Oral streptococcus with anti-aging effect and its application

Abstract

The present invention relates to the field of microbial technology, and specifically to oral streptococci with anti-aging effects and applications thereof. A strain of oral streptococci was isolated from a child's face, and its deposit number is CCTCC NO: M20221853. Experimental results show that its fermentation product contains a relatively high concentration of spermidine. The application of its fermentation product in skin protection can promote the synthesis of desmocollin 2 (DSC2) responsible for skin barrier function, ATP binding cassette subfamily A member 12 (ABCA12), and type I collagen α1 chain (COL1A1), one of the main components of the extracellular matrix, to achieve the purpose of strengthening the skin barrier, enhancing skin elasticity and anti-aging. At the same time, the oral streptococci are non-cytotoxic, safe and reliable, and can be used in cosmetics.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to oral streptococci with anti-aging effects and applications thereof. Background Art

[0002] Probiotics are a general term for microorganisms that are beneficial to the human body. At present, the research on the efficacy of probiotics at home and abroad has become mature, and has been widely used in the field of cosmetics. With the development of bio-fermentation technology, the "microecological skin care era" of maintaining the balance of the skin's "ecosystem", making the skin's "wall" more solid, and repairing the skin's microecology as a whole, thus reshaping healthy skin has arrived. At the same time, with the research and development of skin care ingredients, anti-aging effects are becoming younger and more and more popular in the market. According to Euromonitor data, the scale of anti-aging skin care products in the Chinese skin care market in 2020 reached 64.6 billion yuan, accounting for 28.8% of the skin care market share. According to iResearch data, in terms of focusing on efficacy, the first-ranked efficacy of the overall sample is anti-aging. Consumers are more looking forward to slowing down the aging of the skin and maintaining a youthful state through daily skin care.

[0003] So far, many probiotics have been applied to skin microecological preparations. Most of the effective probiotics come from the isolation of human intestinal microorganisms. Existing relevant studies have shown that the skin microecological flora is closely related to age. The characteristics of skin microorganisms at different ages are very different. For example, in old skin, there is a low proportion of Propionibacterium and high microbial diversity, and in young skin, there is a high proportion of Propionibacterium and low microbial diversity. Chinese patent CN202110049175.1 discloses a composition for enhancing the skin barrier, moisturizing the skin or anti-aging, which studies the effects of four strains of Streptococcus pneumoniae, Streptococcus infantis, Streptococcus mitis and Streptococcus thermophilus on skin aging. However, there is currently little in-depth research on the relationship between the efficacy of skin flora and skin, and more dominant strain resources need to be developed. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide oral streptococci with anti-aging effect and application thereof.

[0005] The invention provides oral streptococcus JWA714 (Streptococcus oralis JWA714) with a deposit number of CCTCC NO: M 20221853.

[0006] The oral streptococcus of the present invention is separated from the face of a child, and is confirmed to be oral streptococcus (Streptococcus oralis) through TSB culture medium, PCR amplification and sequence comparison. It is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 20221853.

[0007] Furthermore, the present invention provides a bacterial community including the oral streptococcus of the present invention. The bacterial community of the present invention includes a bacterial community formed by a single or multiple bacteria that do not have a mutual antagonistic or competitive relationship with the oral streptococcus, and the present invention is not limited to this.

[0008] Furthermore, the present invention provides a bacterial agent containing the oral streptococcus or the bacterial flora. The dosage form of the bacterial agent includes granules, liquid and dry powder, which is not limited by the present invention. In the present invention, the bacterial agent also includes the fermentation liquid, bacterial cells, supernatant and active substances contained therein of the oral streptococcus, which is not limited by the present invention.

[0009] The invention provides application of the oral streptococcus in preparing a product containing spermidine.

[0010] In some specific embodiments of the present invention, two strains of oral Streptococcus were isolated, and the spermidine concentration in the fermentation broth of the oral Streptococcus of the present invention was higher than that of the other strain, and the spermidine content in the fermentation broth reached 2.59 ng / ml.

[0011] The present invention provides a method for preparing spermidine, which comprises culturing the oral streptococcus as described in the present invention to obtain a culture containing spermidine.

[0012] The present invention provides the use of the oral streptococcus, and / or the bacterial flora, and / or the bacterial agent, and / or the culture containing spermidine prepared by the preparation method according to claim 5 in preparing a product for improving skin quality.

[0013] Furthermore, the improvement of skin quality includes at least one of repairing the skin barrier or enhancing skin elasticity.

[0014] In the present invention, the oral Streptococcus can repair the skin barrier, and the repair of the skin barrier includes promoting the expression of skin barrier function genes, and the skin barrier function genes include but are not limited to DSC2FLG, GBA and ABCA12, which are not limited by the present invention. In some specific embodiments of the present invention, the fermentation filtrate of oral Streptococcus can promote the expression of DSC2 gene, ABCA12 gene FLG gene, and / or GBA gene in HSF cells, thereby promoting the repair of the skin barrier.

[0015] The oral streptococcus of the present invention has a strong ability to repair the skin barrier. In some specific embodiments of the present invention, the fermentation liquid of the oral streptococcus has a significantly higher effect on the expression of genes related to repairing the skin barrier in HSF cells than other strains.

[0016] In the present invention, the oral streptococcus can enhance skin elasticity, and the enhancement of skin elasticity includes promoting the expression of genes related to extracellular matrix synthesis, and the genes related to extracellular matrix synthesis include but are not limited to COL1A1. In some specific embodiments of the present invention, the fermentation filtrate of the oral streptococcus can promote the expression of the COL1A1 gene in HaCaT cells, thereby enhancing skin elasticity.

[0017] The oral streptococcus of the present invention is safe and non-toxic. In some specific embodiments of the present invention, the oral streptococcus fermentation liquid has no cytotoxicity to fibroblasts when the concentration is below 4% (v / v); has a proliferation-promoting effect on fibroblasts when the concentration is below 2% (v / v); and has no cytotoxicity to keratinocytes when the concentration of the fermentation liquid is below 5% (v / v).

[0018] The present invention provides a product for improving skin quality, wherein the raw materials thereof include the oral streptococcus described in the present invention, and / or the bacterial flora described in the present invention, and / or the bacterial agent described in the present invention, and / or the culture containing spermidine obtained by the preparation method described in the present invention.

[0019] Furthermore, the products described in the present invention include medicines or cosmetics, which are not limited in the present invention.

[0020] Furthermore, the products described in the present invention include cosmetics, and the cosmetic dosage forms include: at least one of creams, emulsions, water-based agents, gels, oils, powders, loose powders, muds, aerosols, patches, films, and freeze-dried agents.

[0021] The present invention also provides a method for improving skin condition, which comprises using the product of the present invention. The method of using includes applying, external application or injection, which is not limited by the present invention.

[0022] The present invention provides the use of the oral streptococcus in promoting the expression of DSC2 gene and ABCA12 gene in HSF cells, and / or COL1A1 gene in HaCaT cells.

[0023] The oral streptococcus of the present invention is safe and non-toxic. The fermentation liquid thereof is used in an eye irritation experiment, and the result shows that the oral streptococcus is non-irritating to the eyes. The patch experiment also proves that the oral streptococcus of the present invention has no toxic side effects on the skin.

[0024] The present invention isolates a strain of oral streptococcus from the face of a child, and the deposit number thereof is CCTCC NO: M20221853. Experimental results show that the fermentation product thereof contains a relatively high concentration of spermidine, and the fermentation product thereof can be applied to skin protection to promote the synthesis of desmocollin 2 (DSC2), ATP binding cassette subfamily A member 12 (ABCA12), and type I collagen α1 chain (COL1A1), one of the main components of the extracellular matrix, which is responsible for the skin barrier function, so as to achieve the purpose of enhancing the skin barrier, enhancing skin elasticity and anti-aging. At the same time, the oral streptococcus is non-cytotoxic, safe and reliable, and can be applied to cosmetics.

[0025] Biological Deposit Description

[0026] Oral Streptococcus JWA714 (Streptococcus oralis JWA714) was deposited in the China Center for Type Culture Collection on November 30, 2022, at: Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M20221853. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Shown is the standard curve of spermidine;

[0028] Figure 2 Shows the effects of 5# and 20# oral streptococci on keratinocyte gene expression;

[0029] Figure 3 Shown is the effect of 5# oral Streptococcus on gene expression in fibroblasts. DETAILED DESCRIPTION

[0030] The present invention provides oral streptococci with anti-aging effects and their applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve them. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0031] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0032] The present invention will be further described below in conjunction with embodiments:

[0033] Example 1 Strain Isolation, Identification and Performance Study

[0034] 1. Isolation and identification of strains

[0035] After washing the subject's face with sterile water, spray the sterile water onto TSB and grow it aerobically at 37°C. Collect individual colonies and culture them in liquid TSB medium (tryptone soy medium) in a 96-well plate at 37°C for 72 hours. Collect the culture in two rows, centrifuge at 6000 rpm for 30 minutes, collect the precipitate, extract DNA, and perform PCR amplification using specific primers for S. infantis:

[0036] ST2-F, 5'-gcaagtagaacgctgaaggagg-3' (SEQ ID NO: 8);

[0037] ST2-R, 5'-caacatctcacgacacgagc-3' (SEQ ID NO:9).

[0038]

[0039] Table 1. Isolates

[0040] Latin name Homology Sample No. source Streptococcusoralis 99.08％ 5# Children 1# Face Streptococcusoralis 98.98％ 20# Children 1# Face

[0041] 2. Preparation of fermentation broth

[0042] The purified streptococci were inoculated into TSB medium at a volume fraction of 5%, and cultured in a shaking flask at 150 rpm / min for 24 h to obtain a streptococcal culture. The culture was centrifuged at 6000 rpm / min for 10 min to obtain a supernatant, which was then sterilized by filtration to obtain a sterile streptococcal metabolite sample.

[0043] 3. Quantitative analysis of spermidine in fermentation broth

[0044] Experimental method: Sterile streptococcal metabolite samples can be directly tested. The specific implementation method is carried out according to the instructions of spermidine (SMD) enzyme-linked immunosorbent assay kit (ELKBiotechnology), specifically:

[0045] (1) Before the experiment begins, all reagents should be equilibrated to room temperature and prepared. When diluting reagents or samples, they must be mixed evenly, and foaming should be avoided as much as possible.

[0046] (2) Sample addition: Set up standard wells and sample wells. Add 50 μL of the standard or sample to be tested. Be careful not to have bubbles. Add the sample to the bottom of the well of the ELISA plate, and try not to touch the well wall. Then add the biotin conjugate (1 × )50 L, shake gently to mix, cover the ELISA plate or cover it with film, and incubate at 37℃ for 1 hour.

[0047] (3) To ensure the validity of the experimental results, a new standard solution was used in each experiment.

[0048] (4) After incubation for 1 hour, discard the liquid in the wells, spin dry, and wash the plate 3 times, adding washing solution (1 × )200μL, soak for 1 to 2 minutes each time, and shake dry.

[0049] (5) Then add streptomycin-HRP (1 × ) 100 μL, gently shake to mix, cover the ELISA plate, incubate at 37℃ for 1 hour. Discard the liquid in the well, spin dry, wash the plate 5 times, add washing solution (1 × )200μL, soak for 1 to 2 minutes each time, and shake dry.

[0050] (6) Add 90 μL of TMB colorimetric reagent to each well in sequence and color develop at 37°C in the dark for 15 to 20 minutes (within 20 minutes, a clear blue gradient can be seen in the first 3 to 4 wells of the standard, but the gradient is not obvious in the last 3 to 4 wells, and the development can be terminated).

[0051] (7) Add 50 μL of stop solution to each well to stop the reaction (the blue color immediately turns yellow). The order of adding the stop solution should be as consistent as possible with the order of adding the substrate solution. To ensure the accuracy of the experimental results, the stop solution should be added as soon as possible after the substrate reaction time is up.

[0052] (8) Measure the optical density (OD value) of each well in sequence at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) within 5 minutes after adding the stop solution.

[0053] result:

[0054] Use professional curve drawing software to draw the curve (four-parameter logistic curve fitting): equation: y = (AD) / [1 + (x / C) ^ B] + D (A = 228.04177; B = 2.60302; C = 0.11253; D = 1.09873) R 2 =0.99920. The spermidine content in the streptococcal fermentation broth sample is shown in Table 2:

[0055] Table 2. Spermidine content in Streptococcus fermentation broth samples

[0056] OD450nm SMD (ng / ml) sample X Value Y Value 5# (Streptococcus in this protocol) 0.7736 2.59 20# 0.9287 2.03

[0057] The supernatant of 5# and 20# oral streptococcus fermentation broths both contain spermidine, indicating that they have the ability to produce spermidine, and 5# has a stronger spermidine production capacity.

[0058] 4. Cytotoxicity test of 5# streptococcal fermentation broth:

[0059] Different concentrations of streptococcal fermentation broth were co-incubated with keratinocytes and fibroblasts for 24 h, and the cytotoxicity of the culture broth to keratinocytes and fibroblasts was detected by MTT method. 4 The cells were inoculated at a density of 10 cells / well in a 96-well plate. After culturing for 24 hours, fresh culture medium (serum-free) was replaced, and streptococcal fermentation broth was added to treat the cells for 24 hours. The supernatant was discarded, 100 μL of MTT solution was added, and after incubation in an incubator for 4 hours, 110 μL of DMSO was added to dissolve the formazan crystals, and the absorbance was detected at 490 nm. The cytotoxicity results are shown in Tables 3 and 4:

[0060] Table 3. Effects of Streptococcus on HSF toxicity

[0061]

[0062] Table 4. Effect of Streptococcus on HaCaT toxicity

[0063]

[0064] The fermentation broth of 5# oral streptococcus has no cytotoxicity to fibroblasts at a concentration below 4% (v / v), and has a proliferation-promoting effect on fibroblasts at lower concentrations, such as 1% (v / v) and 2% (v / v). The fermentation broth has no cytotoxicity to keratinocytes at a concentration below 5% (v / v).

[0065] 5. Evaluation of the expression level of dermal structural genes

[0066] Cell culture and St solution treatment:

[0067] HSF were cultured in a fibroblast culture system, and when the cells were 80% confluent, they were inoculated into 6-well plates and incubated at 37°C in an atmosphere of 5% CO2 for 24 hours. The cells were then washed once with phosphate-buffered saline (PBS), and culture medium containing 5% (v / v) streptococcal fermentation broth and culture medium without supplements were added to the cell wells as experimental and control groups, respectively, and incubated for 24 hours.

[0068] HaCaT was cultured in DMEM medium (supplemented with 10% (v / v) FBS, double antibody), and when the cells were 80% confluent, they were inoculated into 6-well plates and incubated at 37°C, 5% CO2 for 24 hours. The cells were washed once with phosphate-buffered saline (PBS), and culture medium containing 4% (v / v) streptococcal fermentation broth and culture medium without supplement were added to the cell wells as experimental and control groups, respectively, and incubated for 24 hours.

[0069] RNA extraction and real-time fluorescence quantitative PCR:

[0070] Total RNA was isolated from cells according to the manufacturer's instructions (Novagen RNAisolater (Vazyme Cat. R401-01100ml)). cDNA was synthesized from 1 μg of total RNA using a reverse transcription premix (HiScript II 1st Strand cDNA Synthesis Kit (Vazyme)). qPCRSYBR Green Master Mix (Vazyme) was used for real-time fluorescence quantitative PCR amplification reaction to quantify gene expression. The equipment was CFX Real-Time PCR System (BIO-RAD, USA). The reaction conditions were as follows: start at 95°C for 5 minutes, then cycle at 95°C for 10 seconds, 55°C for 20 seconds, and 72°C for 20 seconds, for 40 cycles. β-actin was used as an internal control.

[0071] The primer sequences are as follows:

[0072] Desmoglein 2 (DSC2):

[0073] Forward: 5′-AGTGTGAGTTTGTTCATCACAGGTC-3′(SEQ ID NO:2);

[0074] Reverse: 5′-CCATGGCCTCACAGCCTTTA-3′(SEQ ID NO:3);

[0075] ABCA12:

[0076] Forward: 5′-ACAGGAATGGCCTTCATCAC-3′(SEQ ID NO:4);

[0077] Reverse: 5′-AACATGGTGCCCTGAGAAAC-3′(SEQ ID NO:5);

[0078] Type I collagen alpha 1 (COL1A1):

[0079] Forward: 5′-GAGGGCCAAGACGAAGACATC-3′(SEQ ID NO:6);

[0080] Reverse: 5′-CAGATCACGTCATCGCACAAC-3′(SEQ ID NO:7);

[0081] GBA:

[0082] Forward: 5-GCTAGGCTCCTGGGATCGAG-3′(SEQ ID NO:10);

[0083] Reverse: 5′-GTTCAGGGCAAGGTTCCAGTCA-3′(SEQ ID NO:11);

[0084] FLG:

[0085] Forward: 5′-AGTGCACTCAGGGGGCTCACA-3′(SEQ ID NO:12);

[0086] Reverse: 5′-CCGGCTTGGCCGTAATGTGT-3′(SEQ ID NO:13);

[0087] Such as Figure 2As shown, the culture supernatants of Streptococcus 5# and Streptococcus 20# were used to treat keratinocytes. Compared with the normal group, the expression levels of desmocollin 2 (DSC2) and filaggrin (FLG) responsible for skin barrier function, glucosylceramidase β (GBA) and ATP-binding cassette subfamily A member 12 (ABCA12) (synthesis of lamellar bodies and ceramides can form lipid barriers) in Streptococcus 5# showed an upward trend (GBA data, due to the large data dispersion, the significance analysis was not significant), among which the gene expression of desmocollin 2 (DSC2) and ATP-binding cassette subfamily A member 12 (ABCA12) increased significantly compared with the control. Desmocollin 2 and glucosylceramidase β in Streptococcus 20# were comparable to those in the control group, with no upward trend.

[0088] Streptococcus 5# treatment of fibroblasts also significantly increased the expression of type I collagen α1 chain (COL1A1), one of the main components of the extracellular matrix (e.g. Figure 3 shown).

[0089] 6. Acute eye irritation / corrosion test of 5# streptococcal fermentation broth

[0090] According to Chapter 6 5 Acute eye irritation test of "Safety Technical Specifications for Cosmetics" (2015 edition), 0.1m of fermentation liquid with a concentration of 10% (v / v) was taken as the test substance and dripped into the conjunctival sac of the left eye of the rabbit, closed for 1s, and not rinsed after administration. The right eye was not treated as a self-control, and the animal's eyes were examined 1, 24, 48, and 72 hours after the dripping of the test substance. If no irritation reaction occurred after 72 hours, the test could be terminated. In addition to observing the cornea, iris, and conjunctiva, other damage effects should be recorded and reported. In each inspection, the score of eye irritation reaction should be recorded according to the scoring standard of eye damage in Table 5 of the test basis.

[0091] Table 5. Scoring criteria for eye damage

[0092]

[0093]

[0094] Table 6. Observation results of eye irritation test

[0095]

[0096] After administration of the test substance, the irritation score of the cornea, iris or conjunctiva of the animals at the observation points of 24, 48 and 72 hours was 0. According to the test basis Table 5, the grading of eye irritation reaction of cosmetic raw materials, the eye irritation intensity of the sample (without washing) was non-irritating.

[0097] 7. Patch test of 5# streptococcal fermentation liquid

[0098] According to the "Technical Specifications for Safety of Cosmetics" (2015 edition), the fermentation liquid of 5# streptococcus was subjected to skin occlusion patch test. 20μl of the test substance (fermentation liquid with volume fractions of 1%, 2% and 4% respectively) was dripped into the patch test patch. The patch tester with the test substance was applied to the curved side of the subject's forearm, and the palm was gently pressed to make it evenly applied to the skin for 24 hours. At the same time, a blank patch tester was left as a blank control. After 24 hours, the test substance patch tester was removed, and the skin reaction was observed once at 0.5h, 24h and 48h. The reaction results were recorded according to Table 7 (Standard Table for Grading of Adverse Skin Reactions). Interpretation of the results of the skin occlusion patch test: When the number of subjects with grade 1 adverse skin reactions is more than 5, or the number of subjects with grade 2 adverse skin reactions is more than 2 (the number of subjects with grade 2 reactions in the deodorant product patch test is more than 5), or when any one grade 3 or above grade 3 adverse skin reaction occurs, the test substance is judged to have adverse skin reactions to the human body.

[0099] Table 7. Grading criteria for adverse skin reactions

[0100]

[0101] Table 8. Skin occlusion patch test skin reaction grading standards

[0102]

[0103] A total of 30 volunteers completed the patch test of 3 samples, and no positive reaction was found, indicating that the 5# streptococcal fermentation broth sample is relatively safe.

[0104] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Streptococcus oralis with a deposit number of CCTCC NO: M 20221853.

2. Use of the oral streptococcus according to claim 1 in the preparation of a product containing spermidine.

3. A method for preparing spermidine, comprising culturing the oral streptococcus as claimed in claim 1 to obtain a fermentation supernatant containing spermidine.

4. Use of the fermentation supernatant containing spermidine obtained by the preparation method according to claim 3 in the preparation of a product for improving skin quality; the improvement of skin quality is repairing the skin barrier or enhancing skin elasticity.

5. A cosmetic for improving skin quality, characterized in that: The raw material comprises the fermentation supernatant containing spermidine obtained by the preparation method described in claim 3.

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