CN105505815B - The Lactobacillus mucosae of one plant of anti-senescence function - Google Patents
The Lactobacillus mucosae of one plant of anti-senescence function Download PDFInfo
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Abstract
It is new the invention discloses one plant, Lactobacillus mucosae CCTCC NO:M 2015581 with very strong anti-oxidative activity, it is isolated from good health and a long life old man's excrement, is gram-positive bacteria, there is very strong Scavenging ability, the activity of antioxidase in organism can be enhanced, it delays senescence, while there is outstanding bile tolerance, can develop as microecological microbial agent, it is used as food additives, improves host gastrointestinal tract health.
Description
Technical field
The invention belongs to microorganism field, be related to new Lactobacillus mucosae (Lactobacillus mucosae) bacterial strain and
Its prebiotic function, in particular for removing the function of free radical and anti-aging.
Background technique
Intestinal flora affects development growth, nutrition and the health or even itself and metabolic of human body as virtual " organ "
Disease (intestinal irritable syndrome, inflammatory bowel disease, cardiovascular disease, diabetes, obesity etc.) is closely related, intestinal flora and human body
Health or even long-lived relevance.
High fields village, Liuyang City, Hunan Province geographical location relative closure, villager is not only long-lived, but also generally without chronic disease
Disease.Sharp contrast is formed with this, surrounding area is both without Phenomenon of longevity, and cardiovascular disease, chronic enterogastritis and cancer
Etc. diseases happen occasionally.
Body gastrointestinal tract colony balance is adjusted in lactic acid bacteria, and improving immunity of organisms reduces serum cholesterol, and blood pressure lowering resists
The special physiological activity of oxidizing and depressing tumour generation etc..Bifidobacterium longum BBMN68 is isolated from Bama of Guangxi long lived elder excrement
Just, living bacterial liquid has functions of loosening bowel relieving constipation to Constipation Model mouse, improves immunity.Lactobacillus salivarius FDB86 can alleviate two
Adverse effect of the methylhydrazine to intestinal flora makes intestinal flora level off to normal condition, to the variation of colon cancer Bacteria from Gl Tract of Rats
Adjustment effect.In addition biological oxidation is the important physiology course of body metabolism, but can generate a variety of free radicals during this,
The accumulation of free radical can gradually increase according at vivo oxidation damage, lead to senile chronic disease, and remove excessive free radical
It can delay aging.
So far, scavenging effect and anti-aging of the bacterial strain to free radical of any Lactobacillus mucosae species are not reported
Function.
Summary of the invention
Now, the present inventor's separation identifies a kind of Lactobacillus mucosae ZDY308 bacterial strain, is a kind of anaerobism, asporogenous
Gram-positive bacteria, thallus are in rod-short, are creamy white in MRS Agar Plating transparent, round, smooth bacterium colony.
Lactobacillus mucosae ZDY308 can be grown on MRS broth bouillon, be gram-positive bacteria.
It is to 0.4%H2O2There is higher tolerance, to hydroxyl radical free radical, DPPH free radical, superoxide anion has height
Clearance rate, and have strong total reducing power.
ZDY308 can also improve activities of antioxidant enzymes in the serum and liver of mouse aging, and reducing has in serum and liver
The content of evil metabolite (such as: malonaldehyde), delays the senile symptom of mouse.
Bacterial strain has preferable tolerance to simulate the gastric juice and simulated intestinal fluid.
Lactobacillus mucosae ZDY308 of the invention not only has extremely strong Scavenging ability as probiotics, and has
Good acid and bile salt tolerance can alleviate the ill symptoms of D- galactolipin inducing mouse, extend mouse life, promote host strong
Health.It is the strain of one plant of function admirable.Therefore there is very high researching value and application value.
Lactobacillus mucosae ZDY308 (Lactobacillus mucosae ZDY308) of the present invention, on September 28th, 2015
It is deposited in China typical culture collection center (Wuhan, China Wuhan University), numbers: CCTCC NO:M 2015581.
A kind of microbial bacterial agent contains the Lactobacillus mucosae bacterial strain ZDY308.
Dairy products, vegetable product and beverage product comprising the Lactobacillus mucosae.
A kind of composition including the Lactobacillus mucosae bacterial strain, or passes through the method microbial bacteria obtained
Agent, or the dairy products, vegetable product and the beverage product that are obtained by the method.
The composition is food, health care product or skin care item.
Lactobacillus mucosae of the present invention can be used for making the composition for treating and preventing intestines problem, delay senescence.
Detailed description of the invention
The Scavenging activity of Fig. 1 Lactobacillus mucosae ZDY308 fermented supernatant fluid, bacterial strain and Yoghourt to DPPH free radical
The Scavenging activity of Fig. 2 Lactobacillus mucosae ZDY308 fermented supernatant fluid, bacterial strain and Yoghourt to hydroxyl radical free radical
The Scavenging activity of Fig. 3 Lactobacillus mucosae ZDY308 fermented supernatant fluid, bacterial strain and Yoghourt to superoxide anion
Total reducing power of Fig. 4 Lactobacillus mucosae ZDY308 fermented supernatant fluid, bacterial strain and Yoghourt is evaluated
Tolerance of Fig. 5 Lactobacillus mucosae ZDY308 to simulate the gastric juice
Tolerance of Fig. 6 Lactobacillus mucosae ZDY308 to simulated intestinal fluid
Influence of Fig. 7 Lactobacillus mucosae ZDY308 to the gene expression of enterocyte HT-29
Influence of Fig. 8 Lactobacillus mucosae ZDY308 to the gene expression of enterocyte CaCO-2
Influence of Fig. 9 Lactobacillus mucosae ZDY308 to antioxidase and malonaldehyde (MDA) in D- galactolipin mice serum
Influence of Figure 10 Lactobacillus mucosae ZDY308 to antioxidase and malonaldehyde (MDA) in D- galactolipin mouse liver
Influence of Figure 11 Lactobacillus mucosae ZDY308 to D- galactolipin mouse small intestine gene expression
Influence of Figure 12 Lactobacillus mucosae ZDY308 to the mouse aging service life
Specific embodiment
The present invention is explained further below in conjunction with example, but embodiment is merely to illustrate the present invention, and of the invention answers
It cannot be limited in any way by these embodiments with range.
Embodiment 1: the separation and identification of Lactobacillus mucosae ZDY308
Lactobacillus mucosae ZDY308 is separated from the excrement of good health and a long life old man.1g excrement is taken to be dissolved in 10mL10% albumen
It in peptone solution, sufficiently dissolves, filter paper filters off residue, and filtrate is centrifuged 10min with 5000rmp, discards supernatant, and collects precipitating, uses 1mL
10% peptone water is resuspended.It is inoculated into MRS fluid nutrient medium, is incubated overnight by 1%.Culture solution uses 10 times of gradient dilutions,
Gradient dilution is 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8With 10-9, each gradient takes 1mL to be spread evenly across respectively
On the agar plate of MRS meat soup, 37 DEG C of Anaerobic culturel 48h.Picking single colonie separates in flat lining out, purifying is repeated
Culture selects gram-positive bacteria with Gram's stain observation colonial morphology.After isolated strain liquid culture, put down
Plate culture, the bacterium colony for using oese picking single carry out bacterium colony PCR as template.Positive PCR product is sent to company and is sequenced.
The 16S rRNA base sequence analysis of separated bacteria strain is sequenced, as a result:
GGGGGGGGGTGCCTATACTGCAGTCGAACGCGTTGGCCCAACTGATTGAACGTGCTTGCACGGACTTGA
CGTTGGTTTACCAGCGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCCAAAGCGGGGGATAACATTTGGA
AACAGATGCTAATACCGCATAACAATTTGAATCGCATGATTCAAATTTAAAAGATGGTTTCGGCTATCACTTTGGGA
TGGACCTGCGGCGCATTAGCTTGTTGGTAGGGTAACGGCCTACCAAGGCTGTGATGCGTAGCCGAGTTGAGAGACTG
ATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCA
AGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAGAGAAGAACGTGCGT
GAGAGCAACTGTTCACGCAGTGACGGTATCTAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATAC
GTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGATGTGAAAGCCT
TTGGCTTAACCAAAGAAGTGCATCGGAAACTGTCAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCG
GTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAA
AGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTC
CGCCCTTCAGTGCCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAAT
TGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACAT
CTTGCGCCAACCCTAGAGATAGGGCGTTTCCTTCGGGAACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTG
TCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTCAGTTGGGCACTCTAG
TGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACA
CGTGCTACAATGGACGGTACAACGAGTCGCGAACTCGCGAGGGCAAGCTAATCTCTTAAAACCGTTCTCAGTTCGGA
CTGCAGGCTGCAACTCGCCTGCACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCC
CGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGCAACACCCAAAGTCGGTGGGGTAACCCTTCGGGGAGC
TAGCCGCCTAAAGGTGGACCCAA
Base sequence is compared, and discovery is consistent with the Lactobacillus mucosae sequence of lane database, and is stored in Wuhan
City's China typical culture collection center, deposit number are CCTCC NO:M 2015581.
Lactobacillus mucosae ZDY308 bacterial strain is a kind of anaerobism, and asporogenous gram-positive bacteria, thallus is in rod-short,
It is creamy white in MRS Agar Plating transparent, round, smooth bacterium colony.Lactobacillus mucosae ZDY308 can be in MRS meat
It is grown on soup culture medium, is gram-positive bacteria.
Embodiment 2: tolerance of the bacterial strain to Oxdative stress
The bacterial strain of fresh cultured is inoculated into H containing 0.4mmol/L with 1% ratio2O2MRS fluid nutrient medium in, 37
DEG C Anaerobic culturel 8h measures the OD of 0h and 8h respectively630, bacterial strain is detected to H2O2Tolerance.
The result shows that: Δ OD > 0.9 has extremely strong tolerance.
Embodiment 3: bacterial strain and fermented supernatant fluid scavenging ability of DPPH free radical
It is in 1% access MRS culture medium according to inoculum concentration, anaerobism 37 DEG C of cultures 18h, 6000r/min are centrifuged 10min, point
It Shou Ji not fermented supernatant fluid and thallus.Thallus is washed 2 times with PBS (pH7.2) solution, then is resuspended with PBS solution to lactic acid bacteria number
It is 109cfu/mL.Fermentation supernatant and the thallus of resuspension are as the sample for removing free radical test.
2mL DPPH ethanol solution (0.2mmol/L) is added in 2mL sample to be uniformly mixed, room temperature is protected from light
30min, 6000r/min are centrifuged 10min, and supernatant is taken to measure absorbance value A at 517nmi；Blank group is with isometric anhydrous second
Alcohol replaces DPPH ethanol solution Ao, control group is with isometric blank solvent instead of sample solution Aj, and with isometric distilled water
It returns to zero with alcohol mixeding liquid blank.Clearance rate (%)=100- (Ai-Ao)/Aj×100
Experimental result:
The Yoghourt of Lactobacillus mucosae ZDY308 fermentation as shown in Figure 1 is to the clearance rate highest of DPPH, more than 80%, in fermentation
Clear liquid and bacterial strain also show higher clearance rate to DPPH free radical.
Embodiment 4: the ability of bacterial strain and fermented supernatant fluid removing hydroxyl radical free radical
The Phen (6mmol/L) of 0.5mL, the FeSO of 0.5mL4The PBS solution of solution (6mmol/L) and 1.0mL
(pH7.2) it mixes.0.5mL sample and 0.1% hydrogen peroxide of 0.5mL is added into this system again, is determined total volume with distilled water
Hold to 4.0mL.It is incubated for 1h at 37 DEG C after mixing, reads absorbance under 536nm.The clearance rate of hydroxy radical is counted as the following formula
It calculates: clearance rate (%)=[(As-Ao)/(A-Ao)]×100
As: the absorbance value of sample；Ao: use H2O substitutes sample；A: H is used2O substitutes H2O2And sample
Experimental result:
See Fig. 2, the results showed that, the fermented supernatant fluid of Lactobacillus mucosae ZDY308 to the clearance rate highest of hydroxyl radical free radical,
More than 80%, and Yoghourt has higher clearance rate to hydroxyl radical free radical compared with bacterial strain.
Embodiment 5: the ability of bacterial strain and fermented supernatant fluid removing superoxide anion
A11Group: 1mL pyrogallol solution is added in 2mL Tris-HCl buffer (150mM pH 8.0), adds
0.5mL sample；A10Group: replace pyrogallol solution with 1mL distilled water；A01Group: replace sample with 0.5mL distilled water；A00Group:
Replace 1mL pyrogallol and 0.5mL sample with 1.5mL distilled water；It mixes, measures extinction at 325nm after reacting at room temperature 30min
Value；Record data simultaneously calculate clearance rate:
Clearance rate (%)=[1- (A11-A10)/(A01-A00)]×100
Experimental result:
As shown in figure 3, the ability that the fermented supernatant fluid of Lactobacillus mucosae ZDY308 removes superoxide anion is most strong, fermentation acid
Milk takes second place, and bacterial strain has preferable clearance rate to ultra-oxygen anion free radical.
Embodiment 5: bacterial strain and the total reducing power measurement of fermented supernatant fluid
The potassium ferricyanide solution and 0.5mL sample of 0.5mL 0.1% are added in 0.5mL 0.2M PBS solution (pH 6.6)；
It mixes well, 50 DEG C of heat preservation 20min；10% isometric trichloroacetic acid is added, 3000g is centrifuged 10min；1mL supernatant is taken, is added
0.175mL 0.1%FeCl3Solution is uniformly mixed, and reacts 10min；Light absorption value is measured at 700nm.
As shown in figure 4, total reducing power of the fermented supernatant fluid of Lactobacillus mucosae ZDY308 is significantly larger than Yoghourt and drinks bacterial strain
Total reducing power.
Embodiment 6: bacterial strain evaluates the tolerance of simulate the gastric juice
Simulate the gastric juice: addition pepsin in disodium hydrogen phosphate-citrate buffer solution (pH 2.2) is made final concentration of
3mg/mL, matching while using.It will be to H2O2The high bacterial strain of tolerance, 5000rmp be centrifuged 10min, cell washing twice, cell washing
It is resuspended in simulate the gastric juice afterwards twice, keeps bacteria concentration identical, 37 DEG C of anaerobism are incubated for 30min.It is used respectively in 0min and 30min
MRS solid medium count plate.
Experimental result: as shown in figure 5, Lactobacillus mucosae ZDY308 30min in simulate the gastric juice, the order of magnitude reduces a water
It is flat, it remains to keep higher tolerance to simulate the gastric juice.
Embodiment 7: evaluation of the bacterial strain to the tolerance of simulated intestinal fluid
Simulated intestinal fluid: PBS buffer solution pH is adjusted to 8.0, and adds 0.45% cholate, the pancreas egg of final concentration of 1mg/mL
White enzyme, matching while using.By resistance to H2O2Bacterial strain, 5000rmp is centrifuged 10min, and cell washing is resuspended in simulated intestinal fluid afterwards twice,
Keep bacteria concentration identical, 37 DEG C are incubated for 2h altogether.0h and 2h uses MRS solid medium count plate respectively.Fig. 6 Lactobacillus mucosae
Tolerance of the ZDY308 to simulated intestinal fluid
Experimental result: as shown in fig. 6, Lactobacillus mucosae ZDY308 is in simulated intestinal fluid after 2 hours, the not change of the order of magnitude
Change, the fabulous tolerance simulated intestinal fluid of energy.
Embodiment 8: effect of the bacterial strain to the antioxidant genes of enterocyte
Caco-2 and HT-29 cell cultured in Tissue Culture Flask is incubated for Lactobacillus mucosae ZDY308 altogether respectively
2h mentions the RNA of cell, after reverse transcription, with the expression of q-PCR detection gene.
Experimental result:
As shown in FIG. 7 and 8, after being incubated for altogether with Lactobacillus mucosae ZDY308, in CaCo-2 and HT-29 cell with it is anti-oxidant and
The related gene of cell differentiation raises, wherein gene PRG1 and MT1A are significantly raised in HT-29 cell, in CaCo-2 cell
MT1A and BCL2 are significantly raised, and show that Lactobacillus mucosae ZDY308 has certain antioxidation to body.
Embodiment 9: effect of the bacterial strain to D- galactolipin inducing mouse
5 weeks big BALB/c mouses are raised after a week, wherein 20 D- galactolipins with 50mg/kg are injected intraperitoneally 30 days,
After 30 days, with 10 for one group, experimental group (ZDY308): the total 28d of stomach-filling Lactobacillus mucosae ZDY308, and D- is injected intraperitoneally simultaneously
Galactolipin；Control group (D-gal): the PBS buffer solution of stomach-filling and bacterial strain re-suspension liquid same volume.10 are blank group (NC): abdomen
Chamber injection is with the same dose of physiological saline of D- galactolipin, the PBS buffer solution of stomach-filling and bacterial strain re-suspension liquid same volume after 30 days, together
Shi Chixu intraperitoneal injection, totally 28 days.Terminate second day of stomach-filling, after eye socket blood sampling, strength vertebra dislocation method puts to death mouse, takes liver respectively
Dirty and small intestine is used for subsequent experimental.
Experimental result:
As shown in Figures 9 and 10, the content of antioxidase SOD and GSH-PX increase in serum and liver, and malonaldehyde is reduced,
Illustrate, Lactobacillus mucosae ZDY308 can alleviate D- galactolipin to the oxidative damage of mouse.
Experimental result:
As shown in figure 11, Lactobacillus mucosae ZDY308 improves the expression quantity of mouse enterocyte antioxidant genes, into
One step demonstrates relaxation effect of the Lactobacillus mucosae ZDY308 to oxidative damage from molecular mechanism.
Embodiment 10: retarding action of the bacterial strain to natural aging mice aging
8 weeks big BALB/c mouses are divided to two groups at random, and every group 10, experimental group (ZDY308): stomach-filling Lactobacillus mucosae
ZDY308 is to mice age natural death；Control group: the physiological saline of stomach-filling equal volume to mouse natural death.It counts respectively
The growth service life of two groups of mouse, and compare analysis.
Experimental result:
As shown in figure 12, the service life for continuing the mouse of stomach-filling Lactobacillus mucosae ZDY308 extends 141 days compared with control group, can
See, which has certain retarding action to aging.
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