CN117625498B

CN117625498B - Application of filtrate of bifidobacterium longum subspecies infantis

Info

Publication number: CN117625498B
Application number: CN202410036419.6A
Authority: CN
Inventors: 承静; 张革; 张翌; 鲁旺旺; 杨洋; 周勰; 窦书恒; 潘文婵; 王西; 张桃
Original assignee: Guangzhou Jiyan Cosmetics Technology Co ltd; Sun Yat Sen University
Current assignee: Guangzhou Jiyan Cosmetics Technology Co ltd; Sun Yat Sen University
Priority date: 2024-01-10
Filing date: 2024-01-10
Publication date: 2024-04-19
Anticipated expiration: 2044-01-10
Also published as: CN117625498A

Abstract

The invention relates to the field of cosmetics, in particular to application of filtrate of bifidobacterium longum subspecies infancy. The invention provides a microbial agent, which consists of one or more of fermentation supernatant, fermentation filtrate and fermentation concentrate of a strain; the strain is bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp.infantis), with the preservation number of: CGMCC No.27851. The filtrate of the strain can achieve the effects of resisting aging and removing cutin in the fields of food and cosmetics, has wider effect range and can better play a role in the fields of beauty and skin care.

Description

Application of filtrate of bifidobacterium longum subspecies infantis

Technical Field

The invention relates to the field of cosmetics, in particular to application of filtrate of bifidobacterium longum subspecies infancy.

Background

The skin is divided into epidermis, dermis and subcutaneous tissue; the surface layer of the skin is divided into five layers, namely a basal layer, a spiny cell layer, a granular layer, a transparent layer and a cuticle layer from bottom to top. Skin cells grow from the basal layer, and as they migrate outward, they undergo aging and death processes, and the horny layer is the final product of the continuous regeneration of epidermal cells, and the skin surface is thick, and the skin is shiny, dull, desquamated, wrinkled, and vaccinia grows.

Disclosure of Invention

In view of this, the present invention provides the use of a filtrate of bifidobacterium longum subspecies infancy. The filtrate of the strain provided by the invention can achieve the effects of resisting aging and removing cutin in the cosmetic field, and has more remarkable effect.

The invention provides a microbial agent which consists of one or more of fermentation supernatant, fermentation filtrate and fermentation concentrated solution of a strain;

The strain is bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis), and the preservation number is: CGMCC No.27851.

In some embodiments of the present invention, in the microbial agent described above, the microbial agent comprises: 15-20% of organic acid, 5-10% of amino acid, 5-10% of inorganic acid, 1-10% of inorganic salt and 1-5% of sugar alcohol.

In some embodiments of the present invention, in the microbial agent described above, the microbial agent comprises: 17% of organic acid, 8.5% of amino acid, 6% of inorganic acid, 4.5% of inorganic salt and 3% of sugar alcohol.

In some embodiments of the present invention, in the microbial agent described above, the microbial agent comprises: 17% of glycolic acid, 8.5% of pyroglutamic acid, 6% of citric acid, 4.5% of hydroxylamine and 3% of sorbitol.

The invention also provides a preparation method of the microbial agent, which comprises the following steps:

S1: inoculating and culturing the bifidobacterium longum subsp infantis (Bifidobacterium longum subsp. Infantis) to obtain seed liquid;

S2: and (3) fermenting the seed liquid, centrifuging, and taking supernatant to obtain the microbial agent.

In some embodiments of the invention, the inoculum size of the inoculum depicted in preparation method S1 described above is 5%.

In some embodiments of the invention, the centrifugation in preparation method S2 described above is performed at a rotational speed of 3000 rpm for a time of 30 min.

In some embodiments of the invention, the supernatant of the preparation method S2 further comprises a concentration step.

In some embodiments of the invention, in the above preparation method, the multiple of the concentration is 5-fold.

The invention also provides application of the microbial agent and/or the microbial agent obtained by the preparation method in preparation of anti-aging cosmetics.

The invention also provides application of the microbial agent and/or the microbial agent obtained by the preparation method in preparation of cosmetics for removing cutin.

The invention also provides cosmetics, which comprise the microbial agent and/or microbial agent obtained by the preparation method and auxiliary agents acceptable in the cosmetic field.

The strain provided by the invention is separated from healthy infants fed by breast milk, and test results show that the filtrate of the strain provided by the invention has the effects of resisting aging and removing cutin, and has more remarkable effects.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows strain morphology;

FIG. 2 shows a strain evolutionary tree;

FIG. 3 shows a gram;

FIG. 4 shows no hemolytic ring;

FIG. 5 shows a strain fermentation filtrate preparation process;

FIG. 6 shows a photograph of a strain fermentation filtrate on an isolated milk pig skin to promote exfoliating;

FIG. 7 shows the physical results of strain fermentation filtrate to improve UVB-induced tissue morphology on a 3D epidermis model;

FIG. 8 shows a fluorescent image of strain fermentation filtrate promoting hyaluronic acid production on ex vivo skin;

FIG. 9 shows IHC staining pictures of strain fermentation filtrate to promote collagen production of XVII type on ex vivo skin.

Evidence of biological preservation

Biological material: YSGBifido001,001; classification naming: bifidobacterium longum subsp infancy (Bifidobacterium longum subsp. Infantis); the microorganism strain is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms (China) for type culture Collection of China, with a year of 07 and 10 of 2023; address: the institute of microorganisms at national academy of sciences of China, national academy of sciences, no.1, beichen West way, no. 3, chat.Chao, beijing, city; preservation number: CGMCC No.27851.

Detailed Description

The invention discloses application of a filtrate of bifidobacterium longum subspecies infancy.

It should be understood that the expression "one or more of … …" individually includes each stated object after the expression and various combinations of two or more of the stated objects unless otherwise understood from the context and usage. The expression "and/or" in combination with three or more recited objects should be understood as having the same meaning unless otherwise understood from the context.

The use of the terms "comprising," "having," or "containing," including grammatical equivalents thereof, should generally be construed as open-ended and non-limiting, e.g., not to exclude other unrecited elements or steps, unless specifically stated otherwise or otherwise understood from the context.

It should be understood that the order of steps or order of performing certain actions is not important so long as the invention remains operable. Furthermore, two or more steps or actions may be performed simultaneously.

The use of any and all examples, or exemplary language, such as "e.g." or "comprising" herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Furthermore, the numerical ranges and parameters setting forth the present invention are approximations that may vary as precisely as possible in the exemplary embodiments. However, any numerical value inherently contains certain standard deviations found in their respective testing measurements. Accordingly, unless explicitly stated otherwise, it is to be understood that all ranges, amounts, values and percentages used in this disclosure are modified by "about". As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range.

The strain sources of the invention are as follows:

The DNA sequence is verified to be an exclusive strain, CGMCC No. 27851.

The strain category of the invention:

bifidobacterium longum subsp. Infantis (Bifidobacterium longum subsp. Infantis), bacteria.

The strain introduction of the invention:

YSGBifido001 was isolated from a healthy infant breast fed in Guangzhou of 4 months of age and identified as bifidobacterium longum subspecies infancy by 16S rDNA sequence alignment (Bifidobacterium longum subsp. Infantis). It grows well in MRS culture medium (solid/liquid), and can reach logarithmic phase after 1 day of culture under anaerobic condition. Bacterial colony forms grown on MRS solid culture medium are milky round, and edges are neat. The strain grows on the blood plate without obvious hemolysis ring. After gram staining, the thalli are blue-violet, rod-shaped, and expanded at the two ends with thin middle, which is similar to a barbell.

In the present invention, the BCA protein concentration assay kit (Biosharp Co.) was used for the measurement of total protein.

The primer related to the invention comprises the following components:

TABLE 1

Name of the name	Primer(s)
		27F	3'-AGAGTTTGATCMTGGCTCAG-5' (shown as SEQ ID NO: 1)
1492R	3'-GGTTACCTTGTTACGACTT-5' (shown in SEQ ID NO: 2)
		Strain sequence	Sequence ：3'-TGCAAGTCGAACGGGATCCATCAGGCTTTGCTTGGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATACACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGTTCCAGTTGATCGCATGGTCTTCTGGGAAAGCTTTCGCGGTATGGGATGGGGTCGCGTCCTATCAGCTTGACGGCGGGGTAACGGCCCACCGTGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTATCGGGGAGCAAGCGTGAGTGAGTTTACCCGTTGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGTCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACGATCCCAGAGATGGGGTTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTGTGCCGGGAACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACGCGGCGACGCGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCA-5'（ is shown as SEQ ID NO: 3).
COL I primer	F5'-GTGGCAGTGATGGAAGTGTG-3' (shown as SEQ ID NO: 4) R5'-AGGACCAGCGTTACCAACAG-3' (shown as SEQ ID NO: 5)
		COL III primers	5'-ACCAGGAGCTAACGGTCTCA-3' (shown as SEQ ID NO: 6); r:5'-TCTGATCCAGGGTTTCCATC-3' (shown as SEQ ID NO: 7).
COL IV primer	5'-AGGTGTCATTGGGTTTCCTG-3' (shown as SEQ ID NO: 8); r:5'-GGTCCTCTTGTCCCTTTTGTT-3' (shown as SEQ ID NO: 9).
		COL VII primers	5'-ACTGTGATTGCCCTCTACGC-3' (shown as SEQ ID NO: 10); r:5'-GGCTGTGGTATTCTGGATGG-3' (shown as SEQ ID NO: 11).
Primers for VCAN	5'-GTGTGGGCATTTCTTCCTGTTAG-3' (shown as SEQ ID NO: 12); r:5'-TCAAAGTCATCTTCAGCAGTCACT-3' (shown as SEQ ID NO: 13).
		Primers for elastin	5'-TCCAGGTGTAGGTGGAGCTT-3' (shown as SEQ ID NO: 14); r:5'-GTGTAGGGCAGTCCATAGCC-3' (shown as SEQ ID NO: 15).
Fibronectin primers	5'-AGCCAGTCGAGGATACCTGT-3' (shown as SEQ ID NO: 16); r:5'-GCACCAAAGCCTGAAACCAG-3' (shown as SEQ ID NO: 17).
		Primers for HAS1	5'-ACTCGGACACAAGGTTGGAC-3' (shown as SEQ ID NO: 18); r:5'-TTAGGAAGCTGACCCAGGAG-3' (shown as SEQ ID NO: 19).
NF- κB primer	5'-GGTGCGGCTCATGTTTACAG-3' (shown as SEQ ID NO: 20); r:5'-GATGGCGTCTGATACCACGG-3' (shown as SEQ ID NO: 21).
		Primers for p21	5'-TCTCTGTGTTAGGGGTATATGATGG-3' (shown as SEQ ID NO: 22); r:5'-GAAGGTCGCTGGACGATTTG-3' (shown as SEQ ID NO: 23).
Primer for p16	5'-CTCTGAGAAACCTCGGGAAAC-3' (shown as SEQ ID NO: 24); r:5'-ATGAAAACTACGAAAGCGGG-3' (shown as SEQ ID NO: 25).
		Primers for TIMP-1	5'-CTGTTGTTGCTGTGGCTGAT-3' (shown as SEQ ID NO: 26); r:5'-TCTGGTTGACTTCTGGTGTCC-3' (shown as SEQ ID NO: 27).
LC3-II primer	5'-GAACTGAGCTGCCTCTACCG-3' (shown as SEQ ID NO: 28); R5'-GGGACAACCCTAACACGACC-3' (shown as SEQ ID NO: 29)

The invention is further illustrated by the following examples.

In examples 1 to 2 and verification examples 1 to 4 of the present invention, raw materials and reagents used were commercially available.

EXAMPLE 1 isolation and purification of Bifidobacterium longum subspecies infantis Strain

(1) Experimental method

1) Collecting feces: the cell culture flask was filled with a bacterial frozen stock solution of 20 mL, and the just excreted feces (feces sample from breast-fed infant Guangzhou) was collected with a sterile cotton swab in the flask, and the flask was sealed in an anaerobic bag for storage (in ice box) and transportation.

2) And (3) dilution coating: after shaking, 100. Mu.L of the fecal suspension in step 1) is sucked into 900. Mu.L of PBS buffer, and after blowing and mixing uniformly, 100. Mu.L of the suspension is sucked into 900. Mu.L of PBS buffer, and the like, and the mixture is subjected to gradient dilution for 10 times. And respectively taking 20 mu L of each gradient of diluent, respectively coating the diluent on MRS, BHI, CBA plates, and culturing for 48-72 hours at 37 ℃ in an aerobic or anaerobic environment.

3) Single colony enrichment culture: and randomly picking single colonies of the flat plates with the colony number of 5-100 by using a disposable inoculating loop, blowing and uniformly mixing the single colonies in 20 mu L of PBS buffer solution, then taking 15 mu L of bacterial liquid to coat the same flat plate, culturing for 48-72 hours in an aerobic or anaerobic environment at 37 ℃, and reserving 5 mu L of bacterial liquid for gram staining identification.

4) Bacterial passage: and scraping bacteria growing on the flat plate as much as possible by using a disposable inoculating loop, re-suspending in 200 mu L of PBS, and then re-coating 200 mu L of bacterial liquid on the same flat plate, and culturing for 48-72 hours under the same condition.

5) Bacterial cryopreservation: collecting bacteria on a plate with enough bacterial increment in sterile frozen stock solution prepared by mixing fetal calf serum, BHI culture medium and glycerol, blowing and mixing, and preserving strain at-80deg.C. And meanwhile, a proper amount of bacteria are reserved for extracting bacterial genome DNA.

6) Gram staining identification: and 5 mu L of bacterial liquid prepared when a single colony is selected is dripped into the center of a glass slide, the bacterial liquid is intermittently baked and fixed by an alcohol lamp, crystal violet staining liquid and lugol iodine liquid are respectively dripped to cover 1 min, the glass slide is washed by running water, the glass slide is further covered with decolorized alcohol for 20-30 s, the glass slide is gently shaken during the process, the glass slide is subjected to counter staining by safranine staining liquid for 1 min after the alcohol is washed by running water, the glass slide is washed by running water again, and the bacterial morphology is observed by an optical microscope after the glass slide is dried. If the colony is not pure and more than one bacterial form exists, the sample is subjected to streak inoculation, and single colony is picked again after culture.

7) Bacterial genome DNA extraction: bacterial genomic DNA extraction was performed using a bacterial genomic DNA extraction kit according to the instructions of the tiangen biochemical technology company, and DNA concentration was determined using NanoDrop 2000.

8) 16S fragment amplification:

<1> the 27F/1492R primer set was used, and the following reaction system was used:

TABLE 2

<2> PCR amplification procedure was as follows: pre-denaturation at 95 ℃ 60 s; denaturation at 95 ℃ for 30s, renaturation at 56 ℃ for 30s, extension at 72 ℃ for 90 s, and cycle number of 31; the temperature was set to 72℃and the end temperature was set to 4℃after extension by 7 min.

9) Agarose gel electrophoresis, whether the size is about 1500 bp.

10 16S rRNA sequencing and result comparison, preliminary identification of the type of bacteria.

(2) Experimental results

The nucleotide sequence of 16S rDNA of Bifidobacterium longum subsp. Infantis YSGBifido001,001 is shown in SEQ ID NO. 3. The 16S rDNA has at least two base mutation sites compared with the known bifidobacterium longum strain, and the strain with the closest relation is B.longum subsp. INFANTIS STRAIN ATCC 15697.

EXAMPLE 2 Bifidobacterium longum subspecies infantis Strain filtrate production flow

(1) Preparation of glycerol tube strain

1) Activating original freeze-dried powder strains: transferring the freeze-dried powder to a 100 mL-cycle Kai MRS triangular flask culture medium, performing anaerobic static culture at 37 ℃ for 20-24 hours, and preparing bacterial liquid for later use;

2) Single colonies were isolated: the bacteria liquid MRS solid culture medium is subjected to flat streak separation, anaerobic static culture at 37 ℃ for 48 hours, and the bacteria liquid MRS solid culture medium is grown for later use;

3) Single colony activation: single colony is transferred to a 100mL liquid triangular flask culture medium (shown in table 3), anaerobic static culture is carried out at 37 ℃ for activating for 20 hours to prepare bacterial liquid, and whether the bacterial liquid reaches the standard OD 10>0.260 and the pH is less than 4.50 is detected by a mirror;

4) Original glycerol tube strain seed preservation: inoculating 5% of the strain (the culture qualification can be judged by detecting OD 10>0.260 after 20-24 hours of culture) to a formula card 1 triangular flask culture medium (shown in table 3), detecting OD 10>0.260 until 20 hours of culture, and ensuring that the strain glycerol tube (0.70 mL strain retention liquid and 0.70 mL strain retention liquid) reaches the standard after microscopic examination;

5) Establishing seed lots: the seed batch system is established according to the relevant regulations of bacterial strain management and quality control for biological product production verification.

(2) Inoculating the strain bifidobacterium longum subspecies of the infants obtained in the step (1) to a culture medium (the formula of the culture medium is shown in table 3), wherein the inoculation amount (the culture qualification can be judged by detecting OD (10) 0.260 after 20-24 hours of culture) is 5%, culturing for 20-24 hours at 37 ℃, centrifuging, precipitating to obtain a supernatant, freeze-drying, concentrating five times to obtain a concentrated fermentation liquor;

TABLE 3 Table 3

(3) The components of the concentrated fermentation broth of the strain obtained in the step (2) are shown in Table 4.

TABLE 4 Table 4

Verification example 1 Total acid, pH, total protein assay

The filtrate obtained in example 2 was examined.

(1) The total acid content was determined by acid-base titration.

(2) The pH was measured using a pH meter.

(3) And (5) measuring the total protein content by using a detection kit.

The experimental results are shown in table 5.

TABLE 5

Verification example 2 exfoliating

1) Test method

And (3) preparing a working solution:

<1> cleaning solution (0.1% Triton X-100) configuration: 0.1 mL Triton X-100 was weighed and dissolved in 100 mL PBS to prepare a 0.1% Triton X-100 solution.

<2> Sodium hydroxide solution (12M) configuration: weighing 4.8 g NaOH solids, adding deionized water to a constant volume of 10 mL, and preparing a NaOH solution of 12M.

Administration:

<1> washing after thawing pigskin, fixing the skin between a supply chamber and a receiving chamber of a Franz cell diffusion cell, with the skin cuticle facing the supply chamber and the dermis facing the receiving chamber; after the suckling pig skin is tightly fixed by adding a receiving liquid (PBS buffer solution) of 7.0 mL into the receiving chamber, 1.5 mL of the receiving liquid is added into the receiving chamber through a sampler, and air is discharged to enable the skin dermis layer to be in close contact with the receiving liquid.

<2> The sample shown in Table 6 was added to the skin surface in the supply chamber, 50. Mu.L of the sample was added to the pigskin surface (PBS buffer was added as a blank), the sample was spread evenly radially from the center of the skin to the edge, and the sample exposure area was 3.14 cm ². 3 replicates of each sample were run in parallel, a constant temperature water bath was maintained at (32.+ -. 1) ℃ and the water bath sandwich was ensured to be bubble free.

<3> After incubation for 24 hours, the sample application area was rubbed with a glove, and after two minutes of rubbing, 0.5 mL wash solution (0.1% Triton X-100) was added to the supply chamber, and the skin surface was rinsed with a blow of keratinocytes.

<4> Transfer the wash to a 1.5 mL centrifuge tube for use.

<5> Photomicrographs: a1.5 mL centrifuge tube containing a wash solution (0.1% Triton X-100) was placed on a mini-mixer (MIX-2500) and oscillated at maximum speed for 2 minutes, after which the wash solution was spotted onto a cell counting plate which was photographed under an inverted microscope at 10 Xmagnification.

And (3) detecting the total protein content:

<1> the collected washing liquid was put into a high-speed centrifuge, 15000 rpm, centrifuged 10 min, the supernatant was discarded, and the pellet was resuspended with 0.5. 0.5 mL deionized water.

<2> 25. Mu.L of NaOH (12M) solution was added to the resuspended liquid of step <1>, keratinocytes were lysed in a water bath (100 ℃) of 30min, 25. Mu.L of concentrated HCl (12M) was added to neutralize the lysate.

<3> Total protein content assay was performed according to BCA protein assay kit instructions.

And (3) result judgment:

and (3) cracking the eluted keratinocytes, detecting the protein content, wherein the total protein content in the cracking liquid is in direct proportion to the number of the eluted keratinocytes, and the higher the total protein content is, the more the eluted keratinocytes are, so that the sample has the effect of exfoliating the horns.

Results statistical analysis:

the results are expressed as mean±sd using GRAPHPAD PRISM plots. Comparisons between groups were performed using t-test statistical analysis. Statistical analysis was double tailed. P <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences.

The experimental results are shown in table 6 and fig. 6, and the average value of the total protein concentration measured by using the in vitro skin model found that 3% of the filtrate had a stronger exfoliating effect on keratin than 5% of mandelic acid or 2% of salicylic acid, and even more exfoliating effect on keratin than 10% of salicylic acid.

TABLE 6

Verification example 3 anti-aging efficacy

(1) DNA damage repair & epidermal atrophy based on 3D epidermal skin model

When the organism is exposed to exogenous UV radiation, DNA damage can occur, the damage can induce the organism to age, and if the damage is not repaired in time, the organism can age.

TABLE 7

Wherein: in the sample group, the lysis and filtrate+lysis were comparative examples, and in the filtrate+lysis, the filtrate was 50% and the lysis was 50%.

1) Test method

Model drug administration:

<1> according to the test group, the model was transferred to a 6-well plate (EpiGrowth broth added in advance in a corresponding group of 0.9 mL), and the test group number was marked on the 6-well plate.

<2> The groups to be subjected to UVB irradiation were subjected to UVB irradiation (irradiation dose: 600mJ/cm ²) in the groups shown in Table 7, and after the irradiation, the sample working solution was uniformly distributed on the surface of the model, and incubation was continued for 24 hours at 37℃with 5% CO ₂.

<3> After the incubation, the test substance remaining on the surface of the model was washed with sterile PBS buffer.

Tissue morphology testing:

Taking a model for tissue morphology detection, fixing with 4% paraformaldehyde for 24 hours, performing H & E staining detection, photographing under a microscope for observation, and collecting and analyzing pictures.

Immunohistochemical detection:

Taking a model for detection, fixing with 4% paraformaldehyde for 24 hours, performing immunohistochemical detection, photographing under a microscope for observation, and collecting and analyzing pictures.

Inhibition rate/turndown rate calculation:

inhibition rate/downregulation rate (%) = (negative control group-sample)/negative control group×100%

Results statistical analysis:

Test results:

①CPD：

TABLE 8 CPD Positive cell Rate

As can be seen from Table 8, CPD positive cells (the smaller the number, the better) and CPD positive cell rate for 5% filtrate was less than for the same concentration of lysates and filtrate+lysates.

TABLE 9 CPD fluorescence intensity statistics

As can be seen from Table 9, the 5% filtrate CPD fluorescence intensity downregulation was better than that of 5% lysis and 5% of Saccharomyces cerevisiae.

② Epidermolysis:

Table 10

As shown in table 10 and fig. 7, 5% sunburn Cells inhibition of the filtrate was comparable to 0.1% retinol, above other samples at the same concentration.

(2) Anti-aging effect test based on fibroblast

TABLE 11

1) Test method

Cell inoculation: after cell resuscitation, cells were inoculated into 6-well plates and incubated overnight at 37℃in 5% CO ₂ when plating rates reached around 60%.

Administration: when the cell plating rate in the 6-hole plate reaches 40% -60%, grouping drug administration is carried out, and 3 compound holes are arranged in each group. 2 mL TGF-beta 1-containing culture solution is added to each hole of the positive control group, 2 mL culture solution is added to each hole of the blank control group, and 2 mL culture solution containing samples to be tested with corresponding concentrations is added to each hole of the sample group. After the completion of the administration, the 6-well plate was placed in a CO ₂ incubator for 24: 24h.

Collecting cells: after the incubation, the old solution was discarded, washed twice with PBS, 1 mL RNAiso Plus was added to each well, and after the lysed cells were blown off, the samples were collected.

And (3) gene expression detection: RNA was extracted, reverse transcribed to cDNA, and then subjected to fluorescent quantitative PCR detection, and the result was calculated by the 2 ^-△△ CT method.

And (5) calculating an up-regulation rate: up-regulation (%) = (sample group-blank group)/blank group×100%.

Results statistical analysis: application software was plotted and the results were expressed as mean±sd. Comparisons between groups were performed using t-test statistical analysis. Statistical analysis was double tailed. P <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences.

2) Test results

①COL I

Table 12

The experimental results are shown in table 12, and the filtrate can significantly up-regulate the expression of the type I collagen gene.

②COL III

TABLE 13

The experimental results are shown in table 13, and the filtrate can significantly up-regulate the expression of type III collagen genes.

③COL IV

TABLE 14

The experimental results are shown in table 14, where the up-regulation of COL IV by the filtrate was higher than that of filtrate + lysis.

④COL VII

TABLE 15

The experimental results are shown in Table 15, and the up-regulation rate of the filtrate on the VII type collagen gene is up to 116%.

⑤VCAN

Table 16

The experimental results are shown in Table 16, and the filtrate can obviously up-regulate the expression of the proteoglycan-related gene VCAN.

Elastin protein

TABLE 17

The experimental results are shown in Table 17, only the filtrate significantly up-regulates elastin expression.

Fibronectin proteins

TABLE 18

The experimental results are shown in table 18, and the filtrate can significantly up-regulate fibronectin expression.

HAS1

TABLE 19

The results of the experiment are shown in Table 19, only the filtrate was able to up-regulate the expression of the hyaluronate synthase HAS 1.

(3) UVA irradiation fibroblast based test

1) Test method-Special administration (UVA continuous irradiation)

Table 20

Table 21

Administration: when the cell plating rate in the 6-hole plate reaches 50% -60%, carrying out UVA irradiation of 30J/cm ² on groups with UVA irradiation, and carrying out grouping drug administration after the irradiation is finished, wherein each group is provided with 3 compound holes. 2 mL TGF-beta 1-containing culture solution is added to each hole of the positive control group, 2 mL culture solution is added to each hole of the blank control group and the negative control group, and 2 mL culture solution containing samples to be tested with corresponding concentrations is added to each hole of the sample group. After completion of the administration, the 6-well plate was placed in a CO ₂ incubator (37 ℃, 5% CO ₂) for 24 hours. The above operation was repeated 3 times.

ELISA test: after the incubation, the cell culture supernatant was collected in a centrifuge tube, frozen and stored at-80℃in a refrigerator, and subjected to detection and analysis according to the instructions of ELISA kit.

And (3) gene expression detection: after incubation, 1 mL/well PBS buffer was washed twice, 1mL RNAiso Plus was added to each well, and after lysis of cells by blowing, samples were collected. RNA was extracted, reverse transcribed to cDNA, and then subjected to fluorescent quantitative PCR detection, and the result was calculated by the 2 ^-△△ CT method.

Up-regulation rate and down-regulation rate calculation:

Up-regulation = (sample group-negative control group)/negative control group×100%

Down-regulation = (negative control group-sample group)/negative control group×100%

Results statistical analysis: the results were plotted using GRAPHPAD PRISM Program software and expressed as mean±sd. Comparisons between groups were performed using t-test statistical analysis. Statistical analysis was double tailed. P <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences.

Wherein: * Indicating significant differences, which are shown to be very significant.

In the sample group, lysis and filtrate+lysis are comparative examples.

Test results:

①NF-κB PCR

Table 22

The results of the experiment are shown in Table 22, and the expression of NF- κB, an inflammation signal pathway related gene, was inhibited by the filtrate, higher than that of the other sample groups.

②p21 PCR

Table 23

The experimental results are shown in Table 23, and the filtrate can significantly down-regulate the expression of senescence-associated gene p 21.

③p16 PCR

Table 24

The experimental results are shown in table 24, and the filtrate can significantly down-regulate the expression of p16 gene.

2) Test methods-routine administration

Table 25

Cell inoculation: after cell resuscitation, cells were inoculated into 6-well plates and incubated overnight in a CO ₂ incubator (5% CO ₂) when the plating rate reached around 60%.

Administration: according to the test group of Table 25, when the cell plating rate in the 6-hole plate reaches 40% -60%, group drug administration is carried out, and 3 compound holes are arranged in each group. 2 mL of culture solution containing the sample to be tested with corresponding concentration is added to each hole of the sample group, 2 mL of culture solution is added to each hole of the blank control group and the negative control group, and 2 mL of culture solution containing TGF-beta 1 is added to each hole of the positive control group. After completion of the administration, the 6-well plate was placed in a CO ₂ incubator (37 ℃, 5% CO ₂) and cultured for 24 hours.

UVA irradiation: according to the groupings of Table 25, UVA irradiation (30J/cm ²) was performed on the remaining groups except the blank. After the irradiation is finished, the culture is continued for 24 hours in a CO ₂ incubator.

ELISA test: after the incubation, collecting the cell culture supernatant in a centrifuge tube, placing in a refrigerator at-80 ℃ for freezing preservation, and carrying out detection and analysis according to the operation instruction of the ELISA kit.

And (3) gene expression detection: after incubation, 1 mL/well PBS was washed twice, 1 mL RNAiso Plus was added to each well, and after cell lysis was performed by blowing, the cells were harvested. RNA was extracted, reverse transcribed to cDNA, and then subjected to fluorescent quantitative PCR detection, and the result was calculated by the 2- ^△△ CT method.

Up-regulation rate and down-regulation rate calculation:

In the sample group, lysis and filtrate+lysis are comparative examples.

Test results:

①TIMP-1 PCR

Table 26

The experimental results are shown in Table 26, the filtrate significantly improved the expression of the matrix metalloproteinase inhibitor TIMP-1, and was better than lysis and filtrate+lysis.

(4) In vitro skin tissue test based on UVA and UVB combined irradiation

Table 27

Wherein: in the filtrate and the lysate, the filtrate accounts for 50 percent, and the lysate accounts for 50 percent.

Table 28

1) Test method

Tissue treatment: immersing the obtained fresh skin tissue in 75% alcohol, washing for 30s, and washing three times by using a sterile PBS buffer solution; after the completion, the skin was cut into tissue pieces of 24±2mm ² with the dermis facing downward and the epidermis facing upward, placed in a culture mold, and then the culture mold was transferred into 6-well plates, each well was added with 3.7 mL culture solution, and cultured in a CO ₂ incubator (37 ℃,5% CO ₂) with daily replacement of the solution.

Administration: after the above-mentioned isolated skin tissue was cultured for 2 days, irradiation and administration were started with reference to the test groups and the corresponding treatment conditions of tables 27 and 28; the irradiation doses were UVA (30J/cm ²) and UVB (50 mJ/cm ²), the irradiation was continued for 4 days, new culture solution was changed after each irradiation was completed, and administration was performed, positive control (VC+VE) was administered under the liquid, and samples to be tested were surface-administered, and each group was repeated 3 times. After 4 days of continuous irradiation, the isolated skin tissue was cultured for 3 further days during which no irradiation was performed and only the sample was administered.

Collagen fiber detection: skin tissues after the end of the administration were fixed with 4% paraformaldehyde, the tissues were embedded, masson stained after slicing, the slicing results were recovered, photographed using a microscope, and analyzed using image-Pro Ⓡ plus image processing software.

Immunohistochemical detection: detection was performed according to the specific procedure of immunohistochemistry.

Immunofluorescence detection: detection is carried out according to specific immunofluorescence operating steps.

And (3) calculating a lifting rate:

improvement rate= (sample group-negative control group)/negative control group×100%

Results statistical analysis: the results are expressed as mean±sd using GRAPHPAD PRISM plots. Comparisons between groups were performed using t-test statistical analysis. Statistical analysis was double tailed. P <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences.

In the sample group, lysis and filtrate+lysis are comparative examples.

2) Test results

① Hyaluronic acid

Table 29

The experimental results are shown in Table 29 and FIG. 8, the filtrate can improve the expression of hyaluronic acid, and the improvement rate is up to 106.82%.

② XVII type collagen

Table 30

The experimental results are shown in table 30 and fig. 9, the filtrate can significantly improve the expression of XVII type collagen, and the improvement rate is far higher than that of the comparative example.

Verification example 4 autophagy-fibroblast-based test

(1) LC3 II gene expression level

There is a one-to-one correspondence between the number of LC3-II and the number of autophagosomes, LC3-II is generally recognized as a marker for mammalian autophagosomes. In the autophagy process, LC3-II on the inner membrane of autophagy is degraded by lysosome, and under the condition of adding a lysosome inhibitor, autophagy flow can be detected by immunofluorescence comparison of the change of the protein amount of LC3-II in the autophagy process, and autophagy activity is reflected.

1) Test method

Cell inoculation: after cell recovery, cells were inoculated into 6-well plates and incubated overnight in a CO ₂ incubator (37 ℃, 5% CO ₂) when the plating rate reached about 60%.

Administration: according to the test group of Table 31, when the cell plating rate in the 6-hole plate reaches 40% -60%, group drug administration is carried out, and 3 compound holes are arranged in each group. 2 mL of culture solution containing the sample to be tested with corresponding concentration is added to each hole of the sample group, and 2 mL of culture solution is added to each hole of the blank control group. After the completion of the administration, the 6-well plate was placed in a CO ₂ incubator for continuous incubation for 24 hours.

Collecting cells: after incubation for 24h, the old solution was discarded, washed twice with PBS buffer, 1 mL RNAiso Plus cells were added to each well, and after lysis cells were blown off, samples were collected.

And (5) calculating an up-regulation rate: up-regulation = (sample group-negative control group)/negative control group×100%

2) Test results

Table 31

The experimental results are shown in table 31, and the filtrate can significantly promote the expression of autophagy occurrence related gene LC3 II.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The preparation method of the microbial agent is characterized by comprising the following steps:

S1: inoculating and culturing the strain to obtain seed liquid; the strain is bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis), and the preservation number is: CGMCC No.27851;

2. The method of claim 1, wherein the inoculation amount of S1 is 5%.

3. The method of claim 2, wherein the centrifugation in S2 is performed at a speed of 3000 rpm for a period of 30 min.

4. A method according to any one of claims 1 to 3, wherein the supernatant obtained in S2 further comprises a step of concentration.

5. The method of claim 4, wherein the concentration is 5-fold.

6. The microbial agent prepared by the preparation method of any one of claims 1-5, comprising: 17% of glycolic acid, 8.5% of pyroglutamic acid, 6% of citric acid, 4.5% of hydroxylamine and 3% of sorbitol.

7. The use of the microbial agent according to claim 6 for the preparation of anti-aging cosmetics.

8. The use of a microbial agent according to claim 6 for the preparation of exfoliating cosmetics.

9. A cosmetic comprising the microbial agent according to claim 6 and an auxiliary agent acceptable in the cosmetic field.