CN105505815A - Lactobacillus mucosae with anti-aging function - Google Patents
Lactobacillus mucosae with anti-aging function Download PDFInfo
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Abstract
The invention discloses novel lactobacillus mucosae CCTCC NO:M 2015581 with high anti-oxidation activity. The lactobacillus mucosae is separated from faeces of healthy and longevous old people, is gram-positive bacteria, has very high free radical scavenging capacity, can improve the activity of antioxidant enzymes in a living body, can delay aging, has excellent bile salt resistance and can be developed into a micro-ecological microbial agent and used as a food additive to improve the gastrointestinal tract of a host.
Description
Technical field
The invention belongs to microorganism field, relate to new Lactobacillus mucosae (Lactobacillusmucosae) bacterial strain and prebiotic function thereof, in particular for scavenging free radicals and the function delayed senility.
Background technology
Intestinal microflora is as virtual " organ ", affect the development growth of human body, nutrition and health, even itself and metabolic disease (irritable bowel syndrome, inflammatory bowel, cardiovascular disorder, diabetes, fat etc.) closely related, the cognation of intestinal microflora and HUMAN HEALTH and even longevity.
High fields village of Liuyang City of Hunan Province geographical position relative closure, its villager is not only long-lived, and does not generally have chronic disease.Form sharp contrast therewith, surrounding area is both without Phenomenon of longevity, and cardiovascular disorder, and the disease such as chronic enterogastritis and cancer happens occasionally.
The adjustable body gastrointestinal tract colony balance of milk-acid bacteria, improves immunity of organisms and reduces serum cholesterol, hypotensive, and the special physiological of the aspects such as anti-oxidant Tumor suppression generation is active.Bifidus longum bb BBMN68 is separated from Bama of Guangxi long lived elder ight soil, and its living bacterial liquid has functions of loosening bowel relieving constipation to Constipation Model mouse, improves immunizing power.Lactobacillus salivarius FDB86 can alleviate the detrimentally affect of dimethylhydrazine to intestinal microflora, makes intestinal microflora level off to standard state, to the regulating effect of colorectal carcinoma Bacteria from Gl Tract of Rats change.Bio-oxidation is the metabolic important physiological process of body in addition, but can produce multiple free radical in this process, the accumulation of free radical, can shine into vivo oxidation damage and increase gradually, cause senile chronic disease to occur, and remove too much free radical and can delay senility.
So far, do not report that the bacterial strain of any Lactobacillus mucosae species is to the scavenging(action) of free radical and the function that delays senility.
Summary of the invention
Now, a kind of Lactobacillus mucosae ZDY308 bacterial strain of the present inventor's isolation identification is a class anaerobism, asporogenous gram-positive microorganism, and thalline is rod-short, is creamy white transparent in MRS Agar Plating, circular, smooth bacterium colony.Lactobacillus mucosae ZDY308 can grow on MRS broth culture, is gram-positive microorganism.
It is to 0.4%H
2o
2have higher tolerance, to hydroxyl radical free radical, DPPH free radical, superoxide anion, has high clearance rate, and has strong total reducing power.
ZDY308 can also improve activities of antioxidant enzymes in the serum of mouse aging and liver, reduces the content of unwanted metabolic products (as: mda) in serum and liver, delays the senile symptom of mouse.
Bacterial strain has good tolerance to simulated gastric fluid and simulated intestinal fluid.
Lactobacillus mucosae ZDY308 of the present invention, as probiotic bacterium, not only has extremely strong Scavenging ability, and has good acid and bile salt tolerance, can alleviate the ill symptoms of D-semi-lactosi inducing mouse, extends mouse life, promotes host health.It is the bacterial classification of a strain excellent property.Therefore there is very high researching value and using value.
Lactobacillus mucosae ZDY308 (LactobacillusmucosaeZDY308) of the present invention, is deposited in China typical culture collection center (Wuhan, China Wuhan University) on September 28th, 2015, numbering: CCTCCNO:M2015581.
A kind of microbiobacterial agent, containing described Lactobacillus mucosae bacterial strain ZDY308.
Comprise the milk-product of described Lactobacillus mucosae, vegetables product and beverage product.
A kind of composition, comprises described Lactobacillus mucosae bacterial strain, or by the microbiobacterial agent that described method obtains, or by the milk-product of described method acquisition, vegetables product and beverage product.
Described composition is food, healthcare products or skin care product.
Lactobacillus mucosae of the present invention can be used for the composition making treatment and prevention of intestinal tract disease, delays senility.
Accompanying drawing explanation
Fig. 1 Lactobacillus mucosae ZDY308 fermented supernatant fluid, bacterial strain and Yoghourt are to the Scavenging activity of DPPH free radical
Fig. 2 Lactobacillus mucosae ZDY308 fermented supernatant fluid, bacterial strain and Yoghourt are to the Scavenging activity of hydroxyl radical free radical
Fig. 3 Lactobacillus mucosae ZDY308 fermented supernatant fluid, bacterial strain and Yoghourt are to the Scavenging activity of superoxide anion
Fig. 4 Lactobacillus mucosae ZDY308 fermented supernatant fluid, total reducing power evaluation of bacterial strain and Yoghourt
Fig. 5 Lactobacillus mucosae ZDY308 is to the tolerance of simulated gastric fluid
Fig. 6 Lactobacillus mucosae ZDY308 is to the tolerance of simulated intestinal fluid
Fig. 7 Lactobacillus mucosae ZDY308 is on the impact of the genetic expression of intestinal epithelial cells HT-29
Fig. 8 Lactobacillus mucosae ZDY308 is on the impact of the genetic expression of intestinal epithelial cells CaCO-2
Fig. 9 Lactobacillus mucosae ZDY308 is on the impact of antioxidase and mda (MDA) in D-semi-lactosi mice serum
Figure 10 Lactobacillus mucosae ZDY308 is on the impact of antioxidase and mda (MDA) in D-semi-lactosi mouse liver
Figure 11 Lactobacillus mucosae ZDY308 is on the impact of D-semi-lactosi mouse small intestine genetic expression
Figure 12 Lactobacillus mucosae ZDY308 is on the impact in mouse aging life-span
Embodiment
Explain the present invention further below in conjunction with example, but embodiment is only for illustration of the present invention, and range of application of the present invention can not be limited by these embodiments by any way.
Embodiment 1: the Isolation and ldentification of Lactobacillus mucosae ZDY308
Lactobacillus mucosae ZDY308 is separated from the ight soil of good health and a long life old man.Get 1g ight soil to be dissolved in 10mL10% peptone solution, fully dissolve, filter paper elimination residue, filtrate with the centrifugal 10min of 5000rmp, supernatant discarded, collecting precipitation, resuspended with 1mL10% peptone water.Be inoculated in MRS liquid nutrient medium by 1%, incubated overnight.Nutrient solution adopts 10 times of gradient dilutions, and gradient dilution is 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8with 10
-9, each gradient is got 1mL and is spread evenly across on the agar plate of MRS meat soup respectively, 37 DEG C of Anaerobic culturel 48h.Picking list bacterium colony, is separated at flat lining out, repeatedly carries out purifying cultivation, observes colonial morphology select gram-positive microorganism by gram staining method.To be separated after the strain liquid that obtains cultivates, slat chain conveyor, with the single bacterium colony of transfering loop picking as template, carry out bacterium colony PCR.Positive PCR primer is delivered to company's order-checking.
The 16SrRNA base sequence analysis of institute's isolate bacterial strain is checked order, result:
GGGGGGGGGTGCCTATACTGCAGTCGAACGCGTTGGCCCAACTGATTGAACGTGCTTGCACGGACTTGACGTTGGTTTACCAGCGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCCAAAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAATTTGAATCGCATGATTCAAATTTAAAAGATGGTTTCGGCTATCACTTTGGGATGGACCTGCGGCGCATTAGCTTGTTGGTAGGGTAACGGCCTACCAAGGCTGTGATGCGTAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAGAGAAGAACGTGCGTGAGAGCAACTGTTCACGCAGTGACGGTATCTAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGATGTGAAAGCCTTTGGCTTAACCAAAGAAGTGCATCGGAAACTGTCAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCCAACCCTAGAGATAGGGCGTTTCCTTCGGGAACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTCAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAACTCGCGAGGGCAAGCTAATCTCTTAAAACCGTTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGCAACACCCAAAGTCGGTGGGGTAACCCTTCGGGGAGCTAGCCGCCTAAAGGTGGACCCAA
Base sequence compares, and find consistent with the Lactobacillus mucosae sequence of lane database, and be stored in Wuhan City's China typical culture collection center, preserving number is CCTCCNO:M2015581.
Lactobacillus mucosae ZDY308 bacterial strain is a class anaerobism, asporogenous gram-positive microorganism, and thalline is rod-short, is creamy white transparent in MRS Agar Plating, circular, smooth bacterium colony.Lactobacillus mucosae ZDY308 can grow on MRS broth culture, is gram-positive microorganism.
Embodiment 2: bacterial strain is to the tolerance of Oxdative stress
By the bacterial strain of fresh culture with 1% ratio be inoculated into containing 0.4mmol/LH
2o
2mRS liquid nutrient medium in, 37 DEG C of Anaerobic culturel 8h, measure the OD of 0h and 8h respectively
630, detect bacterial strain to H
2o
2tolerance.
Result shows: Δ OD>0.9, has extremely strong tolerance.
Embodiment 3: bacterial strain and fermented supernatant fluid scavenging ability of DPPH free radical
Be in 1% access MRS substratum according to inoculum size, anaerobism 37 DEG C cultivates the centrifugal 10min of 18h, 6000r/min, collects fermented supernatant fluid and thalline respectively.Thalline PBS (pH7.2) solution washing 2 times, then with PBS solution resuspended to lactic acid bacteria number be 10
9cfu/mL.The sample that fermentation supernatant and resuspended thalline are tested as scavenging free radicals.
Add 2mLDPPH ethanol solution (0.2mmol/L) in 2mL sample to mix, the centrifugal 10min of room temperature lucifuge reaction 30min, 6000r/min, gets supernatant liquor and measures absorbance A in 517nm place
i; Blank group replaces DPPH ethanol solution A with equal-volume dehydrated alcohol
o, control group replaces sample solution A with equal-volume blank solvent
j, and with equal-volume distilled water and the zeroing of alcohol mixeding liquid blank.Clearance rate (%)=100-(A
i-A
o)/A
j× 100
Experimental result:
The clearance rate of Yoghourt to DPPH of Lactobacillus mucosae ZDY308 fermentation is the highest as shown in Figure 1, and more than 80%, fermented supernatant fluid and bacterial strain also show higher clearance rate to DPPH free radical.
Embodiment 4: bacterial strain and fermented supernatant fluid remove the ability of hydroxyl radical free radical
The phenanthroline (6mmol/L) of 0.5mL, the FeSO of 0.5mL
4solution (6mmol/L) mixes with the PBS solution (pH7.2) of 1.0mL.In this system, add 0.5mL sample and 0.5mL0.1% hydrogen peroxide again, with distilled water, cumulative volume is settled to 4.0mL.At 37 DEG C, hatch 1h after mixing, under 536nm, read absorbancy.The clearance rate of hydroxy radical qiao is calculated as follows: clearance rate (%)=[(A
s-A
o)/(A-A
o)] × 100
A
s: the absorbance of sample; A
o: use H
2o substitutes sample; A: use H
2o substitutes H
2o
2and sample
Experimental result:
See Fig. 2, result shows, the clearance rate of fermented supernatant fluid to hydroxyl radical free radical of Lactobacillus mucosae ZDY308 is the highest, and more than 80%, and Yoghourt has higher clearance rate compared with bacterial strain to hydroxyl radical free radical.
Embodiment 5: bacterial strain and fermented supernatant fluid remove the ability of superoxide anion
A
11group: add 1mL pyrogallol solution in 2mLTris-HCl damping fluid (150mMpH8.0), then add 0.5mL sample; A
10group: replace pyrogallol solution with 1mL distilled water; A
01group: replace sample with 0.5mL distilled water; A
00group: replace 1mL pyrogallol and 0.5mL sample with 1.5mL distilled water; Mixing, measures light absorption value in 325nm place after room temperature reaction 30min; Record data also calculate clearance rate:
Clearance rate (%)=[1-(A
11-A
10)/(A
01-A
00)] × 100
Experimental result:
As shown in Figure 3, the fermented supernatant fluid of Lactobacillus mucosae ZDY308 removes the ability of superoxide anion the most by force, and taking second place of fermented yogurt, bacterial strain has good clearance rate to ultra-oxygen anion free radical.
Embodiment 5: bacterial strain and the total reducing power of fermented supernatant fluid measure
Potassium ferricyanide solution and the 0.5mL sample of 0.5mL0.1% is added in 0.5mL0.2MPBS solution (pH6.6); Abundant mixing, 50 DEG C of insulation 20min; Add isopyknic 10% trichoroacetic acid(TCA), the centrifugal 10min of 3000g; Get 1mL supernatant, add 0.175mL0.1%FeCl
3solution, mixes, reaction 10min; 700nm place measures light absorption value.
As shown in Figure 4, total reducing power of the fermented supernatant fluid of Lactobacillus mucosae ZDY308 drinks total reducing power of bacterial strain far away higher than Yoghourt.
Embodiment 6: bacterial strain is to the tolerance evaluation of simulated gastric fluid
Simulated gastric fluid: make final concentration be 3mg/mL by adding stomach en-in Sodium phosphate dibasic-citrate buffer solution (pH2.2), matching while using.Will to H
2o
2the bacterial strain that tolerance is high, the centrifugal 10min of 5000rmp, thalline washes twice, and is resuspended in simulated gastric fluid after thalline washes twice, and keep bacteria concentration identical, 37 DEG C of anaerobism hatch 30min.MRS solid medium live bacterial count is used respectively at 0min and 30min.
Experimental result: as shown in Figure 5, Lactobacillus mucosae ZDY308 30min in simulated gastric fluid, the order of magnitude reduces a level, still can keep higher tolerance to simulated gastric fluid.
Embodiment 7: bacterial strain is to the evaluation of the tolerance of simulated intestinal fluid
Simulated intestinal fluid: PBS pH of buffer is adjusted to 8.0, and add 0.45% cholate, final concentration is the trypsinase of 1mg/mL, matching while using.By resistance to H
2o
2bacterial strain, the centrifugal 10min of 5000rmp, is resuspended in after thalline washes twice in simulated intestinal fluid, keep bacteria concentration identical, hatch 2h altogether for 37 DEG C.0h and 2h uses MRS solid medium live bacterial count respectively.Fig. 6 Lactobacillus mucosae ZDY308 is to the tolerance of simulated intestinal fluid
Experimental result: as shown in Figure 6, Lactobacillus mucosae ZDY308 after 2 hours, does not have the change of the order of magnitude in simulated intestinal fluid, tolerance simulated intestinal fluid that can be fabulous.
Embodiment 8: bacterial strain is to the effect of the antioxidant genes of intestinal epithelial cells
Caco-2 and HT-29 cell cultured in Tissue Culture Flask is hatched 2h altogether with Lactobacillus mucosae ZDY308 respectively, carries the RNA of cell, after reverse transcription, detect the expression of gene with q-PCR.
Experimental result:
As shown in FIG. 7 and 8, after hatching altogether with Lactobacillus mucosae ZDY308, all raise with genes involved that is anti-oxidant and cytodifferentiation in CaCo-2 and HT-29 cell, wherein, in HT-29 cell, gene PRG1 and MT1A significantly raises, in CaCo-2 cell, MT1A and BCL2 significantly raises, and shows, Lactobacillus mucosae ZDY308 has certain antioxygenation to body.
Embodiment 9: bacterial strain is to the effect of D-semi-lactosi inducing mouse
5 weeks large BALB/c mouse, after raising one week, wherein 20 D-semi-lactosi abdominal injections 30 days with 50mg/kg, after 30 days, be one group with 10, experimental group (ZDY308): gavage Lactobacillus mucosae ZDY308 is 28d altogether, and abdominal injection D-semi-lactosi simultaneously; Control group (D-gal): the PBS damping fluid of gavage and bacterial strain re-suspension liquid same volume.10 is blank group (NC): the physiological saline of abdominal injection and D-semi-lactosi Isodose, the PBS damping fluid of gavage and bacterial strain re-suspension liquid same volume after 30 days, continues abdominal injection, totally 28 days simultaneously.Gavage second day of terminating, after eye socket blood sampling, strength vertebra dislocation method puts to death mouse, gets liver and small intestine respectively, for subsequent experimental.
Experimental result:
As shown in Figures 9 and 10, in serum and liver, the content of antioxidase SOD and GSH-PX all increases, and mda reduces, and illustrate, Lactobacillus mucosae ZDY308 can alleviate the oxidative damage of D-semi-lactosi to mouse.
Experimental result:
As shown in figure 11, Lactobacillus mucosae ZDY308 improves the expression amount of mouse intestinal epithelial cells antioxidant genes, demonstrates the mitigation of Lactobacillus mucosae ZDY308 to oxidative damage further from molecular mechanism.
Embodiment 10: bacterial strain is to the retarding action of natural aging mice aging
8 weeks large BALB/c mouse, often organize 10, experimental group (ZDY308): gavage Lactobacillus mucosae ZDY308 is to mice age natural death by random point two groups; Control group: the physiological saline of gavage equal volume is to mouse natural death.Add up the growth life-span of two groups of mouse respectively, and compare analysis.
Experimental result:
As shown in figure 12, continue gavage Lactobacillus mucosae ZDY308 mouse life-span comparatively control group extend 141 days, visible, this bacterial strain has certain retarding action to aging.
Claims (5)
1. a strain is preserved in Lactobacillus mucosae (Lactobacillusmucosae) the bacterial strain ZDY308 of China typical culture collection center CCTCC, and numbering is hidden in preservation: CCTCCM2015581.
2. a microbiobacterial agent, is characterized in that containing Lactobacillus mucosae bacterial strain ZDY308 described in claim 1.
3. comprise from the milk-product of Lactobacillus mucosae according to claim 1, vegetables product and beverage product.
4. a composition, comprises Lactobacillus mucosae bacterial strain according to claim 1, or by the microbiobacterial agent that method according to claim 2 obtains, or by milk-product, vegetables product and beverage product that method according to claim 3 obtains.
5. composition as claimed in claim 4, is characterized in that it is food, healthcare products or skin care product.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486722A (en) * | 2018-12-26 | 2019-03-19 | 江南大学 | One plant of Lactobacillus mucosae and its application with anti influenza ability |
| CN111979145A (en) * | 2020-08-07 | 2020-11-24 | 上海交通大学 | Human-derived lactobacillus mucosae and application thereof |
| CN112877231A (en) * | 2019-11-29 | 2021-06-01 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Lactic acid bacteria with bacteriostatic and antioxidant activity and application thereof |
| CN115786175A (en) * | 2022-09-30 | 2023-03-14 | 广东省农业科学院动物卫生研究所 | Lactobacillus mucosae and application thereof |
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| WO2011107960A1 (en) * | 2010-03-03 | 2011-09-09 | Italstarter S.R.L. | Compositions comprising lactobacillus mucosae for medical use |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486722A (en) * | 2018-12-26 | 2019-03-19 | 江南大学 | One plant of Lactobacillus mucosae and its application with anti influenza ability |
| CN109486722B (en) * | 2018-12-26 | 2020-08-04 | 江南大学 | Lactobacillus mucosae with anti-influenza capacity and application thereof |
| CN112877231A (en) * | 2019-11-29 | 2021-06-01 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Lactic acid bacteria with bacteriostatic and antioxidant activity and application thereof |
| CN112877231B (en) * | 2019-11-29 | 2023-06-23 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Lactic acid bacteria with antibacterial and antioxidant activities and application thereof |
| CN111979145A (en) * | 2020-08-07 | 2020-11-24 | 上海交通大学 | Human-derived lactobacillus mucosae and application thereof |
| CN115786175A (en) * | 2022-09-30 | 2023-03-14 | 广东省农业科学院动物卫生研究所 | Lactobacillus mucosae and application thereof |
| CN115786175B (en) * | 2022-09-30 | 2023-12-08 | 广东省农业科学院动物卫生研究所 | Lactobacillus mucosae and application thereof |
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