CN104140937B - Lactobacillus plantarum KLDS1001 and application of lactobacillus plantarum KLDS1001 to inhibition of streptococcus mutans - Google Patents
Lactobacillus plantarum KLDS1001 and application of lactobacillus plantarum KLDS1001 to inhibition of streptococcus mutans Download PDFInfo
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Abstract
The invention discloses lactobacillus plantarum KLDS1001 and application of the lactobacillus plantarum KLDS1001 to inhibition of streptococcus mutans, belonging to the technical field of microorganisms. The lactobacillus plantarum KLDS1001 provided by the invention is collected in China general microbiological culture collection center (CGMCC) with the address of institute of microbiology of Chinese academy of sciences, 1# yard, beichen road west, chaoyang district, Beijing; the collection number is CGMCC No.9066; the collection date is April 16th, 2014. The lactobacillus plantarum KLDS1001 has an inhibition effect on the streptococcus mutans, can serve as an additive to be added to health drugs and such oral health products as chewing gum and is used for oral health; the lactobacillus plantarum KLDS1001 can serve as one of ingredients of oral cleaning products to be added into toothpaste and mouth wash for cleaning the oral cavity and reducing the number of streptococcus mutans in the oral cavity; a probiotic therapy method is developed for preventing and treating oral diseases such as decayed teeth.
Description
Technical field
The invention belongs to microbial technology field, it is related to one plant of Lactobacillus plantarum with the prebiotic function in potential oral cavity
(lactobacillus plantarum) and its application.
Background technology
Lactobacillus fermenti (lactobacillus fermentum) y29 has certain suppression to oral cavity Streptococcus mutans
Effect；Boil on the nape opposite the mouth intracavity lysozyme and hydrogen peroxide have certain toleration, can adapt to oral environment.But it does not have with regard to
The acid producing ability of bacterial strain and the correlation analysiss of cariogenic potential, bacterial strain can produce extracellular polysaccharide, has certain tooth surface adhesion
Ability, is potential cariogenic bacteria.
Lactobacillus plantarum ho-69 has weaker tooth surface adhesive capacity, non-acid stronger to the adhesive capacity of oral mucosa
Antibacterial substance has bacteriostasis to Streptococcus mutans and has the fungistatic effect of wide spectrum.But, lack and peace applied for bacterial strain
The evaluation of full property, does not resist the property of medicine, causes the characteristic of mucosal infections to be studied.
Content of the invention
It is an object of the invention to provide one plant of Lactobacillus plantarum inhibited to oral cavity Streptococcus mutans
Klds1001 and its application.
The Lactobacillus plantarum klds1001 that the present invention provides, is preserved in China Committee for Culture Collection of Microorganisms common
Microorganism center (cgmcc), address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation
Numbering is: cgmcc no.9066, and preservation date is on 04 16th, 2014, and the Classification And Nomenclature of strain is Lactobacillus plantarum
(lactobacillus plantarum).Klds1001 is gram-positive short, paired or one-tenth catenation.Put down in mrs
After plate culture, bacterium colony is white, circular, and pillow is smooth, neat in edge.Its thalline and colony morphology characteristic are as Figure 1-3.
The Lactobacillus plantarum klds1001 that the present invention provides is 19.47 to the antibacterial circle diameter of Streptococcus mutans ±
0.50mm, with internal and international on research compared with bacteriostatic activity preferable.By measuring Lactobacillus plantarum klds1001 to variation
Streptococcus form the impact of Mycoderma on teat glass surface, more intuitively find, bacterial strain can effectively reduce variation hammer
Quantity in the middle of Mycoderma for the bacterium.From the point of view of the result of above experiment in vitro, bacterial strain can effectively suppress the life of Streptococcus mutans
Grow and reduce the quantity of Streptococcus mutans in Dental Plaque.Compared with traditional probiotic strain lgg, the plant breast bar that the present invention provides
Bacterium klds1001 bacteriostasis are stronger, and the ability that suppression Streptococcus mutans Mycoderma is formed is close.
The Lactobacillus plantarum klds1001 that the present invention provides can keep good growth shape under conditions of ph is for 3-5
State, keeps the viable count of 4.79 ± 0.49logcfu, illustrates that bacterial strain can well adapt to after culture 1h under conditions of ph is for 2
Change due to ph in the oral cavity causing of taking food；Bacterial strain well-grown under conditions of concentration of hydrogen peroxide is for 1mm/l, in 5mm/
After culture 6h under conditions of l, the bacterium number of survival is the peroxide in 3.29 ± 0.07logcfu boil on the nape opposite the mouth intracavity and dental care products
Change hydrogen and there is stronger toleration, oral environment can be well adapted to.
As new oral cavity probiotic bacteria, lactic acid bacteria is likely to be of following harm during application: (1) is due to having relatively
Strong acid producing ability and have certain pathogenic；(2) drug resistance gene may be carried, there is drug resistance gene horizontal transfer
Harm；(3) infection of oral mucosa may be caused.Lactobacillus plantarum klds1001 cultivates 72h and decomposes hydroxyapatite generation calcium
Measure as 0.29 ± 0.02 μ g/ml, only there is slight degradation capability, this degradation amount similar to traditional probiotic bacteria lgg (0.27 ±
0.01 μ g/ml), illustrate that bacterial strain decomposes enamel and leads to the ability of dental caries weak.Bacterial strain does not have hemolytic it is impossible to cause in oral cavity
The infection of mucosa.Bacterial strain has drug resistance to vancomycin, clindamycin and streptomycin.
The Lactobacillus plantarum klds1001 that the present invention provides can be as oral health-care product, such as: chewing gum, health care medicine
A kind of adding ingredient, for oral cavity health；Toothpaste can be added to as one of composition of oral-cavity cleaning products, collutory is worked as
In, for the quantity of Streptococcus mutans in oral cavity cleaning and minimizing oral cavity；Exploitation probiotic therapy is used for preventing and treating dental caries
Etc. oral disease.
Preservation information:
Depositary institution's full name: China Committee for Culture Collection of Microorganisms's common micro-organisms center；
Depositary institution's abbreviation: cgmcc；
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences；
Preservation date: on 04 16th, 2014；
Deposit number: cgmcc no.9066.
Brief description
Fig. 1 is klds1001 colonial morphology；
Fig. 2 observes form for klds1001 bacterium colony under high power microscope；
Fig. 3 is thalli morphology after klds1001 Gram’s staining；
Fig. 4 is the impact that bacterial strain klds1001 and lgg is formed to Streptococcus mutans Mycoderma, bacterial strain klds1001 and lgg energy
Enough effectively suppression Streptococcus mutans Mycodermas are formed, and bacterium and probiotic bacteria lgg have similar bacteriostasis；
Fig. 5 be bacterial strain klds1001, lgg and Streptococcus mutans degradation capability to hydroxyapatite, bacterial strain klds1001 with
Lgg is respectively provided with weaker hydroxyapatite degradation capability；
Fig. 6 is the impact that different ph grow to bacterial strain klds1001；
Fig. 7 is the hydrogen peroxide impact that bacterial strain klds1001 is grown of variable concentrations, and bacterial strain klds1.0659 is in low ph
Culture a period of time under conditions of existing with hydrogen peroxide, all can keep necessarily surviving bacterium number, stronger to the resistivity of environment.
Specific embodiment
Below in conjunction with the accompanying drawings technical scheme is further described, but is not limited thereto, every to this
Inventive technique scheme is modified or equivalent, without deviating from the spirit and scope of technical solution of the present invention, all should cover
In protection scope of the present invention.
The Lactobacillus plantarum klds1001 that the present invention provides separates from the yogurt of Xinjiang and obtains, and its separation method is such as
Under: take the yoghurt example of a certain amount of Xinjiang natural fermentation, be enriched with degreasing milk medium, line mrs flat board
(ph6.5) in, 37 DEG C of culture 24h, picking is circular, and white milky feature bacterium colony carries out Gram’s staining, picking gram
The thalline of positive bacillus carries out point pure.Bacterial strain after purification carries out indole test, nitrate reduction test, catalase examination
Test, hydrogen sulfide production test, test is the bacterial strain of negative reaction and is initially identified as Lactobacillus.Then adopt sugared fermenting experiment
With the method for 16s rdna, bacterial strain is carried out with the identification of kind of level.
The Physiology and biochemistry qualification result of the Lactobacillus plantarum klds1001 in the present invention is shown in Table 1:
Table 1
| Nitrate reduction is tested | - | Gelatin liquefaction | - |
| Produce hydrogen sulfide experiment | - | Catalase is tested | - |
| Indole is tested | - |
The Lactobacillus plantarum klds1001 sugar alcohol fermenting experiment that the present invention provides the results are shown in Table 2:
Table 2
| Title | klds1001 | Title | klds1001 |
| Mannose | + | Sucrose | + |
| Fructose | + | 6-(.alpha.-D-galactosido)-D-glucose. | + |
| Lactose | + | D- ribose | + |
| Galactose | + | Xylose | - |
| Fiber two pool | + | Mannitol | + |
| Maltose | + | Sorbitol | - |
| Trehalose | + | Raffinose | + |
| Salicin | + | Melezitose | - |
| Glucose | + | Gluconate | + |
| Amygdaloside | + | Esculin | + |
| Arabinose | - | L- rhamnose | + |
The 16s rdna sequencing identification result of Lactobacillus plantarum klds1001 is as follows:
ggggcagggcggcagctataatgcagtcgacgaactctggtattgattggtgcttgcatcatgatttacatttgagt
gagtggcgaactggtgagtaacacgtgggaaacctgcccagaagcgggggataacacctggaaacagatgctaatac
cgcataacaacttggaccgcatggtccgagcttgaaagatggcttcggctatcacttttggatggtcccgcggcgta
ttagctagatggtggggtaacggctcaccatggcaatgatacgtagccgacctgagagggtaatcggccacattggg
actgagacacggcccaaactcctacgggaggcagcagtagggaatcttccacaatggacgaaagtctgatggagcaa
cgccgcgtgagtgaagaagggtttcggctcgtaaaactctgttgttaaagaagaacatatctgagagtaactgttca
ggtattgacggtatttaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgt
tgtccggatttattgggcgtaaagcgagcgcaggcggttttttaagtctgatgtgaaagccttcggctcaaccgaag
aagtgcatcggaaactgggaaacttgagtgcagaagaggacagtggaactccatgtgtagcggtgaaatgcgtagat
atatggaagaacaccagtggcgaaggcggctgtctggtctgtaactgacgctgaggctcgaaagtatgggtagcaaa
caggattagataccctggtagtccataccgtaaacgatgaatgctaagtgttggagggtttccgcccttcagtgctg
cagctaacgcattaagcattccgcctggggagtacggccgcaaggctgaaactcaaaggaattgacgggggcccgca
caagcggtggagcatgtggtttaattcgaagctacgcgaagaaccttaccaggtcttgacatactatgcaaatctaa
gagattagacgttcccttcggggacatggatacaggtggtgcatgggttgtcgtcagctcgtgtcgtgagatgttgg
gtttaagt.
The comprehensive appraisal procedure for oral cavity probiotic strain at present, the present invention is from the bacteriostatic activity of bacterial strain, oral environment
Adaptability, three aspects of application security, are comprehensively evaluated to bacterial strain, wherein specifically include that and are surveyed using Odontothrips loti
Determine the bacteriostatic activity to cariogenic bacteria Streptococcus mutans for the bacterial strain；Domestic at present usual using research probiotic strain and Streptococcus mutans
Speculating the impact to Streptococcus mutans Mycoderma Forming ability for the bacterial strain, research in this respect is relatively fewer for copolymerization effect.Using
Glass surface, as external model, measures the impact that bacterial strain is formed to Streptococcus mutans Mycoderma；Measure the peroxidating of variable concentrations
The hydrogen and ph resistivity to poor environment in bacterial strain oral cavity；Using measuring bacterial strain hydroxyapatite degradation capability, evaluate cariogenic energy
Power；Measure bacterial strain for the drug resistance of major antibiotics and hemolytic, measure the danger that bacterial strain whether there is Detection of insecticide resistance transfer
Evil and the harm causing mucosal infections.And the fungistatic effect of bacterial strain is contrasted with traditional probiotic bacteria lgg, compare the antibacterial effect of bacterial strain
The quality of fruit, its result is as shown in figs. 4-7.
(1) Odontothrips loti detects the bacteriostatic activity of bacterial strain
Concentration is 1 × 109The bacterium solution of cfu/ml accesses in 20ml mrs fluid medium with 2% inoculum concentration, 37 DEG C
Culture 24h, makes bacterial strain reach stable trophophase.In 12000r, it is centrifuged 10min under conditions of 4 DEG C, collects supernatant with sterile test tube
Liquid.Filtered with 0.22 μm of filter membrane, removed thalline and other impurity.The supernatant collection that preparation is completed is managed in 2.0ml ep
In, put into standby in 4 DEG C of refrigerators.
The bhi culture medium that 20ml is contained 2% agar is poured in aseptic flat board, naturally dries.Take 0.5ml variation hammer
Bacterium bacterium solution (1 × 108Cfu/ml), it is spread evenly across in bhi flat board, aseptic operating platform stands 2-3h.In plate center position
Put and take at 3 points, gently place Oxford cup (Oxford cup diameter: 6.00mm) after 3 sterilizings with tweezers.It is upper that absorption 200ul prepares
Clear liquid is slowly added in Oxford cup, then steadily moves to flat board in 37 DEG C of incubators, cultivates 18h.Take out cattle with tweezers
Tianjin cup, uses vernier caliper measurement antibacterial circle diameter.
Result shows, the antibacterial circle diameter of Lactobacillus plantarum klds1001 is significantly higher than rhamnose for 19.47 ± 0.50mm
The antibacterial circle diameter of lactobacilluss lgg is 17.11 ± 0.26mm, has preferable fungistatic effect.
(2) impact that bacterial strain is formed to Streptococcus mutans Mycoderma
Take after adding 2.5ml mrs culture medium to mix with 2.5mlbhi fluid medium in 5ml small test tube, add 5%
Sucrose, in 121 DEG C, 15min sterilizes.Prepare MES (mes) buffer, filtered with 0.22 μm of filter membrane, remove
Remove thalline and impurity.To in culture medium, during experiment, in aseptic operating platform, add 0.5% mes buffer.
By Streptococcus mutans (1 × 109Cfu/ml) it is linked into 2% inoculum concentration in the culture medium of test, in 37 DEG C
After shaking table culture 12h, access the Lactobacillus plantarum of equivalent, continue culture 12h under the same conditions.Take out culture, pour out
Fluid medium, adds 5ml pbs buffer, the slight thalline shaking, removing unstable adhesion.After washing 3 times, add
5mlpbs buffer, test tube is placed in supersound process 30min in ultrasonic cleaning instrument, so that thalline is dispersed in buffer.Ladder
After degree dilution, coat in bhi solid medium, 37 DEG C of culture 48h, according to the difference of Streptococcus mutans and lactobacilluss colony characteristicses
Different, respectively colony counting is carried out to lactobacilluss in plate and Streptococcus mutans.
Result shows, Streptococcus mutans single culture 24h formed Mycoderma in Streptococcus mutans quantity be 8.49 ±
0.05longcfu, after co-culturing with lactobacilluss klds1001 and lgg, in Mycoderma, the quantity of Streptococcus mutans drops to respectively
7.72 ± 0.11 and 7.66 ± 0.09logcfu.The hammer it follows that bacterial strain klds1001 can effectively suppress to make a variation in Mycoderma
The quantity of bacterium, and there is the inhibition similar to probiotic bacteria lgg.
The quantity of Lactobacillus plantarum klds1001 can be able to detect that with Streptococcus mutans in the middle of Mycoderma when co-culturing
For 7.21 ± 0.08logcfu.It follows that Lactobacillus plantarum klds1001 can keep a fixed number in tooth surface biomembrane
Amount.
(3) mensure of hydroxyapatite degradation capability
Weigh the pure caco of analysis3Powder 2g, in weighing botle, after 105 DEG C dry 4h, takes out weighing botle, is placed in exsiccator
It is cooled to room temperature.Weigh the caco after 0.10g is dried3, add 0.5n hc1 so that it is completely dissolved, plus distilled water be fixed to 100ml
Hold, be configured to the calcium stock solution that concentration is 400 μ g/ml.Calcium stock solution is configured to the calcium working solution that concentration is 10 μ g/ml.
Take 6 10ml test tubes, add calcium working solution and distilled water according to the dosage shown in table 3, every test tube adds 5ml adjacent
Cresolphthalein developer, concussion mixes, and measures light absorption value under 570nm wavelength condition.After adding developer, carry out in 15min
Colorimetric, concentration and obtained od value according to calcium draw standard curve.
Reagent dosage needed for table 3
| Test tube is numbered | 1 | 2 | 3 | 4 | 5 | 6 |
| Ca working solution (ml) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1.0 |
| Distilled water (ml) | 1.0 | 0.8 | 0.6 | 0.4 | 0.2 | 0 |
Take 100 μ l Lactobacillus plantarum bacterium solution (1 × 108Cfu/ml), access the 4ml containing 0.10g hydroxyapatite powder
In bhi (ph7.0 ± 0.2) fluid medium.0,3,6,9,12,18,24,36,48,72h is cultivated respectively under the conditions of being placed in 37 DEG C,
Take out test tube, under the conditions of 12000r, be centrifuged 10min, collect supernatant.1ml supernatant is taken to add in the middle of clean test tube, plus
Enter 5ml o-cresolphthalein developer, under 570nm wavelength condition, carry out colorimetric, measure its od value, according to standard calcium curve, calculate
Calcium burst size in lactobacilluss incubation.
Measurement result shows, Lactobacillus plantarum klds1001 has slight degradation capability to hydroxyapatite.With culture
The increase of time, the degradation amount of hydroxyapatite is continuously increased, and after culture 9h, degradation amount tends to balance.After culture 72h, produce calcium
Measure as 0.34 ± 0.01 μ g/ml.After culture 72h, the amount that Streptococcus mutans produce calcium with lgg degraded hydroxyapatite is respectively 0.70
± 0.01 μ g/ml and 0.27 ± 0.01 μ g/ml.Therefore, bacterial strain klds1001 has similar degradation capability to lgg, and much
Degradation capability less than Streptococcus mutans.
(4) impact to strain growth state for the low ph
Lactobacillus plantarum is accessed in mrs fluid medium with 2% inoculum concentration, 37 DEG C of culture 20h are so as to reach stable
Trophophase.Be centrifuged 10min under conditions of 6000r, with and the pbs buffer solution three times of ph7.2 after, thalline is resuspended in
In pbs buffer, and with pbs buffer, bacterial concentration is adjusted to 108cfu/ml.Take this bacterium solution of 1ml to be added to 9ml ph to divide
Not Wei in 2.0,3.0,4.0 and 5.0 mrs fluid medium, under the conditions of 37 DEG C, respectively culture 15,30,45,60min, meter
Calculate the bacterium number of survival.
Result shows, Lactobacillus plantarum klds1001 can well grow under conditions of ph is for 3-5, the bar being 2 in ph
After culture 60min under part, the bacterium number of survival drops to 4.79 ± 0.49logcfu by 7.16 ± 0.14logcfu.Illustrate that bacterial strain exists
Remain under conditions of low ph value keep a number of survival bacterium number.
(5) impact to strain growth state for the hydrogen peroxide
Lactobacillus plantarum is accessed in mrs fluid medium with 2% inoculum concentration, 37 DEG C of culture 20h are so as to reach stable
Trophophase.Culture is centrifuged under conditions of 6000rmp 10min, with and the pbs buffer solution three times of ph7.2 after, by bacterium
Body weight is suspended from pbs buffer, and is adjusted bacterial concentration to 1 × 10 with pbs buffer8cfu/ml.Draw 1ml with liquid-transfering gun
This bacterium solution is added in 9ml mrs fluid medium, make final concentration of hydrogen peroxide be respectively 1.0,2.5,5.0mm/l.In 37
Under the conditions of DEG C, culture 2,4,6h, calculate survival bacterium number.
Result shows, Lactobacillus plantarum klds1001 well-grown under conditions of concentration of hydrogen peroxide is for 1mm/l,
After culture 6h under conditions of 5mm/l, the bacterium number of survival is in 3.29 ± 0.07logcfu boil on the nape opposite the mouth intracavity and dental care products
Hydrogen peroxide has stronger toleration, can well adapt to oral environment.
(6) mensure of antibiotics sensitivity
Will be mould to ampicillin, vancomycin, tetracycline, chloromycetin, erythromycin, kanamycin, gentamycin, crin
Element, streptomycin are configured to the stock solution that concentration is 10280 μ g/ml in its suitable solvent.
Carry out the mensure of antibiotics sensitivity using constant broth dilution method, take 13 test tubes, first addition 3.6ml
Mrs culture medium, remaining adds 2ml culture medium, number consecutively.Draw 0.4ml antibiotic solution to be added in first test tube, shake
After swinging several seconds mixing, separate out 2ml and be added in next test tube, concussion mixes, carry out this operation successively.By Lactobacillus plantarum
Liquid culture adjustment bacterial concentration be about 1 × 107Cfu/ml, draws this bacterium solution of 2ml and is separately added in the middle of above-mentioned test tube, in
Cultivate 8h under the conditions of 37 DEG C, measure od value under the conditions of wavelength is for 600nm.
Measurement result shows, Lactobacillus plantarum klds1001 to vancomycin, clindamycin and streptomycin drug resistance, to other
Several antibiotic sensitive (table 4).
The antibiotics sensitivity of table 4:klds1001
(7) hemolytic measures
Picking bacterial strain single bacterium colony percutaneous puncture-inoculation, in the agar plate containing blood, causes oxygen-free environment, 37 DEG C of culture 48h,
Experimental strain is made to grow in flat board and react.Take out flat board, amplify 60 times under the microscope and observed.Puncture thing agar week
Cross existing transparent circle for α haemolysis, represent and be completely dissolved erythrocyte；β haemolysis represents part lysed erythrocyte；Fine jade around culture
Fat no haemolysis circle appears as γ haemolysis, represents not haemolysis.
Measurement result shows, Lactobacillus plantarum klds1001 is γ haemolysis, does not have hemolytic.Control strain suppuration chain
Coccus haemolysis circle border is clearly more α haemolysis.
Claims (3)
1. Lactobacillus plantarum klds1001, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, bacterium
Planting deposit number is: cgmcc no.9066.
2. application in oral health-care product for the Lactobacillus plantarum klds1001 as claimed in claim 1.
3. application in oral-cavity cleaning products for the Lactobacillus plantarum klds1001 as claimed in claim 1.
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| CN106497835B (en) * | 2016-10-26 | 2018-05-04 | 江南大学 | One plant of lactobacillus fermenti for suppressing Streptococcus mutans propagation |
| CN107325995A (en) * | 2017-09-06 | 2017-11-07 | 光明乳业股份有限公司 | A kind of streptococcus salivarius bacterial strain and its application |
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| CN108728381A (en) * | 2018-06-07 | 2018-11-02 | 内蒙古农业大学 | It is a kind of to have to the lactobacillus plantarum of clindamycin stability, its evaluation method and application |
| CN108486022B (en) * | 2018-07-02 | 2020-08-28 | 四川大学 | Anti-caries lactobacillus plantarum and application thereof |
| CN109957530B (en) * | 2019-03-19 | 2021-12-07 | 江西仁仁健康产业有限公司 | Lactobacillus plantarum and application thereof |
| CN110037157A (en) * | 2019-04-22 | 2019-07-23 | 江南大学 | A method of improving lactobacillus plantarum stability in chewing gum preparation and storage |
| CN111000874A (en) * | 2019-07-25 | 2020-04-14 | 江南大学 | Lactobacillus plantarum with dental caries prevention and treatment effect |
| CN112852686B (en) * | 2021-04-14 | 2022-11-01 | 四川高福记生物科技有限公司 | Lactobacillus plantarum LP220 capable of preventing dental caries and application thereof |
| CN113957006B (en) * | 2021-09-27 | 2022-09-30 | 微康益生菌(苏州)股份有限公司 | Lactobacillus plantarum N13 and application thereof in preventing or treating dental caries and periodontitis |
| CN115960740A (en) * | 2022-05-31 | 2023-04-14 | 青岛蔚蓝生物股份有限公司 | Lactobacillus plantarum and application thereof in preventing or treating oral diseases |
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