CN108048353A - The Lactobacillus reuteri HI120 of one Expression of Plant Height linoleate isomerase LAI and its application - Google Patents
The Lactobacillus reuteri HI120 of one Expression of Plant Height linoleate isomerase LAI and its application Download PDFInfo
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- CN108048353A CN108048353A CN201711415835.3A CN201711415835A CN108048353A CN 108048353 A CN108048353 A CN 108048353A CN 201711415835 A CN201711415835 A CN 201711415835A CN 108048353 A CN108048353 A CN 108048353A
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- 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
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Abstract
The invention discloses the Lactobacillus reuteri HI120 of an Expression of Plant Height linoleate isomerase LAI, which is preserved in Guangdong Province's Culture Collection on November 21st, 2016, and deposit number is GDMCC NO：60119.The expression of the linoleate isomerase of Lactobacillus reuteri HI120 is higher than Lactobacillus reuteri standard bacteria and other probiotics, can be in vivo and in vitro conjugated linoleic acid by linoleic acid.The invention also discloses a kind of microorganism live bacteria preparations containing above-mentioned Lactobacillus reuteri HI120 and the microorganism live bacteria preparation to prepare the application in having effects that prevention and/or auxiliary treatment obese diabetes and having effects that the food additives, food or drug of prevention and/or auxiliary treatment colorectal cancer.
Description
Technical field
The invention belongs to Tiny ecosystem active bacteria formulation technical fields, and in particular to an Expression of Plant Height linoleate isomerase LAI's
Lactobacillus reuteri HI120 and its application.
Background technology
Disease serious threat human health caused by the Anomalous lipid metablisms such as obesity, hyperlipidemia, fatty liver.Long-term lipid metaboli
It is abnormal inevitably to trigger the " three high " diseases such as hyperlipidemia, hypertension and hyperglycaemia, be it is further trigger cardiovascular and cerebrovascular it is unexpected,
The principal element of the serious diseases such as hepatic sclerosis, diabetes and malignant tumour, it is very big to human health damage.
Most of Anomalous lipid metablism is all caused by undesirable dietary structure and habits and customs, is the intake of energy
Caused by unbalanced depletion.Wherein, it is most important factor that fatty acid uptake is excessively out of proportion with Fatty acid compositions.Diet
The proper proportion (5-8: 1) of middle ω -6/ omega-fatty acids (unsaturated bond is located on the 3/6th carbon atom of methyl end) is to maintain body
The necessary condition of health.The intake of ω -6 aliphatic acid is excessive or the out of proportion of ω -6/ omega-fatty acids can all cause lipid metaboli
It is abnormal.For animal oil based on saturated fatty acid, long-term consumption easily causes the Anomalous lipid metablisms such as obesity.The plant of daily consumption
Oil, in peanut oil, rapeseed oil, corn oil and soya-bean oil, although based on unrighted acid, ω -6 content of fatty acid is most
More, omega-fatty acid content is few.Wherein ω -6 aliphatic acid is again most with linoleic acid (Linoleic acid, LA) content, accounts for total
The 35-70% of weight, long-term consumption can equally cause the Anomalous lipid metablisms such as obesity.Only a small number of vegetable oil, such as linseed oil, purple
Soviet Union's oil, which waits, contains a certain amount of omega-fatty acid alpha-linolenic acid (ALA).But these vegetable oil are small, fuel-displaced due to planting area
Rate is low, limited source.20-30% docosahexaenoic acids (DHA C22 are rich in deep sea fish oil, algal oil:And 20 light dydrocarbons 6)
Olefin(e) acid (EPA C20:5) omega-fatty acids such as.But with the exhaustion of marine resources, deep sea fish oil limited source, price is too
Height, can not be as the conventional edible oils of general public.
ω -6 aliphatic acid is the raw material of inflammatory mediator synthesis, in vivo mainly by the way that long-term chronic inflammation is caused to cause fat generation
Thank exception and complication.ω -6 aliphatic acid is converted mainly into arachidonic acid (AA) in vivo, and AA is synthesis of prostaglandins E2
(PGE2), the raw material of the most strong inflammatory mediator such as thromboxane element A2 (TXA2) and leukotriene B4 (LTB4).PGE2, LTA4, LTB4 and
TXA2, TXB2 are the most strong inflammatory mediators of activity in vivo, participate in atherosclerosis, thrombosis, islet damage and inflammation hair
The pathological changes such as raw, and stimulate cellular proliferation, the effect of induced tumor.
Conjugated linoleic acid (CLA) has the functions such as very strong Weight-reducing and lipid-lowering, hypoglycemic be anti-inflammatory.Conjugated linoleic acid (CLA)
It is the isomer of ω -6 Fatty Acids Linoleic acids (LA), it is similar to LA structures, but function is absolutely opposite with LA.CLA
It is originally found in milk.Some bacteriums in ruminant tumor gastric, as Butyrivibrio fibrisolvens, Megasphaera elsdenii have it is higher
LA isomerases (LAI) activity.Double bond in LA chemical constitutions is all cis.A cis-double bonds allosteric in LA can be by LAI
Trans double bond forms conjugated double bond linoleic acid, i.e. CLA.Most common CLA existence forms are cis-9, trans-11C18:2
(c9t11) and trans-10, cis-12C18:2(t10c12).Many probiotics, such as lactobacillus reuteri, Lactococcus lactis
And bifidobacterium breve, there are LAI genes in genome, but LAI activity is not high, and the efficiency that allosteric LA is CLA is very low.
CLA can be finally converted into vivo as the compounds such as PGD3, TXA1/3, LTA5 and prostacyclin I3 (PGI3),
These compounds are similar to the structure of inflammatory mediator PGE2, TXA2 and LTB4 etc., but act on just on the contrary, playing Competitive assays inflammation
The effect of reaction.For example, PGI3 plays the role of expanding blood vessel, reduces platelet aggregation, thrombosis is can inhibit, reduces heart and brain blood
Manage unexpected risk.
CLA can also be combined with nuclear receptor, be directly entered nucleus and adjusted the expression of some genes to play weight-reducing, lipid-loweringing
Function.CLA can be combined with a variety of Pexoxisome proliferator activated receptors (PPAR), regulate and control the table of series of genes
It reaches：(1) inhibit lipoprotein lipase (lipoprotein lipase) activity of adipocyte, prevent apolipoprotein B and the drop of A
Solution promotes the transhipment and mobilization of fat, inhibits the synthesis of fat, plays effect for reducing fat.(2) cyclooxygenase 2 is lowered
The expression of (Cyclooxygenase 2, COX-2).COX-2 is the key enzyme that conversion of arachidonic acid (AA) is converted to PGE2.Under
Adjust COX-2 expression that can inhibit inflammatory reaction.(3) nitricoxide synthase (iNOS) activity is raised, promotes the generation of PGF2 α, it is competing
Strive the effect for inhibiting PGE2.(4) tumor suppressor genes such as P53, P21, P27 and pro apoptotic protein Bax expression are raised, enhance Guang protease
(caspase) 3,9 activity lowers the expression of cycle element (cyclin) D, E and anti apoptotic protein bcl -2, inhibits Insulin-Like life
The expression secretion of long factor II (IGF- II).Thus there is inhibition cancer cell multiplication, promote the effect of Apoptosis.The study found that
CLA can directly inhibit tumor cell proliferation, inducing cell apoptosis in vitro.
There is studies have shown that obesity (OB) mouse feeding CLA, can significantly raise adipocyte PPAR γ expression, mobilize fat
Fat decomposes, and improves insulin resistance.Clinical test shows, takes the mixing CLA of 3-4g daily, normal adult, obese patient and
The fat content of middle-aged and elderly people has to be declined to a certain degree, and blood sugar in diabetic patients reduces, and the sensibility of insulin is improved.
Although CLA just defended planning commission's approval by China early in 2009 and is included in new food register.But chemical synthesis or biosynthesis
CLA is expensive, it is impossible to general food or food additives as general public.
LA allosterics can be t10c12CLA by propionibacterium acnes LAI genes.Studies have shown that takes orally propionibacterium acnes LAI
Genetic transformation lactic acid bacteria viable can significantly change the composition of mouse adipose tissue and liver fat acid, improve enteron aisle and other
The omega-fatty acids contents such as adipose tissue EPA and DHA.But the huge spherical shape of propionibacterium acnes, Butyrivibrio fibrisolvens, Erichsen
The bacteriums such as bacterium are conditioned pathogens, and there are no edible mushroom register is included in, viable bacteria cannot directly eat.And existing regulation does not allow
The recombinant lactic acid bacteria for carrying LAI genes is listed as functional food.
The content of the invention
It is an object of the invention to provide the Lactobacillus reuteri HI120 of an Expression of Plant Height linoleate isomerase LAI, the Roys
The expression of the linoleate isomerase (LAI) of lactobacillus HI120 is higher than Lactobacillus reuteri standard bacteria and other probiotics, can be
Linoleic acid (LA) is converted into conjugated linoleic acid (CLA) in vivo and in vitro.
The present invention also aims to provide a kind of microorganism live bacteria preparation, which contains above-mentioned Roy breast bar
The bacterium is prepared into the active bacteria formulations such as viable bacteria suspension, dairy products, viable bacteria bacterium powder or bacterium piece by bacterium HI120, can be in human body intestines after taking orally
The LA in part of the food source can be converted into CLA by road internal breeding and enrichment.
Final object of the present invention is that providing mentioned microorganism active bacteria formulation is preparing with prevention and/or auxiliary
Treatment obese diabetes effect and have effects that prevent and/or the food additives of auxiliary treatment colorectal cancer, food or
Application in drug.
First purpose of the present invention is achieved through the following technical solutions：One Expression of Plant Height linoleate isomerase
The Lactobacillus reuteri HI120 of LAI, the bacterial strain are preserved in Guangdong Province's Culture Collection on November 21st, 2016,
Its deposit number is GDMCC NO：60119.
Lactobacillus reuteri HI120 is isolated from the excrement of healthy infants, and the expression of linoleate isomerase (LAI) compares sieve
Her lactobacillus standard bacteria DSM 20016 (DSM) and elongated Bifidobacterium are high 6~10 times (as shown in Figure 2), can in vivo and body
It is outer that linoleic acid (LA) is converted into conjugated linoleic acid (CLA).
Specifically, the Classification And Nomenclature of Lactobacillus reuteri HI 120 is：Lactobaccillus reuteri, depositary institution
For：Guangdong Province's Culture Collection, preservation address are：5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100 Guangdong Province
Institute of microbiology, preservation date are：On November 21st, 2016, deposit number are：GDMCC NO：60119.
Second object of the present invention is achieved through the following technical solutions：A kind of microorganism live bacteria preparation, contains
The Lactobacillus reuteri HI120 of above-mentioned high expression linoleate isomerase LAI.
The microorganism live bacteria preparation can be dry for viable bacteria suspension formulation, dairy products, viable bacteria bacterium powder, freeze-dried powder preparation, spraying
The active bacteria formulations such as dry enteric active bacteria formulation, bacterium piece, can be in human body intestinal canal internal breeding and enrichment after taking orally, can be by part of the food source
LA be converted into CLA.
Third object of the present invention is achieved through the following technical solutions：Above-mentioned microorganism live bacteria preparation is being made
It is standby that there are prevention and/or auxiliary treatment obese diabetes and there is prevention and/or auxiliary treatment colorectal cancer
Food additives, the application in food or drug.
The microorganism live bacteria preparation can be applied to the prevention of the diseases such as obese diabetes, malignant intestinal tumour and auxiliary is controlled
It treats.
Compared with prior art, the invention has the advantages that：
(1) the Lactobacillus reuteri HI 120 in the present invention is isolated from the excrement of healthy infants, linoleate isomerase (LAI)
Expression is 6~10 times higher than Lactobacillus reuteri standard bacteria (DSM 20016) and other probiotics, can be in vivo and in vitro by Asia
Oleic acid (LA) is converted into conjugated linoleic acid (CLA)；
(2) Lactobacillus reuteri HI 120 is prepared into the viable bacterias such as viable bacteria suspension, dairy products, viable bacteria bacterium powder or bacterium piece by the present invention
The LA in part of the food source can be converted into CLA by preparation after oral in human body intestinal canal internal breeding and enrichment；
(3) in obesity, hyperlipidemia, diabetes, enteritis, malignant tumour, (enteron aisle swells microorganism live bacteria formulation application of the present invention
Knurl) and the diseases such as gastrointestinal dysfunction prevention and auxiliary treatment.
Description of the drawings
Fig. 1 is the separation of Lactobacillus reuteri HI 120 and colony characteristics, A in embodiment 1：That is turned out in child's excrement detests
Oxygen lactic acid bacteria bacterium colony；B：HI120 bacterial clumps after isolating and purifying；C：The Gram-stained morphosis of HI120 bacterium；
Fig. 2 is the comparisons by LA allosterics for CLA efficiency in vitro with reference culture of Lactobacillus reuteri HI 120 in embodiment 1,
BFL：Bifidobacterium longum NCC2705, DSM：Lactobacillus reuteri DSM20016, HI120：Lactobacillus reuteri HI120；
Fig. 3 is 120 strain 16S rRNA sequences of Lactobacillus reuteri HI and other lactobacillus reuteris 16S in embodiment 1
RRNA gene orders BLAST comparison results in U.S.'s ncbi database；
Fig. 4 is that GC-MS combined instruments detect and identify the chemical property that HI120 bacterium vitro conversions LA is CLA in embodiment 1,
A：19 kinds of mixed methyl aliphatic esters；B：HI120 bacterium tunning methylates；C：Scheme B mark peak mass spectral analysis figures；
Fig. 5 is to DB diabetes models appetite, weight and blood after Lactobacillus reuteri HI120 bacterium viable bacterias take orally in embodiment 3
The influence of fat, A：HI120 bacterium group and the comparison for compareing bacterium group mouse appetite；B：HI120 bacterium group is with compareing bacterium group mouse weight
Compare C：HI120 bacterium group is with compareing the periphery blood triglyceride (TG) of bacterium group mouse weight and the ratio of T-CHOL (TC) concentration
Compared with wherein BC：Blank control；DSM：20016 reference cultures of DSM；HI120：HI120 bacterium, similarly hereinafter；
Fig. 6 is to DB diabetic mices blood glucose and oral after Lactobacillus reuteri HI120 bacterium viable bacterias take orally in embodiment 3
The influence of sugar tolerance, A：HI120 bacterium group and the comparison for compareing bacterium group mouse fasting plasma glucose concentration, B：HI120 bacterium groups are with compareing bacterium
The comparison of change of blood sugar after group Mouse oral glucose；
Fig. 7 is that show that 120 bacterium viable bacteria of Lactobacillus reuteri HI takes orally small to DB diabetes models for oil red O stain in embodiment 3
The influence of mouse fatty liver, A：Blank control (BC) group；B：Standard bacteria (DSM) processing group；C：HI120 bacterium processing groups；
Fig. 8 is the therapeutic effect that HI120 bacterium viable bacteria takes orally to primary colon cancer APC mouse in embodiment 4, left figure：Knot
Intestinal cancer model mice, right figure：HI120 bacterium viable bacteria prevents colon cancer curative effect.
Specific embodiment
The separation and identification of 1 lactobacillus reuteri of embodiment
1st, the separation of lactobacillus reuteri bacterial strain
Healthy infants fresh excreta 0.5g is taken, inserts in 9.5mL MRS culture mediums, after abundant mixing, is diluted to 100
Times.0.1mL is taken to be coated on MRS solid mediums, puts 37 DEG C of culture 48h of anaerobic culture box.Select milky, median rise, week
Side rounding and shinny bacterium colony is inoculated into respectively in MRS fluid nutrient mediums, 37 DEG C of Anaerobic culturels for 24 hours, are then collected bacterium and are carried out
CLA transformation experiments screen linoleate isomerase (LAI) high activity bacterial strain and carry out strain idenfication (Fig. 1).
2nd, high activity linoleate isomerase (LAI) bacterial strain is screened
The bacterium colony selected and lactobacillus reuteri reference culture DSM20016 bacterium are inoculated into containing 1mg/mLTween- respectively
80 MRS culture mediums, bacterial density reach OD600When 0.8, LA to 1mg/mL is separately added into, when suspension shaken cultivation 12 is small.It receives
Collect bacterium solution centrifuging and taking supernatant.1mL culture solution+2mL isopropanol+1.5mL n-hexanes extract 3min, centrifuge 5min through 6000r/m, inhale
Upper strata n-hexane layer is taken, stratification takes upper strata n-hexane layer extraction aliphatic acid.Using ultraviolet specrophotometer in 234nm ripples
The absorbance of long measurement sample, the CLA contents in bacterium solution is calculated according to the standard curve of CLA, with CLA in every milliliter of bacterium solution
Content divided by the LA total amounts being added in every milliliter of bacterium solution, obtain the efficiency that each bacterial strain is converted into CLA.With reference culture
DSM20016 bacterium are as control.
It is in Fig. 2 the results show that HI120 bacterial strains be converted into the average efficiency of CLA respectively than DSM20016 bacterium (DSM) and
Elongated Bifidobacterium NCC2705 (BFL) is high 6.1 and 9.1 times (Fig. 2), so, 120 bacterium of HI are converted into CLA than other bacteriums
Efficient 6-9 times.
3rd, the 16S rRNA gene magnifications of LAI high activities bacterial strain and sequencing identification
Bacterium solution is collected, lysozyme lysis bacterium is added to extract genomic DNA.Bacterial 16 S rRNA universal primers 7F, 1540R are
Primer, using genomic DNA as template, PCR amplification 16S RNA carry out PCR reactions using bacterium solution as template, and reaction condition is：95℃
Pre-degeneration 4min：The reaction each cycled 94 DEG C of 45s, 55 DEG C of 45s, 72 DEG C of 1min, 30 Xun Huans；72 DEG C of extension 10min.PCR
Product send Shanghai Sangon Biotech Company to be measured identification.
4th, sequence alignment and analysis
In U.S.'s ncbi database, using BLAST software tools to HI120 strains 16S rRNA gene orders and other sieve
Yi Shi lactobacillus 16S rRNA gene orders carry out sequence alignment.
120 strain 16S rRNA gene orders of the results show Lactobacillus reuteri HI and lactobacillus reuteri standard in Fig. 3
The homology of strain DSM 20016 and other lactobacillus reuteris 16S rRNA gene orders is up to 99% (Fig. 3).
5th, gas chromatography-mass spectrum (GC-MS) combined instrument determines the general chemical structure for the CLA that HI120 bacterium are converted into
It takes fatty acid extract 1mL, adds in 1mL HCL- methanol (10%), after mixing, 80 DEG C of water-bath 1h.It is cooled to room temperature
Afterwards, 1mL boron trifluoride methanols mixed liquor (BF3) mixing is added in.50 DEG C of water-bath 10min.3mL n-hexanes, vortex are added in after cooling
Mixing is stored at room temperature overnight, takes n-hexane layer for detecting.Internal standard method for gas chromatography instrument (GC-MS) testing conditions：Chromatography
Column DB-5MS fused-silica capillary columns (0.5 μm of 30m × 0.25mm)；250 DEG C of injector temperature；Split sampling, shunt ratio
10.0；Flow velocity：1.0mL/min；Carrier gas is high-purity helium (99.999%)；1 μ L of sample size；Solvent delay 3min.Mass spectrum item
Part：Level Four bar temperature is 250 DEG C；Ionization mode is electron impact ion source (EI)；Ion source temperature is 250 DEG C；Electron energy
70eV；Mass range 30-6000mAu；Sweep spacing 0.2s/scan；Electron multiplier voltage 0.90kV.Gradient increased temperature program：0~
50min, 60 DEG C~300 DEG C；Rate:10℃/min；300 DEG C of running temperature afterwards；Run time 3min afterwards.According to 19 kinds of aliphatic acid
The chromatographic results of methacrylate calibration with reference to the eluting peak of mass spectral analysis purpose sample, check CLA in the sample in mass spectrometric data
Structure type.
It is in Fig. 4 the results show that HI120 bacterium by LA be converted into general chemical structure be c9t11 CLA (Fig. 4).
2 microorganism live bacteria preparation of embodiment
1st, the culture of living bacterial liquid
With MRS culture medium recovery Lactobacillus reuteri HI120 strains, after bacterium reaches a certain concentration, by a certain percentage (1:
It 100-500) is inoculated into MRS culture mediums, reaches OD600 when bacterial densities to collect bacterium during 0.6-1.0.
2nd, the preparation of viable bacteria suspension
For Bacteria Culture to after certain density, 5000g-10000g centrifuges 5-10 minute collection bacteriums, and edible sugar salt is added to mix
It closes solution or beverage is resuspended, put 4-8 DEG C of refrigerator and preserve.
3rd, the preparation of freeze drying viable microorganism preparation
To after certain density, 5000g-10000g is centrifuged 5-10 minute and is collected bacterium Bacteria Culture, by a certain percentage plus is prevented
Freeze protection solution (15% skimmed milk power, 5% glycerine, 0.9%NaCl) and mixing is resuspended, be uniformly dispersed, it is pre- to be put into ultra low temperature freezer
Freeze to fully charge, then put people's freeze drier, dried under the conditions of vacuum degree 10.0-12.00Pa to bacterium powder water content<3%.
After lyophilized, bacterium powder is taken out, is dispensed into sterile sealed containers, -20 DEG C preserve for use.
4th, it is spray-dried the preparation of enteric active bacteria formulation
Bacteria Culture collects bacterium for 5-10 minute to after certain density, 5000g-10000g is centrifuged, addition enteric solubility capsule material
It is resuspended with protective agent, sprays under conditions of 100-120 DEG C certain of drying temperature, wind turbine frequency 50HZ, flow rate of liquid 20r/s
It is dried to enteric solubility viable bacteria bacterium powder.4-8 DEG C of refrigerator saves backup.
5th, HI120 bacterium viable bacteria bacterium powder bacterial action measures
0.1g bacterium powders are weighed, are resuspended with 1.0mL MRS culture mediums, by 1:100、1:1000、1:1000 gradient dilutions, respectively
0.1mL bacterium solutions is taken to be coated in MRS solid medium tablets, 37 DEG C of culture 48h of anaerobic culture box is put, calculates every gram of bacterium powder and fall shape
Into unit (CFU/g).
3 biologic live bacteria preparation of embodiment has effects that the food additive of prevention and/or auxiliary treatment obese diabetes preparing
Add the application in agent, food or drug
1st, application of the HI120 viable bacterias in DB obese diabetes are prevented
12 DB mouse are randomly divided into 2 groups：Respectively oral DSM20016 bacterial strains group (DSM groups) and oral HI120 bacterial strains
Group (HI120 groups).DSM groups and HI120 groups are fed with 0.1mL (about 6 × 109cfu) DSM20016 and HI120 bacterium powders respectively.Daily
Once, mouse is intervened 4 weeks altogether.The appetite and weight of mouse are measured every other day, measures fasting blood-glucose weekly, detect oral Portugal every two weeks
Grape carbohydrate tolerance test (OGTT).Mouse fasting 12h after 4 weeks is plucked after eyeball takes peripheral blood, and the neck that breaks is put to death, and takes liver, pancreas, intestines
Pipe and intestinal contents.
2nd, the fasting plasma glucose of mouse
Respectively first week, second week, the 3rd week, 4th week, respectively each group take 6 mouse, more than fasting 12h, clip
Rat-tail measures its corresponding fasting blood sugar with blood glucose meter, and records, and with SPSS softwares, statistical analysis is carried out to data, and right
The average of data carries out drawing description (Fig. 6) with standard deviation.
3rd, the common carbohydrate tolerance test of OGTT mouse
After mouse fasting plasma glucose is completed, after the weight for weighing mouse, with the standard of 2g/kg glucose, difference
Every group of gavage mouse, after 30min, 60min, 90min, 120min, clip mouse rat-tail, the blood glucose value of measurement each group mouse,
In 0min, 30min, 60min, 120min is detected with blood glucose meter and is recorded the blood glucose value (Fig. 6) of mouse respectively.It is united with SPSS13.0
Meter software calculates its area under a curve (AUC) to reflect the sensibility of insulin.
It is in Fig. 6 the result shows that, compared with blank control (BC) and standard bacteria DSM20016 (DSM), Lactobacillus reuteri
HI120 can significantly reduce mouse fasting blood-glucose, significantly improve glucose tolerance in mice.
4th, the biochemistry detection of blood plasma
After treatment one month, experiment mice before processing fasting 12h plucks eyeball and takes blood, and 2000rpm low-temperature centrifugation 10min take
Blood plasma is stored in 4 DEG C of refrigerators.The biochemical indicators such as plasma triglyceride level (TRIG) and cholesterol (CHOL) content using it is micro it is complete from
Dynamic bio-chemical detector is detected, each sample upper machine measure (Fig. 5) after diluting 3 times with distilled water.
It is in Fig. 5 the result shows that, compared with blank control (BC) and standard bacteria DSM20016 (DSM), Lactobacillus reuteri
HI120 can significantly reduce the content of the triglycerides TRIG and cholesterol CHOL in mouse appetite, weight and blood.
5th, the oil red O stain detection of liver
Fresh liver organization or -80 DEG C of hepatic tissues freezed are taken, are placed in liquid nitrogen container, (10 μm are cut rear embedded section
Piece is adhered fixed on anticreep glass slide), natural air drying；Distilled water embathes, and 60% isopropanol embathes 2min；Oil red O stain
10min (37 DEG C of incubators)；60% isopropanol toning 25S (micro- Microscopic observation color change)；Haematoxylin is redyed after distillation washing
1.5min；Again indigo plant is returned with tap water flushing；It finally (should be by aqueous mounting medium in 60 DEG C of warm water before mounting with gelatin glycerine mounting
In be heated to liquid just can mounting), conventional light microscope is observed and taken pictures (Fig. 7).
It is in Fig. 7 the result shows that, compared with blank control (BC) and standard bacteria DSM20016 (DSM), HI120 can significantly subtract
Light mouse fatty liver, liver red lipochondrion (oil red O stain is positive) become smaller, and structure is more regular.
4 biologic live bacteria preparation of embodiment has effects that the food additive of prevention and/or auxiliary treatment colorectal cancer preparing
Add the application in agent, food or drug
1st, colorectal cancer APCmin/+Model construction
(1) APC is selectedmin/+Male mouse and C57BL female mices are mated in every male three female mice ratios of mouse of 1, cage.Suckling mouse to be given birth to
After 2 weeks, toe mark, one small truncation bar of clip or small pieces ear are cut.Conventional method extracts tissue gene group DNA, according to Nanjing
The method of model animal resources bank expands target DNA fragment with polymerase chain reaction (PCR) method, identifies APCmin/+Heterozygote.
It is wild-type mice that PCR product, which only has 700bp bands,；Only 300bp bands are no mutant homozygote mouse；Simultaneously have 300bp and
700bp bands are APCmin/+Hybrid mice.
(2) 6-8 weeks APC is takenmin/+Male and female modeling, every mouse inject the AOM of 1mg/mL, injection dosage 10 according to weight
μL AOM/g.Then feeding high lipid food, observation mouse state and stool situation, every 3 days measurement mouse weights, treat that mouse occurs
It has blood in stool or even rectal prolapse phenomenon occurs, then modeling success.
2nd, HI120 viable bacterias oral intestinal canal approach prevention primary mouse junction cancer
APC model mices are randomly divided into 6 groups, every group minimum 6.Experiment on therapy selects the successful APC of modelingmin/+, treatment
Group feeding 0.1ml (about 6 × 109Cfu) HI120 bacterium powders suspension (HI120 groups), daily feeding is once；Negative control group feeding etc.
Measure DSM20016 bacterium bacterium powder (DSM groups).Prevent the APC of the non-modeling of group selectionmin/+Hybrid mice, feeding HI120 and
Colon cancer modeling is carried out while DSM20016 bacterium powders.Every other day measure mouse appetite and weight, pays attention to observe mouse state with
Stool situation waits blank controls (BC) the group mouse rectal prolapse that occurs having blood in stool serious, mouse is put to death when weight loss is apparent.Prohibit before putting to death
12h is eaten, is plucked after eyeball takes peripheral blood, the neck that breaks is put to death, and measures the length of intestinal tube, the size for splashing knurl body in intestinal tube, simultaneously
Mouse lymphonodi mesenterici is detected, whether liver, lung have transfer stove, cut intestinal tube tissue and intestinal contents saves backup (figure
8)。
It is in Fig. 8 the result shows that, left figure：Model of colon cancer mouse, it is seen that intraperitoneal huge colorectal cancer, four limbs axillary lymph
It carries down shifting, with liver, splenomegaly.Right figure：HI120 bacterium viable bacteria prevents colon cancer curative effect, with blank control (BC) and standard bacteria
DSM20016 (DSM) is compared, and HI120 bacterium substantially reduce the quantity and size of colon carcinoma body, and an only knurl body has also been opened
Beginning atrophy.
A part of specific embodiment is enumerated above, and the present invention will be described, it is necessary to which indicated herein is specific real up and down
It applies example and is served only for that the present invention is further illustrated, do not represent limiting the scope of the invention.Other people are according to this hair
The nonessential modification of bright some made and adjustment still fall within protection scope of the present invention.
Sequence table
<110>Guangzhou monarch Wei Sheng bio tech ltd
<120>The Lactobacillus reuteri HI120 of one Expression of Plant Height linoleate isomerase LAI and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1521
<212> DNA
<213>Lactobacillus reuteri (Lactobaccillus reuteri)
<400> 1
tcctggctca ggatgaacgc cggcggtgtg cctaatacat gcaagtcgta cgcactggcc 60
caactgattg atggtgcttg cacctgattg acgatggatc accagtgagt ggcggacggg 120
tgagtaacac gtaggtaacc tgccccggag cgggggataa catttggaaa cagatgctaa 180
taccgcataa caacaaaagc cgcatggctt ttgtttgaaa gatggctttg gctatcactc 240
tgggatggac ctgcggtgca ttagctagtt ggtaaggtaa cggcttacca aggcgatgat 300
gcatagccga gttgagagac tgatcggcca caatggaact gagacacggt ccatactcct 360
acgggaggca gcagtaggga atcttccaca atgggcgcaa gcctgatgga gcaacaccgc 420
gtgagtgaag aagggtttcg gctcgtaaag ctctgttgtt ggagaagaac gtgcgtgaga 480
gtaactgttc acgcagtgac ggtatccaac cagaaagtca cggctaacta cgtgccagca 540
gccgcggtaa tacgtaggtg gcaagcgtta tccggattta ttgggcgtaa agcgagcgca 600
ggcggttgct taggtctgat gtgaaagcct tcggcttaac cgaagaagtg catcggaaac 660
cgggcgactt gagtgcagaa gaggacagtg gaactccatg tgtagcggtg gaatgcgtag 720
atatatggaa gaacaccagt ggcgaaggcg gctgtctggt ctgcaactga cgctgaggct 780
cgaaagcatg ggtagcgaac aggattagat accctggtag tccatgccgt aaacgatgag 840
tgctaggtgt tggagggttt ccgcccttca gtgccggagc taacgcatta agcactccgc 900
ctggggagta cgaccgcaag gttgaaactc aaaggaattg acgggggccc gcacaagcgg 960
tggagcatgt ggtttaattc gaagctacgc gaagaacctt accaggtctt gacatcttgc 1020
gctaacctta gagataaggc gttcccttcg gggacgcaat gacaggtggt gcatggtcgt 1080
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgttact 1140
agttgccagc attgagttgg gcactctagt gagactgccg gtgacaaacc ggaggaaggt 1200
ggggacgacg tcagatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga 1260
cggtacaacg agtcgcaaac tcgcgagagt aagctaatct cttaaagccg ttctcagttc 1320
ggactgtagg ctgcaactcg cctacacgaa gtcggaatcg ctagtaatcg cggatcagca 1380
tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgggagtttg 1440
taacgcccaa agtcggtggc ctaaccttta tggagggagc cgcctaaggc gggacagatg 1500
actggggtga agtcgtaaca a 1521
Claims (3)
1. the Lactobacillus reuteri HI120 of an Expression of Plant Height linoleate isomerase LAI, it is characterized in that：The bacterial strain is in 2016 11
It is preserved within 21st Guangdong Province's Culture Collection the moon, deposit number is GDMCC NO：60119.
2. a kind of microorganism live bacteria preparation, it is characterized in that：Contain the high expression linoleate isomerase LAI described in claim 1
Lactobacillus reuteri HI120.
3. the microorganism live bacteria preparation described in claim 2 has effects that prevention and/or auxiliary treatment obese diabetes in preparation
And there is application in prevention and/or the food additives of auxiliary treatment colorectal cancer, food or drug.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114269897A (en) * | 2019-09-26 | 2022-04-01 | 精密生物集团有限公司 | Lactobacillus reuteri |
| CN114561313A (en) * | 2021-08-26 | 2022-05-31 | 广州维生君生物科技有限公司 | Lactobacillus gasseri and application thereof |
| CN115558620A (en) * | 2022-09-30 | 2023-01-03 | 微康益生菌(苏州)股份有限公司 | Lactic acid bacteria freeze-drying protective agent and preparation method and application thereof |
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| CN105567586A (en) * | 2015-12-21 | 2016-05-11 | 南昌大学 | Lactobacillus plantarum having anti-diabetic function and application thereof |
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| CN105567586A (en) * | 2015-12-21 | 2016-05-11 | 南昌大学 | Lactobacillus plantarum having anti-diabetic function and application thereof |
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| SARA E JONES ET AL.: "Probiotic Lactobacillus reuteri biofilms produce antimicrobial and anti-inflammatory factors", 《BMC MICROBIOL》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114269897A (en) * | 2019-09-26 | 2022-04-01 | 精密生物集团有限公司 | Lactobacillus reuteri |
| CN114561313A (en) * | 2021-08-26 | 2022-05-31 | 广州维生君生物科技有限公司 | Lactobacillus gasseri and application thereof |
| CN115558620A (en) * | 2022-09-30 | 2023-01-03 | 微康益生菌(苏州)股份有限公司 | Lactic acid bacteria freeze-drying protective agent and preparation method and application thereof |
| CN115558620B (en) * | 2022-09-30 | 2023-10-20 | 微康益生菌(苏州)股份有限公司 | Lactic acid bacteria freeze-drying protective agent and preparation method and application thereof |
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