606125 Bacteroides Species 0.000 description 1
- 241000252983 Caecum Species 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 108010040721 Flagellin Proteins 0.000 description 1
- 241000186712 Lactobacillus animalis Species 0.000 description 1
- 241000186606 Lactobacillus gasseri Species 0.000 description 1
- 240000006024 Lactobacillus plantarum Species 0.000 description 1
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 1
- 241000186604 Lactobacillus reuteri Species 0.000 description 1
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 1
- 208000001145 Metabolic Syndrome Diseases 0.000 description 1
- 101100412401 Rattus norvegicus Reg3a gene Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000786103 Steatomys pratensis Species 0.000 description 1
- 201000000690 abdominal obesity-metabolic syndrome Diseases 0.000 description 1
- 230000001741 anti-phlogistic effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 210000004027 cell Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
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- 230000006020 chronic inflammation Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
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- 230000029087 digestion Effects 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 244000005709 gut microbiome Species 0.000 description 1
- 210000003405 ileum Anatomy 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 210000001630 jejunum Anatomy 0.000 description 1
- 229940072205 lactobacillus plantarum Drugs 0.000 description 1
- 229940001882 lactobacillus reuteri Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
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- 210000000664 rectum Anatomy 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
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- 239000007787 solid Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
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Classifications
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- 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
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- 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
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- 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
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
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Abstract
The present invention has carried out Primary Study using modern biology technology and bioinformatic analysis to the sequence and function of Bacillus acidi lactici NK318.1, it was found that gut flora is changed in mouse Reg3 γ models, Firmicutes are found by gut flora sequence analysis, lactobacillus genus bacteria showed increased, normal mouse is significantly lower than using the Mouse Weight more than lactic acid bacteria Selective agar medium screening discovery Bacillus acidi lactici NK318.1, show that the bacterium has the function of suppressing body weight increase, NK318.1 bacterium can be designed to the medicine that loses weight by future, with potential huge applications value.
Description
Technical field
The present invention relates to biology field, especially Bacillus acidi lactici NK318.1 and application thereof.
Background technology
With the continuous improvement of living standard, obesity annoyings the life of people more and more.Obesity belongs to a kind of slow
Property metabolic disease, is mainly characterized by the increase of fat cell, a large amount of accumulation of adipose tissue and the inflammation related to metabolism
The factor increases；The accumulation of wherein interior fat is the danger signal for developing into metabolic syndrome.Increasing research table
Bright, low chronic inflammation have impact on the pathology generating process of adiposis patient metabolic disorder.Master of the fat cell in aspect of inflammation
The infiltration with immunocytes such as macrophages is characterized as, causes the liter of proinflammatory inflammation factor and fatty acid concentration in the circulatory system
It is high.In turn, inflammation further promotes the deterioration of fat and its complication, induces insulin resistance and type ii diabetes.
In the subcutaneus adipose tissue of health, the ratio that adipose tissue macrophage (ATMs) is occupied is for about 5%.And in drink
Eat in obese patient's body of induction, the ratio of adipose tissue macrophage is increased to 20%-30%.According to cell surface molecule,
ATMs can be divided into M1 and the classes of M2 two.Macrophage in fatter body fat mass in human body tissue is generally M1 type macrophages, is formed
A kind of proinflammatory microenvironment.The macrophage of lath shows as M2 type macrophages, plays antiphlogistic effects.
In the last few years, numerous researchs point out metabolic disease and body gut flora composition, body immune system have it is close not
The association that can divide.
Enteron aisle is the vitals of digestive system, since stomach, is followed successively by duodenum, jejunum, ileum, caecum, knot
Intestines, rectum.Whole enteron aisle mainly performs digestion food, absorbs the function of nutrition.10 are there are about in mammalian gut13To 1014It is individual
Bacterium is deposited with host's symbiosis, and they regulate and control enteron aisle by metabolite (SCFA etc.) and surface antigen (flagellin etc.)
Immune system and metabolic system.Research finds have identical partially thin or obesity phenotype in same ovum and fraternal twin, and
Kinsfolk has sharing to flora.There is research to show that flora plays a role can be relatively independent of host gene type.Identical feeding
Under the conditions of supporting, the aseptic mouse body weight of the fat mouse excrement of inoculation increases the aseptic mouse apparently higher than the healthy mouse excrement of inoculation.This grinds
Study carefully and allow the deeper important function for recognizing that gut flora is fat to body of people.This group of researcher had found later, obese patient's
Intestinal microflora is significantly different with having for healthy person, and bacteroid is reduced in obese patient's body, and heavy wall increases by bacterium.Due to enterobacteriaceae
Group difference, obese patient perhaps can more effectively assimilate food in nutrition and stimulate fat generation.
The content of the invention
The technical problems to be solved by the invention are to provide Bacillus acidi lactici NK318.1.
Another technical problem to be solved by this invention is the new application for providing above-mentioned Bacillus acidi lactici NK318.1.
In order to solve the above technical problems, the technical scheme is that：
The gene order (V1-V6 sequences) of Bacillus acidi lactici NK318.1, its 16s rRNA is sequence table 400<1>Shown sequence
Row.
Above-mentioned Bacillus acidi lactici NK318.1 has the purposes for suppressing body weight increase.
Preferably, the purposes of above-mentioned Bacillus acidi lactici NK318.1, the Bacillus acidi lactici NK318.1 be used for prepare diet food,
Slimming health product or fat-reducing medicine.
Applications of the above-mentioned Bacillus acidi lactici NK318.1 in terms of diabetes model organisms are manufactured.
Applications of the above-mentioned Bacillus acidi lactici NK318.1 in terms of insulinoma medicine is prepared.
The beneficial effects of the invention are as follows：
The invention provides new applications of the Bacillus acidi lactici NK318.1 in terms of body weight increase is suppressed, for the medicine that loses weight
The research and development of thing have important application value.
Brief description of the drawings
Fig. 1 is systematic evolution tree collection of illustrative plates；
Fig. 2 is six kinds of difference of Bacillus acidi lactici content in Lactobacillus in two kinds of stool in mice；
Fig. 3 be Bacillus acidi lactici NK318.1 with control group simultaneously gavage and feeding high lipid food after glucose tolerance test and
Insulin sensitivity is tested；
Fig. 4 is Bacillus acidi lactici NK318.1, control bacterium reuteri and control group gavage simultaneously body after feeding high lipid food simultaneously
The comparison diagram of weight.
Specific embodiment
In order that those skilled in the art is better understood from technical scheme, it is below in conjunction with the accompanying drawings and specific real
Mode is applied to be described in further detail technical scheme of the present invention.
Embodiment 1
The determination of Bacillus acidi lactici NK318.1 bacterial strains
Chow diet is fed：Wild C57 mouse and each 6 of huREG3 γ transgenic mices, same sex of the same age；
High lipid food is fed：Wild C57 mouse and each 6 of huREG3 γ transgenic mices, same sex of the same age.
After feeding three months, take stool in mice and extract bacterial genomes DNA, analyzed by 16s rRNA, it is determined that
The ratio of Lactobacillus substantially increases in huREG3 γ transgenic mice gut floras.Reg3 γ mouse fresh excretas are taken,
Even spread on MRS lactic acid bacteria selective mediums, 37 DEG C of culture 24hrs.In more than 200 single bacterium colony of growth, there is super
The bacterium colony for crossing 35% is same bacterium.The picking single bacterium colony, by 16s rRNA sequencing identifications, this plant of bacterium sequence close to
Taiwanensis Bacillus acidi lacticis (Fig. 1), are named as Bacillus acidi lactici NK318.1 (sequence tables 400<1>Shown sequence).While C57/
The bacterium colony in BL6WT mouse only less than 5%.NK318.1 bacterium in the excrement of huREG3 γ transgenic mice groups are increased, together
When analyze other kind of Lactobacillus reuteri, Lactobacillus gasseri of Lactobacillus,
Lactobacillus animalis, Lactobacillus rhamnosus and Lactobacillusplantarum do not have
Significant change even reduces (Fig. 2).Immunofluorescence shown except in excrement, the intestinal mucosa mucus of huREG3 γ transgenic mices
NK318.1 bacterium are similarly increased in layer.
Embodiment 2
The preparation of Bacillus acidi lactici NK318.1 bacterium solutions
By Bacillus acidi lactici NK318.1 in MRS fluid nutrient mediums Zengjing Granule, 37 DEG C culture 24hrs, taken after gradient dilution
Appropriate bacterium solution coating MRS solid mediums, 37 DEG C of culture 24hrs, count the colony counts on flat board, according to count of bacteria result
The bacterium solution of culture is diluted to 1*109/100ul。
Embodiment 3
With Bacillus acidi lactici NK318.1 gavage wild type C57 mouse, and raise with high lipid food, observe control group and experimental group
The bodyweight difference of mouse.
Experimental group：6 wild type C57 mouse, every mouse perfusion 0.5ml contains 1*109Individual Bacillus acidi lactici NK318.1's
PBS；
Bacterial controls group：6 wild type C57 mouse, every mouse perfusion 0.5ml contains 1*109Individual Bacillus acidi lactici reuteri
PBS；
Negative control group：6 wild type C57 mouse, every mouse irrigates aseptic 0.5mlPBS buffer solutions.
One week 3 times perfusions, are replaced by high lipid food, and carry out within 3 days a body weight record after one week by mouse feed.
There is body weight attenuating in experimental mice, and glucose tolerance test and insulin sensitivity experiment are below control group (figure
3, Fig. 4).Experiment confirms that Bacillus acidi lactici NK318.1 has and suppresses the function that Mouse Weight increases.
To sum up, the function of Bacillus acidi lactici NK318.1 is ground using modern biology technology and bioinformatic analysis
Study carefully, it is found that Bacillus acidi lactici NK318.1 increases the increasing number for causing M2 phagocytes in each internal organs in mouse intestinal, and then find
Mouse Weight more than Bacillus acidi lactici NK318.1 is significantly lower than normal mouse, shows that the bacterium has the function of suppressing body weight increase, can
The medicine that NK318.1 bacterium are designed to lose weight, with potential huge applications value.Simultaneously as Bacillus acidi lactici
NK318.1 glucose tolerance tests be less than control group, it is possible to use Bacillus acidi lactici NK318.1 manufacture diabetes model organisms with
The modeling mode of existing destruction pancreas is substituted, because the experiment of Bacillus acidi lactici NK318.1 insulin sensitivities is less than control group, therefore
There is certain therapeutic action for insulinoma.
The above-mentioned detailed description carried out to Bacillus acidi lactici NK318.1 and application thereof with reference to specific embodiment, is explanation
Property rather than limited, several embodiments can be included according to limited scope, therefore of the invention overall not departing from
Changing and modifications under design, should belong within protection scope of the present invention.
SEQUENCE LISTING
<110>Nankai University
<120>Bacillus acidi lactici NK318.1 and application thereof
<130> 2017
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1019
<212> DNA
<213>Stool in mice
<220>
<221>Bacillus acidi lactici NK318.1
<222> (1)..(1019)
<223>16s rRNA V1-V6 sequences
<400> 1
caccttagac ggctggctcc aaaggttacc ccaccggctt tgggtgttac aaactctcat 60
ggtgtgacgg gcggtgtgta caaggcccgg gaacgtattc accgcggcat gctgatccgc 120
gattactagc gattccgact tcatgtaggc gagttgcagc ctacaatccg aactgagaac 180
ggctttaaga gatttgctaa acctcgcggt cttgcgactc gttgtaccgt ccattgtagc 240
acgtgtgtag cccaggtcat aaggggcatg atgatttgac gtcatcccca ccttcctccg 300
gtttgtcacc ggcagtcttg ctagagtgcc caacttaatg ctggcaacta acaataaggg 360
ttgcgctcgt tgcgggactt aacccaacat ctcacgacac gagctgacga caaccatgca 420
ccacctgtca ttttgtcccc gaagggaaag tcctatctct aggattgtca aaagatgtca 480
agacctggta aggttcttcg cgttgcttcg aattaaacca catgctccac cgcttgtgcg 540
ggcccccgtc aattcctttg agtttcaacc ttgcggtcgt actccccagg cggaatgctt 600
attgcgttag ctgcagcact gaagggcgga aaccctccaa cacttagcat tcatcgttta 660
cggcgtggac taccagggta tctaatcctg tttgctaccc acgctttcga acctcagcgt 720
cagttacaga ccagagagcc gctttcgcca ctggtgttct tccatatatc tacgcatttc 780
accgctacac atggagttcc actctcctct tctgcactca agtctcccag tttccaatgc 840
actactccgg ttaagccgaa aggctttcac atcagactta aaagaccgcc tgcgttccct 900
ttacgcccaa taaatcccgg ataacgcttg ccacctacgt attaccgcgg ctgctggcac 960
gtagttagcc gtgctttctg gttagatacc gtcgaaacgt gaacagttac tctcacgca 1019
Claims (5)
1. Bacillus acidi lactici NK318.1, it is characterised in that：The gene order of the Bacillus acidi lactici 16s rRNA is sequence table 400<1>
Shown sequence.
2. Bacillus acidi lactici NK318.1 described in claim 1 has the purposes for suppressing body weight increase.
3. the purposes of Bacillus acidi lactici NK318.1 according to claim 2, it is characterised in that：The Bacillus acidi lactici NK318.1 is used
In prepare diet food, slimming health product or fat-reducing medicine.
4. applications of the Bacillus acidi lactici NK318.1 described in claim 1 in terms of diabetes model organisms are manufactured.
5. applications of the Bacillus acidi lactici NK318.1 described in claim 1 in terms of insulinoma medicine is prepared.
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| CN201710269312.6A CN106883999A (en) | 2017-04-24 | 2017-04-24 | Bacillus acidi lactici NK318.1 and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710269312.6A CN106883999A (en) | 2017-04-24 | 2017-04-24 | Bacillus acidi lactici NK318.1 and application thereof |
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| Publication Number | Publication Date |
|---|---|
| CN106883999A true CN106883999A (en) | 2017-06-23 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6872565B2 (en) * | 2003-01-29 | 2005-03-29 | Biogaia Ab | Product containing Lactobacillus reuteri strain ATTC PTA-4965 or PTA-4964 for inhibiting bacteria causing dental caries |
| CN104921140A (en) * | 2015-06-23 | 2015-09-23 | 宣海燕 | Weight losing and body shaping nutrition food |
| CN106148254A (en) * | 2015-03-26 | 2016-11-23 | 南京华贞生物医药科技有限公司 | A kind of transformant for Weight-reducing and lipid-lowering and construction method thereof and application |
-
2017
- 2017-04-24 CN CN201710269312.6A patent/CN106883999A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6872565B2 (en) * | 2003-01-29 | 2005-03-29 | Biogaia Ab | Product containing Lactobacillus reuteri strain ATTC PTA-4965 or PTA-4964 for inhibiting bacteria causing dental caries |
| CN106148254A (en) * | 2015-03-26 | 2016-11-23 | 南京华贞生物医药科技有限公司 | A kind of transformant for Weight-reducing and lipid-lowering and construction method thereof and application |
| CN104921140A (en) * | 2015-06-23 | 2015-09-23 | 宣海燕 | Weight losing and body shaping nutrition food |
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