CN107502576A - A kind of Lactobacillus pentosus and its application in terms of salmonella is suppressed - Google Patents
A kind of Lactobacillus pentosus and its application in terms of salmonella is suppressed Download PDFInfo
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- CN107502576A CN107502576A CN201710868073.6A CN201710868073A CN107502576A CN 107502576 A CN107502576 A CN 107502576A CN 201710868073 A CN201710868073 A CN 201710868073A CN 107502576 A CN107502576 A CN 107502576A
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- salmonella
- lactobacillus pentosus
- lactobacillus
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- pentosus
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- 241000607142 Salmonella Species 0.000 title claims abstract description 90
- 241000186684 Lactobacillus pentosus Species 0.000 title claims abstract description 75
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 11
- 241000894006 Bacteria Species 0.000 claims description 24
- 239000007787 solid Substances 0.000 claims description 8
- 241000186660 Lactobacillus Species 0.000 claims description 5
- 229940039696 lactobacillus Drugs 0.000 claims description 5
- 239000002207 metabolite Substances 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 244000005700 microbiome Species 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 3
- 239000000853 adhesive Substances 0.000 claims description 2
- 230000001070 adhesive effect Effects 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000007884 disintegrant Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims description 2
- 235000013305 food Nutrition 0.000 claims description 2
- 230000036541 health Effects 0.000 claims description 2
- 239000000314 lubricant Substances 0.000 claims description 2
- 239000000047 product Substances 0.000 claims description 2
- 239000000080 wetting agent Substances 0.000 claims description 2
- 239000000969 carrier Substances 0.000 claims 1
- 241000699666 Mus <mouse, genus> Species 0.000 abstract description 31
- 230000012010 growth Effects 0.000 abstract description 4
- 210000003736 gastrointestinal content Anatomy 0.000 abstract description 3
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 210000000056 organ Anatomy 0.000 abstract 1
- 238000003304 gavage Methods 0.000 description 28
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 21
- 206010039438 Salmonella Infections Diseases 0.000 description 9
- 239000012228 culture supernatant Substances 0.000 description 9
- 210000004185 liver Anatomy 0.000 description 9
- 206010039447 salmonellosis Diseases 0.000 description 9
- 210000000952 spleen Anatomy 0.000 description 8
- 238000003501 co-culture Methods 0.000 description 7
- 230000000112 colonic effect Effects 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 230000004913 activation Effects 0.000 description 5
- 238000001994 activation Methods 0.000 description 5
- 230000003115 biocidal effect Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- 241001572175 Gaza Species 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 230000002265 prevention Effects 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 241000252983 Caecum Species 0.000 description 3
- 210000004534 cecum Anatomy 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 229960000448 lactic acid Drugs 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 210000000481 breast Anatomy 0.000 description 2
- 230000020411 cell activation Effects 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 210000003405 ileum Anatomy 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 230000009545 invasion Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 206010002383 Angina Pectoris Diseases 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 101100028791 Caenorhabditis elegans pbs-5 gene Proteins 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- 241000917009 Lactobacillus rhamnosus GG Species 0.000 description 1
- 241000270322 Lepidosauria Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 238000001530 Raman microscopy Methods 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 241001607429 Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344 Species 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 229940040526 anhydrous sodium acetate Drugs 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- CEAZRRDELHUEMR-UHFFFAOYSA-N gentamicin Chemical class O1C(C(C)NC)CCC(N)C1OC1C(O)C(OC2C(C(NC)C(C)(O)CO2)O)C(N)CC1N CEAZRRDELHUEMR-UHFFFAOYSA-N 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 150000002972 pentoses Chemical class 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
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
- C12N1/205—Bacterial isolates
-
- 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
- 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
- A23V2400/167—Pentosus
-
- 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
-
- 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
Abstract
A kind of application the invention discloses Lactobacillus pentosus and its in terms of salmonella is suppressed, belongs to microbial technology field.The invention provides a kind of pharmaceutical composition, described pharmaceutical composition contains Lactobacillus pentosus (Lactobacillus pentosus) AT6 and its active component, can be used in suppressing Salmonella growth；Lactobacillus pentosus AT6 can reduce salmonella to the sticking of the cells of HT 29, attack, reduce carrying capacity of the salmonella in mouse intestinal content and organ, and slow down salmonellal dead mouse.
Description
Technical field
A kind of application the present invention relates to Lactobacillus pentosus and its in terms of salmonella is suppressed, belong to microbial technique neck
Domain.
Background technology
Salmonellosis (Salmonellosis) is that have the disease of various animals and people caused by Salmonella bacteria
General name.The host of Salmonella includes mammality, birds, reptile class, fish and people etc., and host is extensive, can trigger various each
The disease of sample.The order of severity of disease is from chronic to acute, and from local infection to systemic sepsis, serious causing is dead
Die.Salmonellosis is as global disease, and location distribution is quite varied, and there is the more salmonella state of an illness countries in the world
Occur, bring serious economic loss and potential safety hazard.
Acute infection case is often more common in mankind's salmonella infection, shows as angina, diarrhoea, Nausea and vomiting and hair
Heat etc..Its occurring degree is relevant with Salmonella serogroup and infective dose.
Have at present it is a variety of be used for treat medicine such as antibiotic of pathogenic infection etc., can effectively slow down salmonella etc. cause a disease
The metainfective disease symptom of bacterium.Also some Chinese medicine preparations effectively alleviate pathogenic infection symptom.But antibiotic is being controlled
It can also kill substantial amounts of commensal gut bacterium while treating pathogenic infection, cause enteric flora disturbance, and the use of antibiotic
Not only produce side effect in itself to people, can also be flowed into environment and cause antibiotic pressure increase in environment, destroy the ecological balance.
And the action effect for the treatment of by Chinese herbs is often undesirable, and the problem of hysteresis quality be present as antibiosis extract for treating.
The content of the invention
The present invention first purpose be to provide a kind of Lactobacillus pentosus (Lactobacilluspentosus), in
On March 29th, 2017 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is
CGMCCNo.13956, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
Second object of the present invention is to provide a kind of pharmaceutical composition, and described pharmaceutical composition contains the pentose breast bar
Bacterium (Lactobacilluspentosus) and/or its metabolite.
In one embodiment of the invention, content >=1 × 10 of the Lactobacillus pentosus8CFU/mL。
In one embodiment of the invention, content >=1 × 10 of the Lactobacillus pentosus8CFU/g。
In one embodiment of the invention, the metabolite is that Lactobacillus pentosus is seeded in MRS culture mediums,
The supernatant of collected after centrifugation.
In one embodiment of the invention, described pharmaceutical composition by Lactobacillus pentosus microbial inoculum with can pharmaceutically connect
The carrier composition received.
In one embodiment of the invention, the pharmaceutically acceptable carrier is one or more selected from pharmaceutically
Usually used filler, wetting agent, disintegrant, adhesive, the carrier of lubricant or flavouring.
Third object of the present invention is to provide application of the Lactobacillus pentosus in terms of Salmonella organisms amount is suppressed.
In one embodiment of the invention, the Lactobacillus pentosus is seeded in MRS culture mediums, 35~37 DEG C of cultures
After 14~36h, add into the liquid containing salmonella, solid or semisolid.
In one embodiment of the invention, the bacterium of the Lactobacillus pentosus and salmonella is dense than being 1:1~100.
The present invention also provides food or health products prepared by the Lactobacillus pentosus.
Beneficial effect：The Lactobacillus pentosus AT6 culture supernatants of the present invention co-culture with salmonella, can suppress Salmonella
Bacteria growing, its biomass is set to reduce by 82.9%；Lactobacillus pentosus AT6 can reduce salmonella to the sticking of HT-29 cells, invade
Attack, its adherence rate can be made to reduce by 75.0%, invasion and attack rate reduces by 89.3%.Intake Lactobacillus pentosus AT6 makes in mouse liver, spleen
Salmonella recall rate is down to 0%, 0% respectively from 60%, 50%, the salmonella quantity in ileum, caecum, colonic contents
71.0%, 67.7%, 84.3% is reduced respectively.Lactobacillus pentosus AT6 can slow down salmonellal dead mouse, 15 days
When mouse survival rate improved from 40% to 65%.
Biomaterial preservation
Lactobacillus pentosus (Lactobacilluspentosus) provided by the present invention, in preservation on March 29 in 2017
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCCNo.13956, preservation address
For Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
Brief description of the drawings
Fig. 1 be Lactobacillus pentosus AT6 culture supernatants in LB culture mediums to the inhibition figure of salmonella；Wherein,
AT6 groups are that Lactobacillus pentosus AT6 co-cultures with salmonella；LGG groups are that Lactobacillus rhamnosus LGG co-cultures with salmonella；
MRS groups are blank control group, and MRS culture mediums co-culture with salmonella；HCl-3.81 groups are that pH to 3.81 MRS is adjusted with hydrochloric acid
Co-cultured with salmonella；LA-3.81 groups are to adjust pH to 3.81 MRS and salmonella to co-culture with DL-LACTIC ACID；
Fig. 2 is that Lactobacillus pentosus AT6 suppresses the effect that salmonella sticks HT-29 cells；Wherein, CON groups are control group,
1640 blank cultures are added in six orifice plates, add 1.0 × 10 afterwards8CFU salmonellas；LGG groups represent to add into six orifice plates
1.0×108CFU Lactobacillus rhamnosus LGG, 1.0 × 10 are added afterwards8CFU salmonellas；AT6 groups represent to add into six orifice plates
1.0×108CFU Lactobacillus pentosus AT6,1.0 × 10 are added afterwards8CFU salmonellas；
Fig. 3 is the effect that Lactobacillus pentosus AT6 suppresses salmonella invasion and attack HT-29 cells；Wherein, CON groups are control group,
1640 blank cultures are added in six orifice plates, add 1.0 × 10 afterwards8CFU salmonellas；LGG groups represent to add into six orifice plates
1.0×108CFU Lactobacillus rhamnosus LGG, 1.0 × 10 are added afterwards8CFU salmonellas；AT6 groups represent to add into six orifice plates
1.0×108CFU Lactobacillus pentosus AT6,1.0 × 10 are added afterwards8CFU salmonellas；
Fig. 4 is influences of the Lactobacillus pentosus AT6 to salmonella carrying capacity in salmonella infection mouse liver and spleen；Its
In, liver-con represents model group liver salmonella carrying capacity；Liver-LGG represents Lactobacillus rhamnosus LGG treatment group livers
Salmonella carrying capacity；Liver-AT6 represents Lactobacillus pentosus AT6 treatment group liver salmonella carrying capacity；Spleen-con is represented
Model group spleen salmonella carrying capacity；Spleen-LGG represents Lactobacillus rhamnosus LGG treatment group spleen salmonella carrying capacity；
Spleen-AT6 represents Lactobacillus pentosus AT6 treatment group spleen salmonella carrying capacity；
Fig. 5 is influences of the Lactobacillus pentosus AT6 to salmonella carrying capacity in salmonella infection mouse intestinal content；Its
In, Ileum-con represents salmonella carrying capacity in model group ileal contents；Ileum-LGG is represented at Lactobacillus rhamnosus LGG
Salmonella carrying capacity in reason group ileal contents；Ileum-AT6 represents sramana in Lactobacillus pentosus AT6 treatment group ileal contents
Salmonella carrying capacity；Cecum-con represents salmonella carrying capacity in model group cecal content；Cecum-LGG represents rhamnose breast bar
Salmonella carrying capacity in bacterium LGG treatment group cecal contents；Cecum-AT6 represents Lactobacillus pentosus AT6 treatment group caecal contents
Salmonella carrying capacity in thing；Colon-con represents salmonella carrying capacity in model group colonic contents；Colon-LGG represents mouse
Salmonella carrying capacity in Lee's sugar lactobacillus LGG treatment group colonic contentses；Colon-AT6 represents Lactobacillus pentosus AT6 treatment groups
Salmonella carrying capacity in colonic contents；
Fig. 6 is that Lactobacillus pentosus AT6 intake improves the survival rate of mouse after Salmonella Typhimurium Infection；Wherein,
Control group gavages PBS；Model group gavage salmonellas；The first gavage Lactobacillus rhamnosus LGG of LGG groups, rear gavage Salmonella
Bacterium；The first gavage Lactobacillus pentosus of AT6 groups, rear gavage salmonella.
Embodiment
MRS fluid nutrient mediums (per 1L)：Tryptone 10g, beef extract 10g, dusty yeast 5g, glucose 20g, citric acid
The ammonium 2g of hydrogen two, anhydrous sodium acetate 5g, dipotassium hydrogen phosphate 2g, epsom salt 0.5g, manganese sulfate monohydrate 0.25g, Tween 80 1mL,
1000mL is added water to, pH is adjusted to 6.2-6.4,115 DEG C, 20min moist heat sterilizations.
MRS solid mediums (per L)：Agar powder 15g/L is added in fluid nutrient medium.
LB culture mediums (per L)：Dusty yeast 5.0g, tryptone 10.0g, sodium chloride 5.0g.Add water 1L, 121 DEG C of 15min wet
Heat sterilization.
Actication of culture：With oese from a small amount of bacterium solution of picking in tube is protected, rule in MRS solid plates.It is placed on 37 DEG C
Cultivated in constant incubator, until growing single bacterium colony.
Spawn incubation：Dropped down onto with the single bacterium on oese picking MRS solid mediums in MRS fluid nutrient mediums, 37 DEG C quiet
Put culture 14-18h.Afterwards, 1% inoculum concentration is transferred in liquid MRS culture mediums, 37 DEG C of quiescent culture 14-18h.Activation every time,
All it is inoculated with 1% inoculum concentration, 37 degrees Celsius of culture 14-18h.
Bacterium carrying capacity determines：Mouse anesthesia, after putting to death, intestines, caecum, colonic contents are fetched, is weighed, gradient dilution, counted；
Liver, spleen are taken, is homogenized, gradient dilution, is counted.
Method of counting：Sample is coated with the maconkey agar flat board containing 50mg/L streptomysins, and 37 DEG C are cultivated 12h or so
To growing bacterium colony.
Lactobacillus pentosus (Lactobacilluspentosus) provided by the present invention is abbreviated as penta in the examples below
Sugared lactobacillus AT6.Suppress the document report of salmonella on Lactobacillus pentosus due to having no at present, so that Salmonella can be suppressed
The Lactobacillus rhamnosus of bacterium is control.
Embodiment 1：Lactobacillus pentosus AT6 culture supernatants co-culture with salmonella
(1) preparation of Lactobacillus pentosus AT6 culture supernatants
After Lactobacillus pentosus AT6, Lactobacillus rhamnosus LGG (i.e. Lactobacillus rhamnosus ATCC53103) passage, 37 DEG C of trainings
Support 18h after centrifuge, survey culture supernatant pH, while with hydrochloric acid or DL-LACTIC ACID regulation MRS fluid nutrient mediums to AT6 cultures
The identical pH of supernatant (pH3.81) is as a control group.Culture supernatant and the sterilised membrane filter that control group aperture is 0.22 micron filter
It is stand-by.
(2) preparation of Salmonella cultures
Salmonella typhimurium SL1344 culture medium is LB culture mediums, and salmonella is with 2% (2mL/100mL) inoculation
After amount carries out activation culture in LB culture mediums, 37 DEG C of 225rpm concussion and cultivates 12h.
(3) Lactobacillus pentosus AT6 culture supernatants co-culture with salmonella
50 μ L Lactobacillus pentosus AT6, Lactobacillus rhamnosus LGG culture are separately added into 5mLLB fluid nutrient mediums
Supernatant or control group MRS culture mediums, after be separately added into 50 μ L2.0 × 109CFU/mL salmonella is co-cultured.37 DEG C quiet
Culture 24h is put, the OD of coculture is surveyed in different time600。
As a result it is as shown in Figure 1：When cultivating 4h, addition AT6 culture supernatants, LGG culture supernatants, MRS culture mediums, use
MRSs of the HCL regulation pH for 3.81, the MRS groups with DL-LACTIC ACID tune pH to 3.81 OD600Respectively 0.026,0.037,0.574,
0.278、0.042；AT6, LGG, MRS, HCl-3.81, LA-3.81 group OD during 12h600Respectively 0.038,0.178,0.705,
0.525、0.233；AT6, LGG, MRS, HCl-3.81, LA-3.81 group OD when cultivating 24h600Respectively 0.133,0.315,
0.781st, 0.603,0.246, illustrate that Lactobacillus pentosus AT6 can significantly inhibit the growth of salmonella, its inhibition is even
Better than the effect of organic acid.
Embodiment 2：Lactobacillus pentosus AT6 sticks the inhibitory action of HT-29 cells to salmonella
(1) by after Lactobacillus pentosus AT6 and Lactobacillus rhamnosus LGG passages, cultivated in MRS culture mediums in 37 DEG C
Centrifuge after 18h, washed three times with PBS, and thalline is resuspended to 1.0 × 10 with 1640 culture mediums (buying from Gibco)8CFU/mL。
(2) after salmonella carries out activation culture in LB culture mediums, quiescent culture 12h, washed three times, be used in combination with PBS
Thalline is resuspended to 1.0 × 10 1640 culture mediums8CFU/mL。
(3) preparation of HT-29 cells：Culture medium needed for HT-29 growths is 1640 culture mediums, adds 5% (v/v) tire ox blood
(FBS) clearly, without adding penicillin/streptomycin.After HT-29 cell activations, grown in 6 orifice plates to individual layer, it is stand-by.
(4) suppress to stick measure：HT-29 cells are washed three times with PBS, add 1mL Lactobacillus pentosus AT6,1mL sandlwoods per hole
Sugared lactobacillus LGG or 1640 culture mediums (control group), 1h is cultivated in CO2gas incubator；1mL steps 2 are added into every hole again
The salmonella suspension of culture, 1h is cultivated in CO2gas incubator；PBS is washed three times, per Kong Zhongjia 0.5%TritonX-100,
Blown and beaten and mixed with pipettor after 20min；Gradient dilution, with add the LB solid mediums of 50mg/L streptomysins to pour into, bacterium to be grown
Fall behind and count.
CON groups are control group, before adding salmonella, add 1mL1640 culture mediums per hole in 6 orifice plates；LGG groups are in gaza's door
Add 1mL1.0 × 10 per hole into 6 orifice plates before Salmonella8CFU/mL Lactobacillus rhamnosus LGG bacteria suspensions；AT6 groups are in gaza's door
Add 1mL1.0 × 10 per hole into 6 orifice plates before Salmonella8CFU/mL Lactobacillus pentosus AT6 bacteria suspensions.
As a result as shown in Fig. 2 LOG value of CON, LGG, AT6 group per hole cell adhesion salmonella quantity respectively 6.20,
5.98、5.81。
Embodiment 3：Lactobacillus pentosus AT6 attacks the inhibitory action of HT-29 cells to salmonella
(1) by after Lactobacillus pentosus AT6 and Lactobacillus rhamnosus LGG passages, cultivated in MRS culture mediums in 37 DEG C
Centrifuge after 18h, washed three times with PBS, and thalline is resuspended to 1.0 × 10 with 1640 culture mediums (buying from Gibco)8CFU/mL。
(2) after salmonella carries out activation culture in LB culture mediums, quiescent culture 12h, washed three times, be used in combination with PBS
Thalline is resuspended to 1.0 × 10 1640 culture mediums8CFU/mL。
(3) preparation of HT-29 cells：Culture medium needed for HT-29 growths is 1640 culture mediums, adds 5% (v/v) tire ox blood
(FBS) clearly, without adding penicillin/streptomycin.After HT-29 cell activations, grown in 6 orifice plates to individual layer, it is stand-by.
(4) Matrigel is suppressed：HT-29 cells are washed three times with PBS, add 1mL Lactobacillus pentosus AT6,1mL sandlwoods per hole
Sugared lactobacillus LGG or 1640 culture mediums (control group), 1h is cultivated in CO2gas incubator；1mL steps 2 are added into every hole again
The salmonella suspension of culture, 1h is cultivated in CO2gas incubator；PBS is washed three times, per Kong Zhongjia 1mL gentamicins, 37 DEG C
Handle 45min；PBS is washed three times；Blown and beaten and mixed with pipettor after per Kong Zhongjia 0.5%TritonX-100,20min；Gradient is dilute
Release, with adding the LB solid mediums of 50mg/L streptomysins to pour into, counted after bacterium colony is grown.
CON groups are control group, before adding salmonella, add 1mL1640 culture mediums per hole in 6 orifice plates；LGG groups are in gaza's door
Add 1mL1.0 × 10 per hole into 6 orifice plates before Salmonella8CFU/mL Lactobacillus rhamnosus LGG bacteria suspensions；AT6 groups are in gaza's door
Add 1mL1.0 × 10 per hole into 6 orifice plates before Salmonella8CFU/mL Lactobacillus pentosus AT6 bacteria suspensions.
As a result as shown in figure 3, LOG value of CON, LGG, AT6 group per hole cell adhesion salmonella quantity respectively 6.04,
5.68、5.49。
Embodiment 4：Lactobacillus pentosus AT6 is to mouse intestinal content bacterium carrying capacity after salmonella infection and liver, spleen
The influence of salmonella recall rate
Mouse is C57BL/6 strains, female, 6-8w, SPF levels, 10/group, is divided into 3 groups.
Model group --- PBS gavages 10 days, 0.1mL/ days, the 11st day gavage salmonella 1.0 × 106CFU/mouse；
Prevention group --- Lactobacillus pentosus AT6 gavages 10 days, 0.1mL (5 × 108CFU)/day, the 11st day gavage Salmonella
Bacterium 1.0 × 106CFU/mouse；
Control group --- Lactobacillus rhamnosus LGG gavages 10 days, 0.1mL (5 × 108CFU)/day, the 11st day gavage sramana
Salmonella 1.0 × 106CFU/mouse
Comprise the following steps that：
(1) after Lactobacillus pentosus AT6, Lactobacillus rhamnosus LGG activation passage, centrifuged after 37 DEG C of culture 18h, use PBS
Wash three times, be resuspended to 5 × 109CFU/mL。
(2) mouse is handled：Model group PBS gavages, prevention group Lactobacillus pentosus AT6 gavages, control group Lactobacillus rhamnosus
LGG gavages, handle 10 days.
(3) the 11st days gavage salmonellas, 1.0 × 106CFU/mouse。
Mouse after handling 2 days is put to death, determines bacterium carrying capacity.
Liver and spleen bacterium carrying capacity result are as shown in figure 4, liver-con, liver-LGG, liver-AT6, spleen-
Con, spleen-LGG, spleen-AT6 liver bacterium carrying capacity be respectively 7.7CFU, 0.7CFU, 0CFU, 3.9CFU, 1.6CFU,
0CFU。
Ileum, caecum, colonic contents bacterium carrying capacity as shown in figure 5, Ileum-con, Ileum-LGG, Ileum-AT6,
Cecum-con, Cecum-LGG, Cecum-AT6, Colon-con, Colon-LGG, Colon-AT6 bacterium carrying capacity LOG values difference
For 2.58,1.04,0.75,2.85,1.32,0.92,2.37,0.86,0.37.
Embodiment 5：After Lactobacillus pentosus AT6 intakes after salmonella infection mouse death rate change
Mouse is C57BL/6 strains, female, 6-8w, SPF levels, 10/group, is divided into three groups.
Blank control group --- PBS gavages 10 days, 0.1mL/ days, the 11st day gavage PBS；
Model group --- PBS gavages 10 days, 0.1mL/ days, the 11st day gavage salmonella 1.0 × 106CFU/mouse；
Prevention group --- Lactobacillus pentosus AT6 gavages 10 days, 0.1mL (5 × 108CFU)/day, the 11st day gavage Salmonella
Bacterium 1.0 × 106CFU/mouse；
Positive controls --- Lactobacillus rhamnosus LGG gavages 10 days, 0.1mL (5 × 108CFU)/day, the 11st day gavage
Salmonella 1.0 × 106CFU/mouse。
Comprise the following steps that：
(1) centrifuged after Lactobacillus pentosus AT6, Lactobacillus rhamnosus LGG, 37 DEG C of culture 18h, washed three times, be resuspended extremely with PBS
5×109CFU/mL。
(2) mouse is handled：Control group and model group PBS gavages, prevention group Lactobacillus pentosus AT6 gavages, positive controls
Lactobacillus rhamnosus LGG, coprocessing 10 days.
(3) salmonella infection：11st day gavage salmonella, 1.0 × 106CFU/mouse。
(4) death rate is observed：Observation each group dead mouse situation daily, and record.
As a result show：Every group of 20 mouse, 15 days after salmonella gavage, dead 0 of control group mice, the death rate is
0%；Model group (model) dead mouse 12, death are survival rate 40% from 60%；Treatment group (Lactobacillus pentosus AT6) is small
Dead 7 of mouse, the death rate are 35% i.e. survival rate 65%；Control group (Lactobacillus rhamnosus LGG) dead mouse 8, the death rate
40% and survival rate 60%.Lactobacillus pentosus AT6 processing makes salmonellal mouse death rate be reduced at 15 days
25%.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention
Enclose being defined of being defined by claims.
Claims (10)
1. a kind of Lactobacillus pentosus (Lactobacillus pentosus), the micro- life of China is preserved on March 29th, 2017
Thing culture presevation administration committee common micro-organisms center, deposit number are CGMCC No.13956, and preservation address is Beijing
The institute 3 of Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica.
2. a kind of pharmaceutical composition, it is characterised in that contain the Lactobacillus pentosus (Lactobacillus described in claim 1
) and/or its metabolite pentosus.
3. pharmaceutical composition according to claim 2, it is characterised in that content >=1 of the Lactobacillus pentosus ×
108CFU/g or 1 × 108CFU/mL。
4. pharmaceutical composition according to claim 2, it is characterised in that the metabolite is that the Lactobacillus pentosus connects
Kind is cultivated into MRS culture mediums, the supernatant of collected after centrifugation.
5. according to any described pharmaceutical compositions of claim 2-4, it is characterised in that described pharmaceutical composition also contains medicine
Acceptable carrier on.
6. pharmaceutical composition according to claim 5, it is characterised in that the pharmaceutically acceptable carrier be it is a kind of or
A variety of carriers selected from pharmaceutically usually used filler, wetting agent, disintegrant, adhesive, lubricant or flavouring.
7. it is a kind of suppress Salmonella organisms amount method, it is characterised in that by the Lactobacillus pentosus described in claim 1 and/
Or its metabolite is added in the liquid containing salmonella, semisolid or solid.
8. according to the method for claim 7, it is characterised in that the Lactobacillus pentosus described in claim 1 is seeded to MRS
In culture medium, after 35~37 DEG C of 14~36h of culture, add into the liquid containing salmonella, solid or semisolid.
9. according to the method for claim 8, it is characterised in that Lactobacillus pentosus and salmonella described in claim 1
Bacterium it is dense than be 1:1~100.
10. the food or health products that are prepared using Lactobacillus pentosus described in claim 1.
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