CN107058171A - Lactobacillus pentosus and application with reduction cholesterol reduction triglycerides effect - Google Patents
Lactobacillus pentosus and application with reduction cholesterol reduction triglycerides effect Download PDFInfo
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- CN107058171A CN107058171A CN201710091198.2A CN201710091198A CN107058171A CN 107058171 A CN107058171 A CN 107058171A CN 201710091198 A CN201710091198 A CN 201710091198A CN 107058171 A CN107058171 A CN 107058171A
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- bacterial strain
- cholesterol
- lactobacillus pentosus
- norcholesterol
- pentosus
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- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- QWIZNVHXZXRPDR-WSCXOGSTSA-N melezitose Chemical compound O([C@@]1(O[C@@H]([C@H]([C@@H]1O[C@@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O)CO)CO)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O QWIZNVHXZXRPDR-WSCXOGSTSA-N 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
- 230000004899 motility Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- NGFMICBWJRZIBI-UJPOAAIJSA-N salicin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UJPOAAIJSA-N 0.000 description 1
- 229940120668 salicin Drugs 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- DGPIGKCOQYBCJH-UHFFFAOYSA-M sodium;acetic acid;hydroxide Chemical compound O.[Na+].CC([O-])=O DGPIGKCOQYBCJH-UHFFFAOYSA-M 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000003445 sucroses Chemical class 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
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
-
- 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
- A23V2400/167—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
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- 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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- Tropical Medicine & Parasitology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses one plant of Lactobacillus pentosus (La.Pentosus) that there is norcholesterol, triglyceride reducing to act on isolated and purified out from the Tibetan mastiff enteron aisle of Gansu Maqu County, preserving number is CGMCC 7426, experiment is proved, bacterial strain has very strong acidproof, bile tolerance ability, cholesterol (TC) can be significantly reduced, and triglycerides (TG) can be reduced, In-vivo test in mice is found, the bacterial strain is without any side effects, therefore, bacterial strain of the present invention can be used for the preparation of lipid-loweringing norcholesterol health medicine and functional food.
Description
【Technical field】
The present invention relates to a kind of Lactobacillus pentosus, particularly a kind of pentose breast acted on norcholesterol triglyceride reducing
Bacillus (La.Pentosus) and its application.
【Background technology】
Cholesterol is nutritional ingredient needed by human, and important physiological function is carry in vivo, but in human serum
Cholesterol level is often too high, a series of angiocardiopathy generally existings caused by serum cholesterol is too high.Modern study is
It was found that, atherosclerosis, venous thronbosis have close correlation with hypercholesterolemia.WTO was once predicted, to the year two thousand thirty
Angiocardiopathy will cause main causes of death, affect the health of the people of the whole world about 2.36 hundred million.Once had been reported that, west
The heart disease incidence of Ou Guojia and the East European countries is due to what hypercholesterolemia was caused.With the horizontal people's faciation of normal lipid
Than there is people with hyperlipidemia the heart disease for being higher by 3 times to induce risk.Triglycerides (Triglyceride, abridge TG) is long-chain
Aliphatic acid and the fat molecule of glycerine formation, are the most lipid of people's in-vivo content, most tissues can utilize glycerine three
Ester catabolite supplies energy.But if triglycerides is excessive, hoards in will subcutaneously make body obesity, hoard in vascular wall then
Artery sclerosis is caused, hoards and may result in cardiomegaly in heart, fatty liver can then be caused in liver by hoarding.Current China has closely
The Hyperlipidemia of 1/3 adult, first stealthy killer of the hyperlipemia now as human health.With entering for society
Step, the enhancing of people's health consciousness, Weight-reducing and lipid-lowering is extremely urgent.
At present, hyperlipemia is mainly controlled by statins, (courage is solid for its energy Reverse transcriptase HMG-CoA reductase
The rate-limiting enzyme of alcohol synthesis) activity reduce the autologous synthesis of cholesterol, specificity reduction serum cholesterol is (T-CHOL TC, low
Density lipoprotein-cholesterol LDL and C-VLDL VLDL), moderate reduction triglyceride levels and rise are highly dense
Lipoprotein levels are spent, the thus preventing and treating to atherosclerosis and coronary heart disease is acted.But this kind of medicine has serious poison
Side effect, easily causes the side effects such as myalgia, headache, upset,gastro-intestinal, blood fat disorder, liver and kidney dysfunction.Statins
A kind of more serious adverse reaction is rhabdomyolysis.Cerivastatin (visiing sthene) is due to serious cause rhabdomyolysis and kidney
Exhaustion, worldwide withdraws in August, 2001, and it has caused the thing of the U.S. and regional 52 patient deaths of Some European
Therefore.Therefore, development and utilization has fat-reducing and antihyperglycemic, and nontoxic health medicine, has weight to protection health
The realistic meaning wanted.
Probiotics be studied as " lived green medicine " prove to have excellent effect, safely, to meet ecology etc. many
Advantage, is more and more applied to the every field of human diseases.
About research of the probiotic lactobacillus in terms of norcholesterol, there is substantial amounts of paper to report and patent achievement both at home and abroad.
CN 104818230A Lactobacillus plantarums (L01), CN 102899276A streptococcus thermophiluses (BLST), CN102604851A lactic acid breast
Coccus, CN101559082A lactobacillus (MA2), CN1186855A urine streptococcus (DM891128) and CN 104789488A sandlwoods
Sugared lactobacillus (SCF 20120718) has certain effect in the effect of norcholesterol, but degradation rate is not high, and pentose
Lactobacillus has no report in the active patent of norcholesterol, and bacterial strain report of the lactic acid bacteria in terms of triglyceride reducing is rarely seen.
【The content of the invention】
It is an object of the invention to provide a kind of lactic acid bacteria acted on norcholesterol, triglyceride reducing and its application.
To achieve the above object, the technical scheme is that：
A kind of lactic acid bacteria for being isolated from Tibetan mastiff enteron aisle, is Lactobacillus pentosus (La.Pentosus), bacterial strain is in 2013 04
The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 07th, and its deposit number is CGMCC
7426, preservation address is：City of BeiJing, China Chaoyang District North Star West Road 1 institute 3.
The present invention is separately cultured by the way that Tibetan mastiff intestinal contents are coated on MRS culture mediums, point pure bacterial strain bacterium colony
For milky, diameter about 2mm, surface is smooth, neat in edge, central uplift, periphery of bacterial colonies has obvious molten calcium circle, microscopy form
For rod-short, pure bacterial strain will be divided to carry out bacterial strain Gram's staining, flat-plate bacterial colony form, bacterial strain to cholesterol in yolk and cholesterol
The degradation rate of standard items, the degradation rate to triglycerides, acid resistance test, bile tolerance experiment, toxicity test (mouse) etc. are tested.Adopt
Molecular Identification is carried out to divided pure bacterial strain with 16S rDNA methods.Its 16S rDNA sequence is in NCBI (National Center
For Biotechnology Information, US National Biotechnology Information center) it is compared in database, as a result
It was found that the gene order of the bacterial strain, in all similar sequences, the homology with Lactobacillus pentosus (La.Pentosus) is
99%, therefore physiological and biochemical property is combined, identification bacterial strain is Lactobacillus pentosus, is named as EAM-ZL016T.
It can be used for further research and development prevention and treatment because cholesterol triglycerides is too high the invention provides one kind
The functional food of caused cardiovascular and cerebrovascular disease and the raw material bacterial strain of health medicine.Surveyed by methods such as phosphorus sulphur-iron ratio color methods
Determine bacterial strain EAM-ZT003T norcholesterol, triglycerides and acidproof, bile tolerance effect, and determine bacterial strain in Mice Body
Inside whether there is toxic side effect.
As a result find：The Lactobacillus pentosus (La.Pentosus) that what the present invention was provided isolate and purify out from Tibetan mastiff enteron aisle,
With good security, and In-vivo test in mice discovery is nontoxic, while there is good cholate, strong acid tolerance, its
Norcholesterol triglyceride reducing ability is strong, with fungistatic effect, can apply in the preparation of functional food and health medicine.
Called function food refers to the food with specific function, is suitable for specific crowd and eats, can adjust the function of body, and not
For the purpose for the treatment of.
【Brief description of the drawings】
Fig. 1 is bacterial strain Gram's staining result of the invention.
Fig. 2 is bacterial strain flat board colonial morphology of the invention.
Fig. 3 is bacterial strain of the invention to cholesterol in yolk and the degradation rate of cholesterol standards.
Fig. 4 is the degradation rate of bacterial strain of the invention to triglycerides.
Fig. 5 is bacterial strain acid resistance test result of the invention.
Fig. 6 is bacterial strain bile tolerance result of the test of the invention.
Fig. 7 is the changes of weight of bacterial strain of the invention to toxicity test mouse.
Fig. 8 is bacterial strain toxicity test result (mouse liver section) of the invention.
Fig. 9 is bacterial strain toxicity test result (mouse kidney section) of the invention.
【Embodiment】
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
The screening of Lactobacillus pentosus EAM-ZL016T provided by the present invention with norcholesterol triglyceride reducing ability
And identification.
Separation, the purifying of 1 strain
0.5g Gansu Maqu County Tibetan mastiff intestinal contents are weighed, are added in the test tube equipped with 4.5ml sterilized waters, it is gradually dilute
Release to 10-6, take 10-4、10-5、10-6Three each 0.1ml of pipe dilution, using rubbing method, are coated on MRS culture mediums and are separated,
37 DEG C of incubated 48h.Picking colony, continuous line is isolated and purified, and finally selects single bacterium colony, through microscopy confirm as it is purebred after,
It is inoculated into after slant medium culture 24h, puts 4 DEG C of refrigerators and save backup.
The formula and condition of MRS solid mediums be：Peptone 10g, beef extract 10g, dusty yeast 5g, glucose 20g, nothing
Water sodium acetate 5g, Tween-80 1mL, lemon acid diamine 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.58g, manganese sulfate 0.25g, carbonic acid
Calcium 20g, agar 18g, distilled water 1000mL, pH6.6~6.8,115 DEG C, sterilize 20min.
Point pure bacterial strain bacterium colony is milky, diameter about 2mm, surface is smooth, neat in edge, central uplift, periphery of bacterial colonies has
Obvious molten calcium circle, microscopy form is rod-short, and (1-1.2) μ m (1-1.5) μm, single, paired, short chain arrangement will divide pure
Strain Designation is EAM-ZL016T.
2 bacterial strain Physiology and biochemistries are identified
Pure bacterial strain will be divided to carry out the experiment such as Gram's staining, motility inspection, oxidizing ferment, catalase, gelatin liquefaction.
Bacterial strain Gram-positive, punctures culture through semisolid culturemedium and finds, bacterial strain is clear along line growth, edge is punctured
It is clear, bacterial strain without motion, amphimicrobian.Bacterial strain does not reduce nitrate, and do not liquefy gelatin, and catalase and oxydase reaction are the moon
Property.
The EAM-ZL016T Physiology and biochemistry qualification results of table 1
| Detection project | As a result | Detection project | As a result | Detection project | As a result | Detection project | As a result |
| Mannitol | + | Lactose | + | Nitrate (reduction) | - | Arabite | + |
| Glucose | + | Aesculin | + | Adonite | - | Sorbierite | - |
| Raffinose | + | Phenylalanine | + | Bile aesculin | - | Xylitol | + |
| Maltose | + | Erythrite | + | Lysine decarboxylase | - | Amarogentin | + |
| Fructose | + | Melezitose | + | Ornithine decarboxylase | - | Tartrate | - |
| Starch | - | Sucrose | + | Arginine decarboxylase | - | Mannose | + |
| Hydrogen sulfide | - | Cellobiose | + | Arginine dihydrolase | - | Melibiose | + |
| Acetate | - | Melampyrin | - | Beta galactose glycosides | + | Salicin | + |
| D-ribose | + | Inositol | - | Methyl red | + | Rhamnose | - |
| Acetamide | - | Arabinose | - | V.P. | - | Galactolipin | + |
| Urea | - | Citrate | - | Gelatin | - | Gill fungus sugar | + |
| Malonate | - | Xylose | - | Sorbose | + | Dextrin | + |
| 40℃ | - | 15℃ | + | 6.5%NaCl | - | 10%NaCl | - |
316S rDNA Molecular Identifications
Molecular Identification is carried out to divided pure bacterial strain using 16S rDNA methods.16S rDNA are expanded and sequencing is raw by treasured
Thing engineering (Dalian) Co., Ltd completes.
Sequencing completes to submit bacterial strain EAM-ZL016T 16S rDNA sequences in NCBI (National Center for
Biotechnology Information, US National Biotechnology Information center) it is compared in database, as a result find
Bacterial strain EAM-ZL016T gene order, in all similar sequences, the homology with Lactobacillus pentosus (La.Pentosus)
For 99%, therefore physiological and biochemical property is combined, identification bacterial strain EAM-ZL016T is Lactobacillus pentosus.This is isolated from Tibetan mastiff enteron aisle
Lactobacillus pentosus (La.Pentosus), bacterial strain was preserved in Chinese microorganism strain preservation management committee on 04 07th, 2013
Member's meeting common micro-organisms center, its deposit number is CGMCC 7426.
Embodiment 2
The drop courage of Lactobacillus pentosus EAM-ZL016T provided by the present invention with norcholesterol triglyceride reducing ability
Sterol effect.
The effect of cholesterol bacterium in 1 drop yolk
Under aseptic condition, a certain amount of egg yolk is added in the culture medium of sterilizing as cholesterol source, sterile uniform packing
Into test tube.Using 2%, (cell concentration is 108Cfu/ml inoculum concentration) adds bacterium solution, 37 DEG C of incubated 24h, in 8000r/
10min is centrifuged under min, takes supernatant according to phosphorus sulphur-iron ratio color method, determines cholesterol level in supernatant.Every group of experiment setting 2
Individual repetition.
Configuration --- the liquor ferri trichloridi of sulphur ferrophosphorus developer：Weigh 5.0g FeCl3·6H2O is dissolved in 200ml concentrated phosphoric acids
In；Phosphorus sulphur ferron (P-S-Fe reagents)：Take 40ml liquor ferri trichloridi concentrated sulfuric acid constant volume to 500mL.
Phosphorus sulphur-iron ratio color method：The amount of trying to please is 10ml centrifuge tube, adds 4.8ml absolute ethyl alcohols and 0.2ml fermented supernatant fluids,
Put in room temperature after 10min, 8000r/min centrifugations 5min.Clean tube separately is taken, supernatant 2ml additions are accurately pipetted with liquid-transfering gun
In test tube, then along tube wall the sulphur ferrophosphorus reagent that 2ml has been configured is slowly added to, after shaking up immediately, room temperature cooling 30min.Wait to give birth to
After colour stable, colorimetric is carried out under 560nm with spectrophotometer, blank zeroing is made with absolute ethyl alcohol, not meet the MRS- of bacterium
Yolk culture medium is compared.
Cholesterol reduced rate is calculated according to below equation：
Cholesterol reduced rate=(C-A)/C × 100%
A：OD value of the nutrient solution at 560nm after strain fermentation
C：To impinging upon the OD values at 560nm
It is 64.84% that cholesterol ability in bacterial strain EAM-ZL016T drop yolk is measured through experiment.
The effect of 2 drop high-purity cholesterol bacterium
By bacterial strain, (cell concentration is 10 using 2%8Cfu/ml inoculum concentration) is inoculated in high cholesterol MRSO-CHOL culture mediums
In, 37 DEG C of incubated 24h centrifuge 10min under 8000r/min, take supernatant according to phosphorus sulphur-iron ratio color method, determine supernatant
Middle cholesterol level.Make blank zeroing with absolute ethyl alcohol, compared with nonvaccinated high cholesterol MRSO-CHOL culture mediums.
Cholesterol reduced rate is calculated according to below equation：
Cholesterol reduced rate=(C-A)/C × 100%
A：OD value of the nutrient solution at 560nm after strain fermentation
C：To impinging upon the OD values at 560nm
High cholesterol MRSO-CHOL culture mediums：In terms of 1000mL MRS fluid nutrient mediums, the glue of cholesterol containing 1.0mg/mL
It is prepared by beam solution：Precise cholesterol 0.1g is put into small beaker, adds 0.2g bovine biles, 0.1g sucrose esters, 1mL Tween 80s
Stir, then pipette 5mL glacial acetic acid dissolve by heating, lysate with after ultrasonication 15min, be added rapidly to prepare
In good MRS fluid nutrient mediums, stir while adding, form it into uniform and stable colloidal solution.
The norcholesterol ability for measuring bacterial strain EAM-ZL016T through experiment is 22%.
Embodiment 3
The drop of Lactobacillus pentosus EAM-ZL016T provided by the present invention with norcholesterol triglyceride reducing ability is sweet
Oily three ester effects.
Under aseptic condition, a certain amount of triglycerides standard items are added in the culture medium of sterilizing, it is aseptic subpackaged to arrive test tube
In.Using 2%, (cell concentration is 108Cfu/ml inoculum concentration) adds bacterium solution (not connecing the test tube of bacterium as blank control), 37 DEG C
Incubated 24h, determines triglycerides degradation rate at 420nm.
Triglycerides reduced rate=(C-A)/C × 100%
A in formula is OD value of the nutrient solution at 420nm after strain fermentation, and C is OD value of the blank control at 420nm.
The triglyceride reducing ability for measuring bacterial strain EAM-ZL016T through experiment is 2.8%.
Embodiment 4
Lactobacillus pentosus EAM-ZL016T's provided by the present invention with norcholesterol triglyceride reducing ability is acidproof
Bile tolerance is tested.
The acid resistance test of 1 bacterial strain
Bacteria suspension is made after bacterial strain is activated, the concentration of bacterial strain is determined using dilution coated plate method.Activated strains are connect respectively
Plant in pH 6.8, pH 3.0, pH 2.0 MRS fluid nutrient mediums, 37 DEG C of incubated 10h utilize gradient dilution flat band method
Viable count is determined, 5 is done and parallel averages.Test result indicates that, bacterial strain has stronger acid resistance, in pH3.0 condition
Lower survival rate is higher, during pH2.0, cultivates 10h, the survival rate of bacterial strain has declined, but remains to reach 106cfu/ml。
2 bacterial strains are determined to the tolerance of 0.3% cholate environment
Bacteria suspension is made after bacterial strain is activated, and (cell concentration is 108Cfu/ml), it is inoculated in and contains respectively by 2% inoculum concentration
In 0.0% bovine bile (i.e. blank), the composite fluid culture medium of 0.3% bovine bile.After 37 DEG C of culture 24h, its OD is determined respectively
Value, do 5 it is parallel average, tolerance of the bacterial strain to cholate according to the following formula.
OD × 100% of cholate tolerance (%)=OD/ blank cultures containing bile salt culture-medium
The cholate tolerance for measuring bacterial strain EAM-ZL016T through experiment is 15%.Cholate content exists in human small intestine
Fluctuated in the range of 0.03%-0.3%, the selected cholate content of experiment is Cmax.
Embodiment 5
Lactobacillus pentosus EAM-ZL016T's provided by the present invention with norcholesterol triglyceride reducing ability is antibacterial
Experiment.
The bacillus subtilis activated, Escherichia coli and S. aureus Inoculate are taken respectively in nutrient broth,
37 DEG C of incubated 24h, 10 are diluted to sterilized water8Cfu/ml, it is standby.
45 DEG C are cooled to after 50mL nutrient agars solid medium is sterilized, with indicator bacteria (bacillus subtilis, large intestine bar
Bacterium and staphylococcus aureus) fresh cultured thing 1000 μ L mix after be down flat plate, after putting the Oxford cup of sterilizing after flat board solidification.
It is 10 to adjust bacteria suspension concentration8Cfu/ml, the bacteria culture fluid and sterilized water for measuring 100 μ L respectively with liquid-transfering gun adds Oxford cup
In, ware lid is covered, flat board is placed in 4 DEG C of refrigerator diffusions, inhibition zone around 37 DEG C of incubated 24h, observation aperture is then placed
Diameter.Each indicator bacteria is cooked two wares, is surveyed antibacterial circle diameter, is averaged.It the results are shown in Table 2.
The antibacterial result of the Lactobacillus pentosus of table 2
Embodiment 6
The mouse of Lactobacillus pentosus EAM-ZL016T provided by the present invention with norcholesterol triglyceride reducing ability
Toxicity test.
20 mouse are randomly divided into 2 groups, A group gavage physiological saline, B group gavage Lactobacillus pentosus suspensions.A group mouse are every
Only every daily gavage of gavage physiological saline 0.5ml, B group mouse contains 1 × 10 daily8The suspension 0.5ml of individual Lactobacillus pentosus.
In order to ensure viable count certain in phage solution, each gavage all uses the bacterial strain of fresh processing.Freely intake, record weekly
Body weight.After administration 42 days, dissection is put to death, by heart, liver, spleen, kidney, lung taking-up normal saline flushing and is wiped
Weighed after net, calculate mice organs index, in addition, paraffin section is done in internal organ separation, whether observation internal organs occur lesion.Experiment knot
Fruit sees Fig. 7,8,9.
The toxicity test mice organs index of table 3 compares
The toxicity test of 42 days by a definite date finds that mouse hair color is bright and clean, and appetite is normal, and gavage does not occur abnormal sign in 42 days,
Increase very fast within 14 days before no abnormal death, mouse weight, tend towards stability within latter 28 days, maintain 27g or so, but process of the test
In do not occur becoming thin, the phenomena of mortality.Internal organ are normal after mouse dissection, and color is ruddy, smooth, and section result is shown, mice organs
Sexually revised without makings.
As a result show that feeding Lactobacillus pentosus EAM-ZL016T does not have toxic side effect to mouse.
Conclusion：The bacterial strain that the present invention is provided has effects that fabulous norcholesterol triglyceride reducing, and with very good
Acidproof, bile tolerance, into after human gastrointestinal tract, with the presence of more viable bacteria, it is ensured that the norcholesterol of bacterial strain drop glycerine three
Ester activity, bacterial strain has fungistatic effect, in lipid-loweringing norcholesterol simultaneously, can play a part of whole intestines and regulation gut flora, and
Bacterial strain does not have any toxic side effect.
The above-mentioned characteristic for the Lactobacillus pentosus EAM-ZL016T that the present invention is provided can be applied to prepare health food
In medicine.
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still may be used
To be modified to the technical scheme described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic.
Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., should be included in the present invention's
Within protection domain.
Claims (3)
1. a kind of Lactobacillus pentosus (La.Pentosus) for being isolated from Tibetan mastiff enteron aisle, bacterial strain is in preservation on the 07th in 04 month in 2013
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is CGMCC 7426.
2. the Lactobacillus pentosus described in a kind of claim 1, applied to lipid-loweringing norcholesterol health medicine.
3. the Lactobacillus pentosus described in a kind of claim 1, the food applied to lipid-loweringing norcholesterol function.
Priority Applications (1)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107929329A (en) * | 2017-07-07 | 2018-04-20 | 西北民族大学 | A kind of prebiotic compound bacteria Chinese medicinal granule of embolism extinguishing lipid-loweringing and preparation method thereof |
| CN109207389A (en) * | 2017-07-07 | 2019-01-15 | 西北民族大学 | A kind of prebiotic compound bacteria traditional Chinese medicine oral liquid of embolism extinguishing lipid-loweringing and preparation method thereof |
| CN113939198A (en) * | 2019-05-14 | 2022-01-14 | 韩国食品研究院 | Strain having liver function improving activity and use thereof |
| CN115710563A (en) * | 2022-09-26 | 2023-02-24 | 重庆第二师范学院 | Lactobacillus pentosus CQZC02 and application thereof in preparation of drugs for treating liver injury |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107929329A (en) * | 2017-07-07 | 2018-04-20 | 西北民族大学 | A kind of prebiotic compound bacteria Chinese medicinal granule of embolism extinguishing lipid-loweringing and preparation method thereof |
| CN109207389A (en) * | 2017-07-07 | 2019-01-15 | 西北民族大学 | A kind of prebiotic compound bacteria traditional Chinese medicine oral liquid of embolism extinguishing lipid-loweringing and preparation method thereof |
| CN113939198A (en) * | 2019-05-14 | 2022-01-14 | 韩国食品研究院 | Strain having liver function improving activity and use thereof |
| EP3970511A4 (en) * | 2019-05-14 | 2023-01-11 | Korea Food Research Institute | Strain showing liver function improving activity, and use thereof |
| CN115710563A (en) * | 2022-09-26 | 2023-02-24 | 重庆第二师范学院 | Lactobacillus pentosus CQZC02 and application thereof in preparation of drugs for treating liver injury |
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| CN107058171B (en) | 2019-12-20 |
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