CN113308420B - Bifidobacterium bifidum BB-20 and application thereof in preparing product for improving skin and reducing color spots - Google Patents
Bifidobacterium bifidum BB-20 and application thereof in preparing product for improving skin and reducing color spots Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, in particular to Bifidobacterium bifidum BB-20 and application thereof in preparing products for improving skin and reducing color spots, wherein the Bifidobacterium bifidum BB-20 is classified and named as Bifidobacterium bifidum, is preserved in China general microbiological culture collection management center at 3-11 months in 2021, and has the preservation number of CGMCC 21893. The bacterial strain has good gastric acid barrier permeability, high affinity to intestinal epidermal cells, excellent colonization effect, is particularly suitable for oral administration, can increase the expression of in vivo superoxide dismutase related genes by activating pathways such as antioxidation and the like, improves the antioxidation level of organisms, breaks up melanin spots and improves skin quality.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bifidobacterium bifidum BB-20 and application thereof in preparing products for improving skin and reducing color spots.
Background
The probiotics is an active microorganism which is beneficial to a host and is formed by changing the flora at a certain part of the host through colonization in a human body, and has great medical value. The existing research shows that the probiotics has exact curative effects on the aspects of regulating intestines and stomach, resisting oxidation, reducing cholesterol, preventing gastrointestinal cancer, regulating immunity and the like.
However, the biological function of probiotics is not limited to their systematic classification, even though there are large biological differences between different strains of the same species. Meanwhile, common commercial strains generally have the factors of low colonization rate after being ingested, low adsorption capacity of intestinal epidermal cells, uncertain probiotic effect and the like. Therefore, whether the strains with the best probiotic functions can be screened or not can be researched, and the definition of the probiotic effect and the therapeutic target becomes the problem which needs to be solved urgently by the microecological preparation industry at present.
Therefore, the provision of Bifidobacterium bifidum with high colonization rate and strong cell affinity has great significance.
Disclosure of Invention
Aiming at the problems of low colonization rate and low intestinal epidermal cell adsorption capacity of common commercial probiotic strains after being ingested, the invention provides a novel Bifidobacterium bifidum strain and application thereof.
On the first hand, the Bifidobacterium bifidum BB-20 provided by the invention is classified and named as Bifidobacterium bifidum, and has been deposited in China General Microbiological Culture Collection Center (CGMCC) at 11.3.2021, with the deposition address of No. 3 of the North Chen West Lu No. 1 of the sunward area in Beijing and the deposition number of CGMCC 21893.
Further, said Bifidobacterium bifidum BB-20 is isolated from Tibet naltrexone healthy infant faeces.
In a second aspect, the invention provides an application of bifidobacterium bifidum BB-20 in preparing a product for improving skin and reducing color spots, wherein the product for improving skin and reducing color spots is an anti-aging product, a whitening product and/or a color spot lightening product.
Furthermore, the product for improving skin and reducing color spots is an oral preparation.
Further, the product for improving skin quality and reducing color spots contains bifidobacterium bifidum BB-20 powder.
Further, the product for improving skin and reducing color spot contains Bifidobacterium bifidum BB-20 0.5 × 109~1.5×109cfu/g。
Further, the preparation method of the bifidobacterium bifidum BB-20 powder comprises the following steps:
inoculating bifidobacterium bifidum BB-20 into a liquid MRS culture medium for culture, and centrifuging and freeze-drying the obtained zymocyte liquid to obtain bifidobacterium bifidum BB-20 powder.
Further, the culture conditions of the bifidobacterium bifidum BB-20 are anaerobic and 35 ℃, and the culture time is 18 hours.
The beneficial effect of the invention is that,
the new bifidobacterium bifidum strain BB-20 with high colonization rate and strong cell affinity has high gastric acid barrier permeability, high affinity to intestinal epidermal cells, excellent colonization effect, capacity of activating antioxidant and other passages, capacity of increasing the expression of SOD related gene, high antioxidant level, capacity of disintegrating melanin spot and capacity of improving skin quality.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 shows the finger prints of BB-20 and model strain CICC-6071;
FIG. 2 is a female Drosophila survival curve under two strain feeding conditions;
FIG. 3 shows the expression of antioxidant and anti-aging related genes of female drosophila melanogaster under feeding conditions of two strains.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 screening and preservation of strains
1. The source of the strain
Tibet naltrexone healthy infant faeces.
2. Separation method
(1) Mixing the healthy infant feces sample with 0.7% saline water, fully oscillating and uniformly mixing to obtain mixed liquid;
(2) dipping the mixed liquid by using a bacteria inoculating ring, inoculating the mixed liquid on a flat plate containing a TPY culture medium for streak culture under the conditions of anaerobism, 37 ℃ and 48 hours, and selecting and collecting single bacterial colonies;
(3) carrying out subculture on a new TPY plate on a bacterial colony from a primary plate under the anaerobic condition at 37 ℃ for 72h, and stopping subculturing after the bacterial colony is stable in shape;
(4) selecting single colony inoculation in liquid MRS culture medium, culturing at 37 deg.C for 18 hr under anaerobic condition for preservation and identification, and giving enterprise number BB-20.
EXAMPLE 2 identification of the strains (systematics)
Collecting BB-20 liquid culture medium bacterial suspension into a sterilized centrifuge tube, cleaning and centrifuging for 2 times, extracting genome DNA by using a rhizobacteria genome extraction kit (DP 302), performing a 16S rDNA PCR test by using a universal primer after extraction is finished, sequencing the obtained amplicon product, and performing blast comparison on the sequencing result and a related sequence in a Gene Bank;
the universal primer comprises:
27f：5'-AGAGTTTGATCCTGGCTCAG-3'，
1492r：5'-CTACGGCTACCTTGTT-3'。
the 16S rDNA gene sequence of the BB-20 strain is as follows:
CTTGGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATGCTCCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGTTCCACATGATCGCATGTGATTGTGGGAAAGATTCTATCGGCGTGGGATGGGGTCGCGTCCTATCAGCTTGTTGGTGAGGTAACGGCTCACCAAGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTGTTTGGGAGCAAGCCTTCGGGTGAGTGTACCTTTCGAATAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGATTTATTGGGCGTAAAGGGCTCGTAGGCGGCTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCGATGGCGAAGGCAGGTCTCTGGGCCGTCACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGACGCTGGATGTGGGGCACGTTCCACGTGTTCCGTGTCGGAGCTAACGCGTTAAGCGTCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACGACGCCAGAGATGGCGTTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCACGTTATGGTGGGAACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAGCGGGATGCGACATGGCGACATGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGGAGCCTGCAACCCGGCTCCGTGAAGGCGGAGTCGCTAGTAATCGCGGATCAGCAACGCCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAAGCCGGTGGCCTAACCCCTTGTGGGATGGAGCCGTCTAAGGTGAGGCTCGT。
EXAMPLE 3 identification of the Strain (Gene map)
The BB-20 genomic DNA obtained in example 2 was used as a template, RAPD amplification was carried out using bacterial universal random primer 5'-GACGGATCAG-3', and the amplification product was detected by agarose gel electrophoresis (2% agarose), and compared with the genomic structure of a Bifidobacterium bifidum model strain (purchased from China center for Industrial culture Collection of microorganisms, accession number: CICC-6071).
By comparison, BB-20 has a different genome structure from the model strain (CICC-6071) and belongs to different strains of the same bacterial species, and the result is shown in FIG. 1.
Example 4 gastric acid barrier tolerance test
Gastric acid and bile acid were simulated manually, the gastric acid barrier permeability of BB-20 was examined, and 5 biological replicates per group were set with cic-6071 as the reference strain to verify its statistical significance.
The test method comprises the following steps:
(1) BB-20 was activated by a cryopreservation tube, and after 5 rejuvenations (10 h), inoculated in 10mL gastric simulated medium (TPY, pH3.0, pH adjusted with hydrochloric acid) for 2 h;
(2) taking 3mL of culture solution, and adopting a LIVE/DEAD Baclight cell activity detection kit to perform differential staining on cell activity;
(3) live and dead cells were observed and counted under an olympus BX51 epifluorescence microscope, and the survival rate was calculated according to the formula survival rate =2h viable cell number/initial viable cell number × 100%.
The results show that the survival rate of BB-20 after 2 hours in liquid medium at pH3.0 was reached (102.4% + -6%), significantly (T-test, p < 0.05) was higher than model strain CICC-6071 (88.9% + -8%), indicating that the gastric acid barrier permeability of this strain is good and that very high delivery efficiency can be achieved in combination with an enteric formulation protocol.
Example 5 intestinal colonization and cell affinity assay
Caco-2 cells are used for simulating the environment of intestinal epidermal cells, the affinity capacity of the intestinal epithelial cells of the BB-20 strain is detected through a co-culture test, a control strain is a model strain CICC-6071, and 5 biological repetitions are set in a colonization test.
The test method comprises the following steps:
(1) and (3) culturing intestinal cells: 1mL of 0.05% Insulin is used for treating a human intestinal cell strain Caco-2, the separation is carried out after 5min, the abundance of the human intestinal cell strain is quantified by a light absorption method, and the human intestinal cell strain is subpackaged on a 96-hole cell plate (10000 cells/plate) and enters an anaerobic workstation for culture;
(2) preparing a strain: activating BB-20 twice with a TPY liquid culture medium, centrifuging at 3000g for 10min to obtain thallus, transferring the thallus to a sterile PBS (pH7.2) buffer solution, washing twice for later use, adding 20 mu L of bacterial liquid into each hole of the cell culture plate in the step (1), and culturing for 1h to be detected;
(3) binding assay: washing the cell culture plate with PBS buffer to remove free lactobacillus, adding 100 μ L10% formalin into each well, and standing the cell plate for 0.5 h;
(4) fixing and dyeing: adding 100 μ L crystal violet, dyeing for 5min, rapidly washing with 75% alcohol, and removing the staining agent on the cells;
(5) counting and observing: the cell culture plate was placed under an inverted phase contrast microscope, 20 random fields were selected, 50 Caco-2 cells were selected, the amount of BB-20 adsorbed thereto was observed, and the average value was calculated.
The results show that the bifidobacterium bifidum BB-20 related to the invention has the advantages of high colonization power (69% + -12%), strong adhesion (T-test, p < 0.05) higher than that of a model strain CICC-6071 (51% + -9%), and the strain has high affinity to intestinal epidermal cells, has excellent colonization effect and is particularly suitable for oral administration.
Example 6 antioxidant and Experimental animal Life cycle assessment
Adopting a premature senility type drosophila melanogaster mutant to accelerate the simulation of female premature senility, wherein the drosophila melanogaster mutant is from the resource center of the drosophila melanogaster in Indiana USA,
numbered 35578, P { TRiP. GL00156} attP2,
the genotype is y 1 sc [ v 1 ]; p { y [ + t7.7] v [ + t1.8] = terip.gl00156 } attP2,
the biological function is a fruit fly aging acceleration model.
Preparing a basic culture medium according to the following components: 40g of cane sugar, 25g of yeast, 67g of corn flour, 9g of soybean flour, 10g of agar powder, 1g of sodium benzoate, 0.25g of methylparaben and 1000mL of water.
The test method comprises the following steps:
(1) fruit fly culture: methods refer to whitech h. Fly Pushing: the Theory and Practice of Drosophila Genetics, Second Edition By Ralph J Greenspan [ J ]. Quarterly Review of Biology, 2004, 36 (3): 1139-1140;
(2) administration and observation: selecting 400 female fruit flies, averagely dividing into a CICC-6071 group and a BB-20 group, respectively filling each group into 10 fruit fly tubes containing 2mL of culture medium, wherein each fruit fly tube contains 20 fruit flies, changing the culture medium every 5 days, observing and recording the death number of the fruit flies every day, and the culture conditions are as follows: alternating light and dark rhythms at 25 ℃ for 12h/12h and 60% humidity;
wherein the culture medium of the CICC-6071 group is a basic culture medium plus 1 x 10 of CICC6071 freeze-dried powder9The culture medium of cells, BB-20 group is basal culture medium + BB-20 lyophilized powder 1X 109cells；
(3) Anti-aging and energy metabolism related gene expression detection: selecting 600 heads of female fruit flies, averagely dividing into three groups, namely a control group, a CICC-6071 group and a BB-20 group, respectively filling each group into 10 fruit fly tubes containing 2mL of culture medium, wherein 20 heads of fruit flies are contained in each fruit fly tube, changing the culture medium every 5 days, and the culture conditions are as follows: alternating light and dark rhythms at 25 ℃ for 12h/12h and 60% humidity;
wherein the culture medium of the control group is basal medium, and the culture medium of the CICC-6071 group is basal medium + CICC6071 lyophilized powder 1 × 109The culture medium of cells, BB-20 group is basal culture medium + BB-20 lyophilized powder 1X 109cells；
After 30 days of culture, the expression of relevant genes such as oxidation resistance, aging resistance and the like of the test fruit flies after BB-20 and CICC-6071 are given is detected by adopting a fluorescent quantitative PCR method, the anti-aging effect of the BB-20 on female fruit flies is considered on the basis, and genes and primer pairs related to the fluorescent quantitative PCR detection are shown in Table 1.
The test results are shown in table 2, fig. 2, and fig. 3. The result shows that the bifidobacterium bifidum BB-20 related in the invention can remarkably prolong the life of the mutant drosophila (Log-rank test p = 0.3001), and can remarkably improve the survival rate of the mutant drosophila within 60 days compared with a reference strain CICC-6071;
secondly, the strain can obviously improve the antioxidant level of a female drosophila organism, compared with a control group, when the female drosophila fed with the BB-20 strain is fed, the sod2 gene is up-regulated by 2.8 times (p = 0.007), and the model strain CICC-6071 which is synchronously referred to does not have similar biological functions;
meanwhile, compared with the model strain CICC-6071, the BB-20 has the advantages that the anti-aging gene and energy metabolism rate (Srl and ATPsyn-d) of female fruit flies are improved remarkably.
TABLE 1 fluorescent quantitative PCR detection of the genes involved and primer pairs
TABLE 2 Drosophila Life Curve Log-rank test under two probiotic feeding conditions
| Bacterial strains | Number of samples | Mean (% survival rate) | Neutral positionNumber (% survival rate) | Timing significance test (vs BB-20) |
| BB-20 | 200 | 43.15 | 33.63 | - |
| CICC-6071 | 200 | 41.01（-4.39%） | 29.31（-8.15%） | P=0.3001 |
Example 7A skin improvement and stain reduction product
A product for improving skin and reducing mottle contains Bifidobacterium bifidum BB-20 powder with content of Bifidobacterium bifidum BB-20 of 1.0 × 109cfu/g, wherein the preparation method of the bifidobacterium bifidum BB-20 powder comprises the following steps:
(1) mixing the healthy infant feces sample with 0.7% saline water, fully oscillating and uniformly mixing to obtain mixed liquid;
(2) dipping the mixed liquid by using a bacteria inoculating ring, inoculating the mixed liquid on a flat plate containing a TPY culture medium for streak culture under the conditions of anaerobism, 37 ℃ and 48 hours, and selecting and collecting single bacterial colonies;
(3) carrying out subculture on a new TPY plate on a bacterial colony from a primary plate under the anaerobic condition at 37 ℃ for 72h, and stopping subculturing after the bacterial colony is stable in shape;
(4) selecting single colony to inoculate in liquid MRS culture medium under anaerobic condition at 35 deg.C for 18 hr;
(5) and (4) centrifuging and freeze-drying the zymocyte liquid obtained in the step (4) to obtain bifidobacterium bifidum BB-20 powder.
Example 8 evaluation of whitening Effect and color Spot lightening Effect
Randomly selecting 100 volunteers (50 male and female, the age is between 30-50 years), wherein the male volunteers are randomly divided into BB-20 group and CICC-6071 group, the female volunteers are randomly divided into BB-20 group and CICC-6071 group, the number of the volunteers in each experimental group is the same, the BB-20 group uses the product for improving skin quality and reducing color spots prepared in example 7, the CICC-6071 group uses the preparation containing CICC-6071, the preparation method is the same as that in example 7, and the dosage used by each person per day is 2g (specification: 1.0 x 10)9cfu/g), after 50 days, adopting a HONKON-TC20 facial skin test system, and recording data; meanwhile, the number of stools and the number of diarrhea of each volunteer within 50 days were recorded, and the short-term diarrhea rate (short-term diarrhea rate = number of diarrhea/number of stools × 100%) was calculated. The statistical results are shown in table 3 below.
TABLE 3 average improvement statistics of skin melanin spots for 50 days after taking probiotic preparation
Note: p <0.05 indicates correlation; p <0.01 indicates significant correlation.
The result shows that BB-20 can increase the expression of the gene related to the superoxide dismutase in vivo, improve the antioxidant level of the organism, disrupt melanin spots and improve the skin quality by activating the antioxidant pathway and the like; experimental data of large-queue volunteers show that the skin quality improving and color spot reducing product taking the strain powder as a core component has a particularly obvious effect on improving the skin quality of women, and the pigment fading level of a tested female volunteer is remarkably higher than that of a model strain after the product is continuously taken for 50 days; meanwhile, the BB-20 strain has a remarkable effect on improving the skin quality of men and removing color spots.
It is noted that in the early period of administration, the model strain CICC-6071 shows higher diarrheal property, and the diarrhea rate of the BB-20 strain is obviously lower than that of the model strain, so that the model strain has higher adaptability.
Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
SEQUENCE LISTING
<110> Shandong Zhongke Jiayi bioengineering Co., Ltd
<120> Bifidobacterium bifidum BB-20 and application thereof in preparing products for improving skin and reducing color spots
<130> 2021
<160> 12
<170> PatentIn version 3.5
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gaaagattct atcggcgtgg gatggggtcg cgtcctatca gcttgttggt gaggtaacgg 180
ctcaccaagg cttcgacggg tagccggcct gagagggcga ccggccacat tgggactgag 240
atacggccca gactcctacg ggaggcagca gtggggaata ttgcacaatg ggcgcaagcc 300
tgatgcagcg acgccgcgtg agggatggag gccttcgggt tgtaaacctc ttttgtttgg 360
gagcaagcct tcgggtgagt gtacctttcg aataagcgcc ggctaactac gtgccagcag 420
ccgcggtaat acgtagggcg caagcgttat ccggatttat tgggcgtaaa gggctcgtag 480
gcggctcgtc gcgtccggtg tgaaagtcca tcgcttaacg gtggatctgc gccgggtacg 540
ggcgggctgg agtgcggtag gggagactgg aattcccggt gtaacggtgg aatgtgtaga 600
tatcgggaag aacaccgatg gcgaaggcag gtctctgggc cgtcactgac gctgaggagc 660
gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta aacggtggac 720
gctggatgtg gggcacgttc cacgtgttcc gtgtcggagc taacgcgtta agcgtcccgc 780
ctggggagta cggccgcaag gctaaaactc aaagaaattg acgggggccc gcacaagcgg 840
cggagcatgc ggattaattc gatgcaacgc gaagaacctt acctgggctt gacatgttcc 900
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tggggatgac gtcagatcat catgcccctt acgtccaggg cttcacgcat gctacaatgg 1140
ccggtacagc gggatgcgac atggcgacat ggagcggatc cctgaaaacc ggtctcagtt 1200
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Claims (8)
1. Bifidobacterium bifidum BB-20 is classified and named Bifidobacterium bifidum, and is preserved in China general microbiological culture collection management center at 3.11.2021 with the preservation address of No. 3 of Beijing Corp-Yang district Beichen Xilu No. 1 and the preservation number of CGMCC 21893.
2. The Bifidobacterium bifidum BB-20 of claim 1, wherein the Bifidobacterium bifidum BB-20 is isolated from Tibet naltrexone healthy infant feces.
3. Use of bifidobacterium bifidum BB-20 as defined in claim 1 for the preparation of a stain reducing product using bifidobacterium bifidum BB-20.
4. The use of claim 3 wherein the stain reducing product is in an oral dosage form.
5. The use of claim 3, wherein the stain reducing product comprises a powder of Bifidobacterium bifidum BB-20.
6. The use according to claim 5,the product for reducing mottle contains Bifidobacterium bifidum BB-20 0.5 × 109~1.5×109cfu/g。
7. The use according to claim 5, wherein the Bifidobacterium bifidum BB-20 powder is prepared by:
inoculating bifidobacterium bifidum BB-20 into a liquid MRS culture medium for culture, and centrifuging and freeze-drying the obtained zymocyte liquid to obtain bifidobacterium bifidum BB-20 powder.
8. The use according to claim 7, wherein Bifidobacterium bifidum BB-20 is cultured under anaerobic conditions at 35 ℃ for 18 hours.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110860480.9A CN113308420B (en) | 2021-07-29 | 2021-07-29 | Bifidobacterium bifidum BB-20 and application thereof in preparing product for improving skin and reducing color spots |
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