CN102690768B - Lactobacillus brevis and application thereof - Google Patents
Lactobacillus brevis and application thereof Download PDFInfo
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- CN102690768B CN102690768B CN201210177785.0A CN201210177785A CN102690768B CN 102690768 B CN102690768 B CN 102690768B CN 201210177785 A CN201210177785 A CN 201210177785A CN 102690768 B CN102690768 B CN 102690768B
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Abstract
The invention discloses a lactobacillus brevis BBE-Y52 separated from an oral cavity, with a preservation number of CCTCC M 2012162. The lactobacillus brevis can generate bacteriocin to inhibit the growth of streptococcus mutans and other oral pathogenic microorganisms, can generate extracellular polysaccharide to provide an essential condition for the copolymerization between lactobacillus and other microorganisms in the oral environment and the formation of bio-membrane, and can further generate hydrogen peroxide so as to not only achieve the sterilization effect but also whiten teeth. Therefore, the lactobacillus brevis has a wide application prospect in substituting antibiotics to solve the common oral health problems.
Description
Technical field
The present invention relates to strain short lactobacillus and an application thereof, particularly a strain separation, from the short lactobacillus with probiotic properties in oral cavity, can produce the short lactobacillus that protects exo polysaccharides, hydrogen peroxide and suppress oral cavity pathogen growth.
Background technology
Food and Argriculture OrganizationFAO and the World Health Organization are defined as a kind of bioactive microorganism that has by probiotic bacterium, healthy and helpful to human body after giving the suitable dosage of human body.Probiotic bacterium of a great variety, modal two large class probiotic bacteriums are respectively lactobacillus class and bifidus bacillus class.
Along with increasingly sharpening of antibiotic overuse and bacterial resistance problem, the advantage of probiotic bacterium aspect control and alleviation infection, inflammation and anaphylactic disease is more and more obvious.A large amount of probiotic bacteriums that studies confirm that can be used for the treatment of the diseases such as oropharynx infection, digestive tract diseases, urogenital infections.Probiotic therapy was also applied to stomatology field gradually in recent years.Common oral disease is as carious tooth, periodontitis and halitosis etc. clinically, all with some pathogenic bacterium or to the disadvantageous microorganism of oral Health the excess growth in oral cavity relevant.Probiotic therapy not only can effectively suppress the growth of pathogenic bacterium, can also help to maintain normal oral cavity micro-ecological bacterial group simultaneously, ensures oral Health.Children that there are some researches show long-term absorption interpolation lactobacillus rhamnosus milk, the sick incidence of dental caries is lower, this is due to the growth that has the lactobacillus rhamnosus of prebiotic property and can suppress pathogenic bacteria of decayed tooth Streptococcus mutans, reduces Streptococcus mutans quantity in oral environment, reduces the sick incidence of dental caries.Also there is research to adopt the medicines such as prebiotic Bacterium lacticum preparation treatment gingival hemorrhage and gingivitis to solve oral problem.
In oral environment, field planting has various microorganisms, and these microorganisms, by effects such as coexistence and competition antagonisms, have formed the complicated ecosystem.One of prerequisite that oral cavity probiotic bacterium plays a role in this ecosystem is and to form microbial film with other microorganism copolymerization collection in oral cavity.In addition the growth that, suppresses pathogenic bacteria by multiple antimicrobial substances such as secretion hydrogen peroxide, bacteriocin, organic acids is also that microorganism is as the screening principle of oral cavity probiotic bacterium.
Summary of the invention
The invention provides a strain short lactobacillus (Lactobacillus brevis) BBE-Y52, on May 9th, 2012, be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC No:M 2012162.
The invention provides a strain oral cavity short lactobacillus, have and produce exocellular polysaccharide, produce hydrogen peroxide and suppress Streptococcus mutans and the probiotic properties of campylobacter jejuni growth.
Described short lactobacillus separation is from oral Health volunteer's plaque.Its cultivation and storage conditions are as follows: from well-grown flat board, picking list bacterium colony accesses in MRS substratum, at 37 ℃ of (5%CO
2) be preserved in-80 ℃ of refrigerators after cultivating 24h.
The culture condition that described short lactobacillus produces exocellular polysaccharide is inoculated in to contain 10%(w/v) in the MRS substratum of sucrose, at 30 ℃ of standing cultivation 48h.Short lactobacillus produces free exocellular polysaccharide and uses phenolsulfuric acid method to measure.
The culture condition that short lactobacillus produces hydrogen peroxide is inoculated in MRS substratum, 37 ℃ of 5%CO
2cultivate 24h, with the content of hydrogen peroxide in superoxide ELISA test strip fermented liquid.
The growth that research short lactobacillus suppresses Streptococcus mutans and campylobacter jejuni adopts Oxford agar diffusion method.
Short lactobacillus provided by the invention (Lactobacillus brevisBBE-Y52), can suppress the growth of Streptococcus mutans and other oral microorganisms; Can produce exocellular polysaccharide, for milk-acid bacteria provides necessary condition with other microorganism copolymerization collection and biomembranous formation in oral environment; Can also produce hydrogen peroxide, there is the effect of sterilization tooth-whitening.Short lactobacillus can, with solving common oral problem, ensure oral Health.
Biomaterial:
The present invention obtains a strain short lactobacillus (Lactobacillus brevis) BBE-Y52, on May 9th, 2012, is preserved in Chinese Typical Representative culture collection center, and deposit number is CCTCC No:M 2012162.
Embodiment
Embodiment 1: the separation of oral cavity Bacterium lacticum
Saliva and plaque diluted sample are coated on to (pH 5.5) on MRS solid medium, at 37 ℃ of (5%CO
2) cultivation 24h.Select gram-positive bacillus rule separation and preservation.
Embodiment 2: the screening of the prebiotic Bacterium lacticum in oral cavity and probiotic properties thereof
The screening of the prebiotic Bacterium lacticum in oral cavity and characteristic are investigated to the growth that comprises product exocellular polysaccharide, produces hydrogen peroxide and the typical oral microorganism of inhibition three strain.
Oral cavity Bacterium lacticum produces the method that exocellular polysaccharide preliminary evaluation adopts bacterium colony wire drawing.By Gram-positive bacillus dibbling to adding 10%(w/v) on the MRS solid medium of sucrose, cultivate 48h for 30 ℃, choosing colony has the Gram-positive bacillus of wire drawing and measures the content that it produces exocellular polysaccharide.The short lactobacillus of choosing is inoculated in and contains 10%(w/v) in the MRS liquid nutrient medium of sucrose, cultivate 48h for 30 ℃, get 10ml fermented liquid, add 2ml 80%(w/v) trichoroacetic acid(TCA), ice bath stir process 30min, 4 ℃ of centrifugal 20min of 15000rpm remove thalline and albumen, in supernatant liquor, add 3 times of freezing dehydrated alcohols of volume, 4 ℃ of refrigerations are spent the night, and polysaccharide is flocks and separates out.In 4 ℃ of centrifugal 20min of 15000rpm, with 10mL distilled water dissolution precipitation.Adopt phenol sulfuric acid process to measure free exocellular polysaccharide content in fermented liquid.First production standard curve, accurately takes standard dextran (or glucose) 20mg, and constant volume is to 500mL, draw respectively 0.4,0.6,0.8,1.0,1.2,1.4,1.6,1.8mL, each is mended to 2.0mL with water, then adds 6% phenol 1.0mL and vitriol oil 5.0mL, static 10min, shake up, after room temperature placement 20min, in 490nm photometry density, the 2.0mL water of usining is by same color operation as blank, and X-coordinate is polysaccharide micrograms, ordinate zou is optical density value, obtains typical curve.Again free exocellular polysaccharide in sample is measured.Draw sample liquid 1.0mL, by above-mentioned steps operation, photometry density, calculates polysaccharide content with typical curve.
The mensuration that oral cavity Bacterium lacticum produces hydrogen peroxide is that catalog number is 91319 with superoxide test strip Peroxide 25(Germany MN company) detect the content of hydrogen peroxide in Gram-positive bacillus fermented liquid.Select the bacterial strain that produces hydrogen peroxide.
Oral cavity Bacterium lacticum adopts Oxford agar diffusion method to the restraining effect of Streptococcus mutans and campylobacter jejuni.Double-Medium is used in experiment, and bottom is 1.5% nutrient agar.Upper strata substratum is 0.75% BHI(Brain Heart Infusion, brain heart medicinal extract substratum) nutrient agar.By temperature, be the upper strata substratum of 42 ℃ of left and right, mix with above-mentioned indicator overnight culture that (final concentration is 10
7cFU/mL), be poured on bottom substratum.Solidify the lactobacillus ferment supernatant liquor that adds 100 μ L in the well of the cup-shaped one-tenth in backward Oxford, overnight incubation is observed bacteriostatic action, selects three strain indicators are all had to inhibiting Bacterium lacticum.
According to the prebiotic property of three aspects:, screen a strain short lactobacillus (Lactobacillus brevis) BBE-Y52, on May 9th, 2012, be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC No:M 2012162.Short lactobacillus BBE-Y52 can produce exocellular polysaccharide, and in fermented liquid, free exocellular polysaccharide content is 106.63mg/L, is conducive to realize copolymerization collection and form microbial film with other oral microorganisms.It produces hydrogen peroxide during the fermentation, and content, between 2-5mg/L, has the effect of sterilization tooth-whitening.In addition the short lactobacillus that, screening obtains can suppress the growth of Streptococcus mutans and campylobacter jejuni.
Embodiment 3 oral care product exploitations
The healthcare products such as toothpaste, mouth-washes and liquid tooth sanitising agent that exploitation contains described bacterial classification, guarantee can not bring disadvantageous effect on sense organ to product after adding short lactobacillus.
Be understandable that, for those of ordinary skills, can be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, and all these changes or replacement all should belong to the protection domain of the appended claim of the present invention.
Claims (3)
1. strain short lactobacillus (Lactobacillusbrevis) BBE-Y52, is preserved in Chinese common micro-organisms culture presevation administrative center on May 9th, 2012, and deposit number is CCTCCNo:M2012162.
2. the application of short lactobacillus claimed in claim 1 in preparing exocellular polysaccharide.
3. the application of short lactobacillus claimed in claim 1 in preparing toothpaste, mouth-washes and liquid tooth sanitising agent.
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| CN107418912B (en) * | 2017-06-20 | 2019-09-17 | 江南大学 | One plant of Lactobacillus brevis for improving old face steamed bun ferment local-flavor and its application |
| CN108813626A (en) * | 2018-06-07 | 2018-11-16 | 广州普正生物科技有限公司 | A kind of compound probiotic chewing soft capsule of tooth-whitening |
| CN111808769B (en) * | 2020-07-07 | 2022-03-18 | 华熙生物科技股份有限公司 | Lactobacillus brevis and application thereof in grapefruit fermentation and cosmetics |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1357038A (en) * | 1999-06-21 | 2002-07-03 | Vsl制药有限公司 | Combination of lactic acid bacteria and its use for prevention and/or treatment of infections and inflammatory conditions |
| CN101144064A (en) * | 2007-09-03 | 2008-03-19 | 江南大学 | Lactobacillus brevis with anti-mutagenesis active and capable of producing exopolysaccharide and use thereof |
| CN101500435A (en) * | 2005-11-23 | 2009-08-05 | 可口可乐公司 | High-potency sweetener composition with dietary fiber and compositions sweetened therewith |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1357038A (en) * | 1999-06-21 | 2002-07-03 | Vsl制药有限公司 | Combination of lactic acid bacteria and its use for prevention and/or treatment of infections and inflammatory conditions |
| CN101500435A (en) * | 2005-11-23 | 2009-08-05 | 可口可乐公司 | High-potency sweetener composition with dietary fiber and compositions sweetened therewith |
| CN101144064A (en) * | 2007-09-03 | 2008-03-19 | 江南大学 | Lactobacillus brevis with anti-mutagenesis active and capable of producing exopolysaccharide and use thereof |
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