CN104894005B - The lactic acid bacteria BL21 and preparation method and application of a kind of high-yield extracellular polysaccharide - Google Patents
The lactic acid bacteria BL21 and preparation method and application of a kind of high-yield extracellular polysaccharide Download PDFInfo
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Abstract
The invention belongs to microorganism fields, and in particular to a kind of lactic acid bacteria of high-yield extracellular polysaccharide, the optimum condition and preparation method of extracellular polysaccharide and generated exocellular polysaccharide and application thereof.The present invention provides bifidobacterium longum BL21, BL21 is by cultivating and extracting acquisition exocellular polysaccharide.The exocellular polysaccharide can be used for preparing to improve the external preparation of the colour of skin.Lactic acid bacteria provided by the invention can high yield have inhibit cell in melanin synthesis and reduce the exocellular polysaccharide of skin splash quantity, and since bifidobacterium longum belongs to probiotics, it is completely harmless or even beneficial to human body using the exocellular polysaccharide of its fermenting and producing, security is excellent, can be consequently used for manufacture cosmetics, skin care item and various Dermatologic preparation compositions.
Description
Technical field
The invention belongs to microorganism fields, and in particular to the lactic acid bacteria BL21 of a kind of high-yield extracellular polysaccharide and produced
Exocellular polysaccharide and application thereof.
Background technology
Skin ecto-entad is divided into epidermis and corium two parts, and the depth of the colour of skin depends on melanin in epidermis and (is also referred to as
Melanocyte) content number.
Melanin is to be catalyzed using tyrosine as precursor through tyrosine enzyme system, and oxidation polymerization process occurs and is formed one group is answered
Miscellaneous pigment composition can be divided into insoluble black 5,6- dihydroxy indole type melanin and soluble higher 5,6- dihydroxy
Indole-carboxylic acid type melanin.Melanin is synthesized and moved in the melanosome generated by the melanocyte in basal layer of epidermis
It moves on in the keratinocyte of surrounding, constantly moves to skin surface with the update of corneocyte, form the colour of skin, and with angle
The update of matter confluent monolayer cells and come off.
Hyperpigmentation in specific region on skin makes the region colour of skin deeper with peripheral region, then forms color spot, often
See that color spot species has chloasma, freckle, inflammatory pigmentation etc..Wherein, chloasma is a kind of color for being common in female middle-aged
Plain calmness phenomenon, shows as that Face and cheek symmetry sheet is light brown or auburn pigmentation spots, pathogenesis are still endless
All clear Chu, current study show that, solarization, sex hormone level are principal elements, skin barrier function imbalance, some drugs, chronic
Disease, inherent cause, skin Dysbiosis, contact heavy metal etc. may also cause chloasma occurs.
Make the colour of skin more pale, removal color spot is main work(of the consumer to cosmetics and skin care item to improve the colour of skin uniformity
One of energy demand.The common colour of skin, which improves ingredient, includes ascorbic acid and its derivative, licorice (glabridin, Radix Glycyrrhizae
Flavones, glycyrrhizic acid etc.), ursin, quinhydrones, kojic acid and its derivative, ferulic acid and its derivatives, tretinoin, azelaic acid class,
Tranexamic acid, N-Acetyl-D-glucosamine, niacinamide, the resorcinol class with alkane side chain, 4- methoxysalicylic acids potassium, promotion
Organic acid (lactic acid, tartaric acid) that horn cell comes off etc..Above each constituents are different in effect, security.Exploitation tool
Have and inhibit that melanin is formed, to reduce color spot, highly safe ingredient all particularly significant to skin care item manufacturer and consumer.
Streptococcus thermophilus (Streptococcus thermophilus) and bifidobacterium longum (Bifidobacterium
Longum the lactic acid bacteria of Yoghourt and fermented dairy product production) is commonly used for, it is safe, it is put into Ministry of Public Health's announcement《It can use
In the strain list of food》(defend and do supervision hair [2010] 65).Some strains of streptococcus thermophilus and bifidobacterium longum bacterial strain exist
Exocellular polysaccharide can be generated in fermentation process.The exocellular polysaccharide that streptococcus thermophilus and bifidobacterium longum generate has on monose and structure
There is strain specificity, and influenced by condition of culture；Yield of extracellular polysaccharide is related with bacterial strain and condition of culture.
As a kind of macromolecule polyalcohol, exocellular polysaccharide has certain viscosity and retentiveness, and the property is in Yoghourt Production
In there is positive effect, therefore can the streptococcus thermophilus of the extracellular polysaccharide of programmed screening and bifidobacterium longum bacterial strain in conventional industries
For Yoghourt Production.
In recent years, the application study on Exopolysaccharides Produced by Lactic Acid Bacteria increasingly increases, and utilizes with generations such as Lactococcus lactis
Method (Japanese Unexamined Patent Publication 9-249524 publications, the Japanese Unexamined Patent Publication 10- that phosphorylated polysaccharide is utilized as moisturizer and whitening agent
No. 251140 publications), method (the Japanese Unexamined Patent Publication 7- that the polysaccharide component generated using lactobacillus strains is utilized as antiphlogistic
No. 70209 publications), contain method (CN 101505721A) of the lactic acid bacteria culture supernatant as wrinkle inhibitor of exocellular polysaccharide；
But there has been no the reports for using bifidobacterium longum and the exocellular polysaccharide of streptococcus thermophilus generation as colour of skin improvement ingredient.
The content of the invention
It is an object of the invention to provide it is a kind of can high-yield extracellular polysaccharide lactic acid bacteria and generated exocellular polysaccharide and its
Purposes.The exocellular polysaccharide can inhibit the synthesis of melanin, play an important role of to improve the colour of skin.
Technical scheme is as follows：
A kind of lactic acid bacteria of high-yield extracellular polysaccharide, entitled BL21 belong to bifidobacterium longum (Bifidobacterium
Longum), China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved in, address is Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, deposit number are CGMCC No.10452, and preservation date is on January 27th, 2015.
The present invention also provides a kind of exocellular polysaccharides produced by above-mentioned lactobacillus-fermented.
The present invention also provides a kind of methods that above-mentioned lactobacillus-fermented produces the exocellular polysaccharide, comprise the steps of：
(1) by BL21 inoculations in being carried out in culture medium, cultivation temperature is 20~50 DEG C, incubation time for 1~60 it is small when, trained
Nutrient solution；(2) exocellular polysaccharide is extracted from the culture solution.
Further, the culture medium includes following components in percentage by weight：0.5% fructose, 1.2% sucrose,
The water of 0.5% oligofructose, 0.5% galactooligosaccharide and surplus.
Further, in the step 1), incubation specifically, during prior to 37-45 DEG C quiescent culture 12-14 small, then
It is cooled to 20-35 DEG C, when culture 36-48 is small.
Further, the step (2) comprises the following steps：A, using the method for press filtration or centrifugation from the culture solution
In isolate the first supernatant；B, the chloroform or trichloroacetic acid of its volume 1/10~1/5 are incorporated as in first supernatant,
Centrifugation or stratification and the second supernatant is extracted after mixing, the step b can be repeated as many times as desired；C, step b is obtained
Second supernatant be mixed and stirred for being not less than 85% ethanol water for the concentration of 1~3 times of its volume, filtering
Separation obtains precipitate；D, the precipitate is carried out with ethanol water eluting repeatedly and dry, obtains exocellular polysaccharide.
Further, in the step b, before chloroform or trichloroacetic acid is added in, the dissolved weight that sterilizes first is added in
Content is the aqueous solution of 0.2% carragheen, and additive amount is the 2-2.1% of the first supernatant weight.
Further, the drying temperature in the step d is 50~80 DEG C, when drying time is 1~24 small.
The present invention also provides the external preparation containing above-mentioned exocellular polysaccharide, the external preparation is used to improve the colour of skin.
Further, the external preparation is toner, lotion, cream, facial mask, Essence, foundation cream, foundation emulsion, the screening flaw
Frost, suncream or externally applied drug.
It, can also be according to normal in addition to addition exocellular polysaccharide is as active ingredient when preparing preparation for external application to skin and skin care item
Rule add in grease, surfactant, polyalcohols, moisturizer, antifoaming agent, thickener, preservative, antioxidant, acid-base accommodation
Agent, essence and flavoring agent, pigment, ultra-violet absorber, all kinds of functional components, powder, water etc..The manufacturing process that can be taken is conventional
The manufacturing process of cosmetics, skin care item or external application pharmaceuticals.
Further, content of the exocellular polysaccharide in the external preparation is 0.1~99.9%.
Further, content of the exocellular polysaccharide in the external preparation is 0.1~5%.
Lactic acid bacteria provided by the invention can high yield have inhibit cell in melanin synthesis and reduce skin splash quantity
Exocellular polysaccharide, and since bifidobacterium longum belongs to probiotics, using its fermenting and producing exocellular polysaccharide to human body entirely without
Harmful or even beneficial, security is excellent, can be consequently used for manufacture cosmetics, skin care item and various Dermatologic preparation compositions.
Description of the drawings
Fig. 1 is to produce melanin situation, Fig. 1 a for the B16 cells of embodiment 3：Control group, Fig. 1 b：Exocellular polysaccharide group
(3.3mg/ml)；
Fig. 2 is the influence that various concentration exocellular polysaccharide forms B16 cell viabilities and melanin.
Specific embodiment
With reference to embodiment, the present invention is further described, and following embodiments are illustrative, be not it is limited,
Protection scope of the present invention cannot be limited with following embodiments.
Embodiment 1：Separation, the screening and identification of bacterial strain
Healthy infants fecal specimens are acquired, with sterile water dissolution, gradient dilution is added in the tablet of sterilizing, is inclined
Enter into 55 DEG C or so of MRS culture mediums, MRS culture medium prescriptions are：Peptone 10.0g, beef extract 8.0g, yeast extract 4.0g,
Lactose 20.0g, dipotassium hydrogen phosphate 2.0g, sodium acetate 5.0g, magnesium sulfate 0.2g, manganese sulfate 0.05g, L-cysteine hydrochloride
1.0g, Triammonium citrate 2.0g, Tween 80 1.0g, agar 20.0g between pH6.0~6.5, add distilled water to 1000mL, and 121
DEG C sterilizing 20min.Under anaerobic, 37 DEG C of culture 72h.It selects the big energetic bacterium colony of molten calcium circle and carries out line purifying training
It supports, is further inoculated into 1%~3% volume ratio in the cow's milk of 10% (W/V) solid content, when 43 DEG C of quiescent cultures 6 are small, stirring
Curdled milk and observation state.
Selecting curdled milk has wire drawing phenomenon, the bacterial strain of curdled milk appearance exquisiteness, and such bacterial strain is mostly that exocellular polysaccharide yield is higher
Bacterial strain.Curdled milk does not generate exocellular polysaccharide or exocellular polysaccharide yield is very low into particle appearance, fineless and smooth bacterial strain.
One plant be isolated to is named as BL21, and the observation under the microscope of the bacterial strain is in wooden club shape, and tapered shape is short
Small, more carefully, energy lactose fermenters, glucose etc. generate lactic acid and acetic acid, a kind of Gram-positive bacillus of anaerobism.It simultaneously should
Bacterium 16S rDNA sequences are compared with type strain, are accredited as bifidobacterium longum, are named as bifidobacterium longum BL21
(Bifidobacterium longum BL21), is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart, deposit number are CGMCC No.10452, and preservation date is on January 27th, 2015.
Embodiment 2：Fermentation and Polyose extraction
The bifidobacterium longum BL21 for screening gained is inoculated into exocellular polysaccharide culture medium, each group in exocellular polysaccharide culture medium
Divide weight percent：0.5% fructose, 1.2% sucrose, 0.5% oligofructose, 0.5% galactooligosaccharide as carbon source and
The water of surplus, prior to 37 DEG C at quiescent culture 12 it is small when, then reduce temperature to 30 DEG C culture 36 it is small when.
Culture solution is handled using angle rotor centrifuge, and 6000rpm is centrifuged 20 minutes, collects the first supernatant.Then to
Add in the OK a karaoke club glue solution of the first supernatant weight 2% in one supernatant, the OK a karaoke club glue solution be it is sterilized after card
Glue is drawn to be dissolved in the water acquisitions, wherein carragheen content is 0.2wt%, after abundant mixing, adds in the chloroform of 1/10 volume, shakes
Make 6000rpm centrifugations 15 minutes after mixing, make its layering, liquid is divided into 3 layers, and upper strata is the second supernatant, and middle level is analysis
The protein layer gone out, bottom are chloroform.
The second supernatant is collected, adds in 95% alcohol of 2 times of volumes, stirring, exocellular polysaccharide is in cotton-shaped precipitation, with 200 mesh
Net filtration is collected.
By the exocellular polysaccharide of collection be placed in 80% alcoholic solution of 1 times of volume impregnate 4 it is small when, take out and squeeze remove wine
Essence when being placed on that drying process 4 is small in 60 DEG C of baking ovens, obtains block-like exocellular polysaccharide.It is used to test after being crushed with mortar.
Bifidobacterium longum BL21 Exopolysaccharide Production From The Fermentation liquid polysaccharide concentrations are up to 5%.
Embodiment 3：The influence that bifidobacterium longum BL21 exocellular polysaccharides form B16 cells melanin
With the DMEM culture solution culture B16 melanoma cells containing 5% hyclone, condition of culture is 37 DEG C, 5%CO2It is permanent
Temperature culture.It takes the logarithm the B16 melanoma cells of phase, discards culture solution, cleaned 2 times with PBS liquid, add in culture solution piping and druming, adjust
Concentration of cell suspension is 5 × 104-10 × 104/ml, is added in 96 orifice plates, and 100 μ l are added in per hole.Culture 12-16 makes when small
Cell attachment sucks culture solution.
The exocellular polysaccharide that embodiment 2 obtains is configured to the culture solution of various concentration, while adds in the CCK-8 reagents of 10 μ l
(CCK-8Cel l Counting Kit, Yeasen) and the method test cell vigor indicated according to kit.Control group is set
(cell+culture solution), each concentration group of exocellular polysaccharide set 5 multiple holes.
Cultivate 56 it is small when after, digested with 0.25% pancreatin, supernatant abandoned with 500rpm centrifugations 5min, under each processing factor
Cell add in 200ul contain 10% dimethyl sulfoxide (DMSO) NaOH (1mol/L) solution, be completely dissolved black when 65 DEG C of water-baths 2 are small
Crude granule, absorbance A value at spectrophotometer measurement 490nm.Melanin formation rate=A experimental groups/A control group × 100%.
Result of the test shows that it is thin that the exocellular polysaccharide that bifidobacterium longum BL21 is generated can substantially reduce B16 under certain concentration
The formation of born of the same parents' melanin, such as it can reduce melanin formation rate up to 50% under 3.3mg/ml concentration.
The photo of B16 cells production melanin situation is shown in attached drawing 1.Cell viability and melanin production statistical chart are shown in attached drawing 2.
Embodiment 4：The preparation of lotion
The exocellular polysaccharide according to the form below 1 that bifidobacterium longum BL21 bacterial strains generate is formulated as lotion.
Table 1
Collocation method is：After whole raw materials are mixed in a reservoir, to 70 DEG C, 300rpm is stirred 40 minutes heating water bath.
During for human test, lotion used in blank control group is added without exocellular polysaccharide.
Embodiment 5：Human test
Volunteer is women of 40 ages at 35-52 Sui.Test group, control group, each 20.
Using the lotion prepared in embodiment 4, the exocellular polysaccharide containing 1%W/W in lotion used in test group, used in control group
Exocellular polysaccharide is free of in lotion.
Before the test begins facial given zone is obtained using VISIA Complexion Analys is face analysis system
Color spot, chloasma quantity in domain obtain the melanin numerical value of skin of face using 580 skinanalysis apparatus of Cutometer MPA.
Volunteer uses lotion respectively once, to be measured using after 8 weeks to color spot quantity and melanin numerical value sooner or later daily.
Statistical result shows that exocellular polysaccharide can substantially reduce color spot, chloasma quantity, reduces dermal melanin numerical value, and concrete outcome is shown in
The following table 2：
Table 2
Claims (2)
1. a kind of lactic acid bacteria of extracellular polysaccharide, which is characterized in that entitled BL21 belongs to bifidobacterium longum
(Bifidobacterium longum), is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, protects
It is CGMCC No.10452 to hide number, and preservation date is on January 27th, 2015.
2. a kind of method that bifidobacterium longum BL21 as described in claim 1 prepares exocellular polysaccharide, which is characterized in that comprising following
Step：Bifidobacterium longum BL21 described in claim 1 is inoculated into exocellular polysaccharide culture medium, each group in exocellular polysaccharide culture medium
Divide weight percent：0.5% fructose, 1.2% sucrose, 0.5% oligofructose, the water of 0.5% galactooligosaccharide and surplus,
When quiescent culture 12 is small at prior to 37 DEG C, then reduce temperature to 30 DEG C culture 36 it is small when；
Above-mentioned culture solution is handled using angle rotor centrifuge, and 6000rpm is centrifuged 20 minutes, collects the first supernatant, then to the
Add in the OK a karaoke club glue solution of the first supernatant weight 2% in one supernatant, the OK a karaoke club glue solution be it is sterilized after card
Glue is drawn to be dissolved in the water acquisitions, wherein carragheen content is 0.2wt%, after abundant mixing, adds in the chloroform of 1/10 volume, shakes
Make 6000rpm centrifugations 15 minutes after mixing, make its layering, liquid is divided into 3 layers, and upper strata is the second supernatant, and middle level is analysis
The protein layer gone out, bottom are chloroform；
The second supernatant is collected, adds in 95% alcohol of 2 times of volumes, stirring, exocellular polysaccharide is in cotton-shaped precipitation, with 200 mesh net mistakes
Filter is collected；
By the exocellular polysaccharide of collection be placed in 80% alcoholic solution of 1 times of volume impregnate 4 it is small when, take out and squeeze remove alcohol, put
When putting that drying process 4 is small in 60 DEG C of baking ovens, block-like exocellular polysaccharide is obtained.
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| CN118272282B (en) * | 2024-06-04 | 2024-10-11 | 微康益生菌(苏州)股份有限公司 | Probiotics containing bifidobacterium longum BL21 strain for promoting development and growth and application thereof |
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