TWI757850B - Lactobacillus salivarius tci153 and use of lactobacillus salivarius tci153 and/ or its bacteriolytic solution for preparing compositions that improve skin conditions - Google Patents

Abstract

Lactobacillus salivarius TCI153 was deposited in the Food Industry Research and Development Institute under the accession number BCRC 910982. Based on this, Lactobacillus salivarius TCI153 and/ or its bacteriolytic solution can be used to prepare compositions that improve skin condition.

Description

Uses of Lactobacillus salivarius TCI153 and Lactobacillus salivarius TCI153 and/or their broken bacteria in the preparation of compositions for improving skin condition

The present invention relates to a Lactobacillus salivarius and its use, in particular to a Lactobacillus salivarius TCI153 (Lactobacillus salivarius TCI153), which can be used to prepare a composition for improving skin condition.

Probiotics are generally considered to refer to ingested microorganisms that have positive benefits to the host (or receptors, such as animals or humans) after ingestion. Its name comes from the Greek "for life" (beneficial to life), also known as "primary health-care bacteria"; and probiotics mainly refer to lactic acid bacteria and some yeasts.

Generally speaking, "Lactic Acid Bacteria" refers to the general term of bacteria that can use carbohydrates to ferment and produce a large amount of lactic acid, which is a rather complex flora. Among many strains of lactic acid bacteria, some strains of lactic acid bacteria have been found to be beneficial to the health of recipients, so these strains are regarded as probiotics. In other words, probiotics are mostly lactic acid bacteria, but only a few strains of lactic acid bacteria that have been shown to be beneficial to the recipient's health can be called probiotics.

For example, common species of lactic acid bacteria that can be used as probiotics include Enterococcus, Lactobacillus, Bifidobacterium (Bifidobacterium), Bacillus (Bacillus), etc., and the yeast species that can be used as probiotics include Saccharomyces.

Lactobacillus is a group of fermentative, facultatively anaerobic, gram-positive bacilli that do not produce spores. Lactobacillus, named for its ability to ferment carbohydrates into lactic acid, is used to make liquid yogurt, solid cheese, sauerkraut, beer, wine, sauerkraut, pickles, and other fermented foods.

In some embodiments, a Lactobacillus salivarius TCI153, wherein Lactobacillus salivarius TCI153 is deposited with the Food Industry Development Research Institute, a consortium, and the deposit number is BCRC 910982.

In some embodiments, the use of a Lactobacillus salivarius TCI153 and/or its breaking solution in the preparation of a composition for improving skin, wherein Lactobacillus salivarius TCI153 is deposited in the Food Industry Development Research Institute of a consortium, and deposited in The number is BCRC 910982.

In some embodiments, Lactobacillus salivarius TCI153 is capable of producing sialic acid.

In some embodiments, Lactobacillus salivarius TCI153 functions as an epidermal growth factor that promotes receptor production.

In some embodiments, Lactobacillus salivarius TCI153 and/or its sterilization fluid has the function of promoting the synthesis of elastin in cells.

In some embodiments, Lactobacillus salivarius TCI153 and/or its disrupted fluid has the function of promoting cell reorganization and aggregation of collagen.

In some embodiments, the composition is used to increase skin elasticity, skin firmness, or a combination thereof in a recipient.

In some embodiments, the composition is used to improve wrinkles, skin pores, skin water dispersion, or a combination thereof in a recipient.

In some embodiments, the composition is used to increase skin radiance, skin hydration, or a combination thereof in a recipient.

In some embodiments, the composition is a pharmaceutical, food, skincare or cosmetic.

To sum up, the Lactobacillus salivarius TCI153 and/or its broken solution of any embodiment can produce sialic acid (also known as bird's nest acid) and improve skin. In some embodiments, Lactobacillus salivarius TCI153 and/or its sterilization fluid can be used to prepare skin-improving compositions.

W0: wrinkles

W8: Wrinkles

P0: Pores

P8: Pores

Fig. 1 is the result graph of the survival rate experiment of Lactobacillus salivarius TCI153 in gastrointestinal simulated environment; Fig. 2 is the experimental result graph of sialic acid content of Lactobacillus salivarius TCI153 and other Lactobacillus strains; Fig. 3 is the experimental data result of sialic acid content Fig. 4 is the relative data result graph of elastin production; Fig. 5 is the data result graph of the relative size of collagen membrane; Fig. 6 is the sialic acid content in blood measured in the 0th week, the 4th week and the 8th week Data result graph; Figure 7 is the result graph of epidermal growth factor content in blood measured in weeks 0, 4 and 8; Figure 8 is the wrinkle data measured in weeks 0, 4 and 8 result graph; Figure 9 is the wrinkle analysis photos in the 0th week and the 8th week: Figure 10 is the pore data result graph measured in the 0th week, the 4th week and the 8th week; Figure 11 is the pore analysis in the 0th week and the 8th week Photos: Figure 12 shows the results of skin gloss data measured in weeks 0, 4 and 8; Figure 13 is a photo of skin gloss at weeks 0 and 8: Figure 14 shows the results of weeks 0 and 4 Figure 15 shows the results of skin moisture content data measured in weeks 0, 4 and 8.

Lactobacillus salivarius TCI153 is a strain of Lactobacillus isolated from human breast milk. Lactobacillus salivarius TCI153 is deposited with the Food Industry Development Research Institute under the accession number BCRC 910982. Lactobacillus salivarius TCI153 was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession number DSM 33503.

Lactobacillus salivarius TCI153 is a Gram-positive bacterium that can grow in an anaerobic environment and is an anaerobic bacterium. Lactobacillus salivarius TCI153 grows at a temperature of 35°C to 37°C, and can survive in an environment with a pH value of 3 to 7.

In some embodiments, Lactobacillus salivarius TCI153 is resistant to gastric bile salts. For example, Lactobacillus salivarius TCI153 had an 83% survival rate in a gastric simulated environment (pH 3-4) and a 93% survival rate in a gut simulated environment (pH 7). Therefore, Lactobacillus salivarius TCI153 can colonize the human gastrointestinal tract environment, thereby producing active ingredients in the human body that can be absorbed by the human body, such as sialic acid (also known as bird's nest acid).

In some embodiments, the Lactobacillus salivarius TCI153 has the function of generating sialic acid, thereby increasing the content of sialic acid in the recipient. In other words, when the recipient took L. salivarius TCI153, L. salivarius TCI153 could increase the sialic acid content in the recipient's blood. In some embodiments, the recipient can be human.

In some embodiments, the Lactobacillus salivarius TCI153 functions to improve the skin condition of the recipient. In other words, when the recipient took L. salivarius TCI153, L. salivarius TCI153 could improve the recipient's skin condition. In some embodiments, improving the skin condition includes at least one of the following: increasing the recipient's skin elasticity, increasing the recipient's skin firmness, improving the recipient's wrinkles, reducing the recipient's skin pores, increasing the recipient's skin radiance , reduce the skin moisture loss of the receptor, and increase the skin moisture content of the receptor.

In some embodiments, the Lactobacillus salivarius TCI153 disrupted solution has the function of improving the skin condition of the recipient. In other words, when the recipient takes the bacteria-breaking solution of Lactobacillus salivarius TCI153 or the composition prepared from the bacteria-breaking solution, the bacteria-breaking solution of Lactobacillus salivarius TCI153 can improve the skin condition of the recipient. In some embodiments, improving the skin condition includes at least one of the following: increasing the recipient's skin elasticity, increasing the recipient's skin firmness, improving the recipient's wrinkles, reducing the recipient's skin pores, increasing the recipient's skin radiance , reduce the skin moisture loss of the receptor, and increase the skin moisture content of the receptor.

In some embodiments, the term "disrupting solution" refers to a solution containing bacterial fragments (cell membranes, etc.) of the disrupted Lactobacillus and intracellular proteins of the Lactobacillus. For example, the breaking fluid of L. salivarius TCI153 includes bacterial fragments of L. salivarius TCI153, the intracellular protein of L. salivarius TCI153, and a lysis buffer for re-solution of L. salivarius TCI153.

In some embodiments, a Lactobacillus salivarius TCI153 breakage can be prepared by culturing Lactobacillus salivarius TCI153 and then collecting Lactobacillus salivarius TCI153 cells. In some examples, Lactobacillus salivarius TCI153 was cultured in liquid Lactobacillus salivarius MRS medium (BD Difco ^™ Lactobacilli MRS Broth) at 37° C. in an anaerobic environment (that is, the oxygen concentration in the culture environment was 1 vol%) for 16 hours, Centrifuge at 5,000×g for 20 minutes to collect the Lactobacillus salivarius TCI153 cells after centrifugation. Next, the centrifuged Lactobacillus salivarius TCI153 cells were disrupted to collect the disrupted solution. In one embodiment, the centrifuged Lactobacillus salivarius TCI153 cells are redissolved in a lysis buffer (Lysis Buffer), and subjected to a DNase reaction to decompose the DNA of Lactobacillus salivarius TCI153. Next, three times of sterilization were performed by a high pressure sterilizer to form a sterilized liquid of Lactobacillus salivarius TCI153.

In some embodiments, Lactobacillus salivarius TCI153 and/or its sterilization fluid has the function of promoting the synthesis of elastin in cells. In other words, when the recipient takes the Lactobacillus salivarius TCI153 live bacteria or takes the Lactobacillus salivarius TCI153 broken bacteria, the skin elasticity of the recipient can be increased.

In some embodiments, the Lactobacillus salivarius TCI153 and/or its disrupted fluid has the function of recombining aggregated collagen. In other words, when the recipient took Lactobacillus salivarius TCI153 or the bacteria-breaking solution of Lactobacillus salivarius TCI153, the skin firmness of the recipient was improved.

In some embodiments, Lactobacillus salivarius TCI153 functions as an epidermal growth factor (EGF) that promotes receptor production. In other words, when the recipient takes Lactobacillus salivarius TCI153, it can increase the EGF content in the recipient's blood, thereby increasing the metabolism of the recipient's skin cells, improving the anti-aging ability of the recipient's skin, improving the anti-wrinkle ability of the recipient's skin, or a combination thereof.

Based on this, in some embodiments, Lactobacillus salivarius TCI153 and/or its breakage can be used to prepare a composition for improving skin condition. Among them, improving skin condition includes at least one of the following Scenarios: increase receptor skin elasticity, increase receptor skin firmness, improve receptor wrinkles, reduce receptor skin pores, increase receptor skin radiance, reduce receptor skin moisture loss, and improve Receptor's skin moisture content.

In some embodiments, any of the foregoing compositions can be a pharmaceutical. In other words, the medicinal product contains an effective content of Lactobacillus salivarius TCI153 and/or its broken solution.

In some embodiments, the aforementioned pharmaceutical products may be manufactured for parenterally, parenterally, orally, or topically using techniques well known to those skilled in the art Dosage form.

In some embodiments, the dosage form for parenteral or oral administration can be, but is not limited to, a tablet, troche, lozenge, pill, capsule , dispersible powder or granule, solution, suspension, emulsion, syrup, elixir, slurry or the like. For example, when the dosage form of the composition is a capsule, the dosage of the composition is 1 x ¹⁰⁹ colony forming units/capsule (CFU/cap) of Lactobacillus salivarius TCI153.

In some embodiments, the dosage form for parenteral or topical administration can be, but is not limited to, an injection, sterile powder, external preparation, or the like. In some embodiments, the injection can be administered by subcutaneous injection, intraepidermal injection, intradermal injection or intralesional injection.

In some embodiments, the aforementioned medicinal products may include those widely used in medicines A pharmaceutically acceptable carrier for manufacturing techniques. In some embodiments, the pharmaceutically acceptable carrier can be one or more of the following carriers: solvent, buffer, emulsifier, suspending agent, disintegrant ( decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent ), gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, and the like. Regarding the type and quantity of the carrier selected, it falls within the scope of the professional quality and routine skills of those who are familiar with the technology. In some embodiments, the solvent as a pharmaceutically acceptable carrier may be water, normal saline, phosphate buffered saline (PBS), or an aqueous solution containing alcohol (aqueous solution containing alcohol). ).

In some embodiments, any of the foregoing compositions can be a food product. In other words, the food contains a specific amount of Lactobacillus salivarius TCI153. In some embodiments, the aforementioned food may be an edible product or a food additive. In some embodiments, edible products can be, but are not limited to, beverages, dairy products, fermented foods, bakery products, health foods, and dietary supplements products (dietary supplements).

In some embodiments, any of the foregoing compositions may be a skin care product or a cosmetic. In other words, the skin care product or cosmetic contains a specific content of Lactobacillus salivarius TCI153 and/or its bacterial breaking liquid.

In some embodiments, the aforementioned skin care products or cosmetics can be in any of the following forms: lotion, gel, jelly, mud film, lotion, cream, lipstick, foundation, pressed powder, powder, makeup remover, makeup remover Milk, facial cleanser, body wash, shampoo, conditioner, sunscreen, hand cream, nail polish, perfume, serum and mask. In some embodiments, the aforementioned cosmetics or skin care products may further include externally acceptable ingredients as needed. In some embodiments, topical acceptable ingredients may be, for example, emulsifiers, penetration enhancers, emollients, solvents, excipients, antioxidants, or combinations thereof.

Example 1: Identification of bacterial species

An appropriate amount of samples were taken from human breast milk and kimchi, and were coated on solid Lactobacillus MRS medium (BD Difco ^TM Lactobacilli MRS Broth with 1.5% agar) and incubated at 37°C in an anaerobic environment (that is, the oxygen concentration in the culture environment was 1% by volume) Incubate for 16 hours until single colonies form. A plurality of single colonies were picked from the solid Lactobacillus MRS medium respectively, and the 16S ribosomal gene (16S rDNA) sequence of the lactobacillus was used to identify the species. The 16S rDNA sequences (ie, SEQ ID NO: 1 to SEQ ID NO: 4) of these single colonies were obtained by polymerase chain reaction (PCR). Then, using the website of the National Center for Biotechnology Information (NCBI), the gene sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4 were sequenced with the 16S rDNA sequences of other Lactobacillus strains. The similarity between the 16S rDNA sequence of a single colony and the 16S rDNA sequence of other Lactobacillus species is shown in Table 1.

Among them, this single colony isolated from human breast milk and having a similarity of 98.96% to 99.15% with other Lactobacillus salivarius subsp. . In addition, this Lactobacillus salivarius TCI153 was deposited with the Food Industry Development Research Institute under the accession number BCRC 910982, and was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession number DSM 33503.

Example 2: Gastric acid bile salt resistance test

Here, Lactobacillus salivarius TCI153 obtained in Example 1 was tested with buffer solution, artificial gastric juice (pH 3) and artificial intestinal juice (pH 7) to confirm the acid-base tolerance of Lactobacillus salivarius TCI153 in the gastrointestinal tract of the organism . The buffer was 0.2 molar (M) potassium chloride (KCL, brand Sigma-Aldrich) and pH 7. The artificial gastric juice was 0.2M potassium chloride and pH 3. The artificial intestinal juice was 0.2M potassium chloride and 0.3% by weight bovine bile salts (brand Difco ^™ Oxgall) and had a pH of 7.

The following activation procedure was carried out with the Lactobacillus salivarius TCI153 obtained in Example 1 as the target strain. In the activation procedure, firstly take 100 μL of the target strain's frozen bacteria solution to solid Lactobacillus MRS medium (BD Difco ^TM Lactobacilli MRS Broth with 1.5% agar) for coating, and at 37 ℃ anaerobic environment (that is, the oxygen concentration in the culture environment) 1 vol%) for 16 hours to grow a single colony of the target strain. Next, pick a single colony of the target strain into 15 mL of liquid Lactobacillus MRS medium (BD Difco ^™ Lactobacilli MRS Broth), and cultivate it in an anaerobic environment at 37° C. for 16 hours to obtain the activated target strain.

Next, the activated target strain was inoculated into 20 ml of the solution to be tested at a concentration of 1% by volume, and then incubated with shaking at 50 rpm at 37 °C for 3 hours in an anaerobic environment. Among them, the solutions to be tested in the three groups are artificial gastric juice (experimental group), artificial intestinal juice (experimental group) and potassium chloride buffer (control group). Take 100 microliters (μL) of the bacterial solution from the cultured bacterial solution and spread it on the plate, and incubate it at 37°C for 3 days in an anaerobic environment. Colony counts on solid Lactobacillus MRS medium).

See Figure 1. The survival rate of Lactobacillus salivarius TCI153 in the graph is relative survival rate, expressed as a percentage (%), and the survival rate of the control group is regarded as 100%. As can be seen from Figure 1, compared with the control group, the survival rate of the artificial gastric juice experimental group was 83%, and the survival rate of the artificial intestinal juice experimental group was 93%. Based on this, Lactobacillus salivarius TCI153 has the function of resistance to gastric acid bile salts, which means that Lactobacillus salivarius TCI153 can colonize the host intestine through the pH pressure of the host gastrointestinal tract.

Example 3: Detection of sialic acid content of Lactobacillus salivarius strains

Here, compared with other strains, the sialic acid-producing ability of Lactobacillus salivarius TCI153 obtained in Example 1 was evaluated. Wherein, the medium used is Lactobacillus MRS medium (BD Difco ^™ Lactobacilli MRS Broth). The used lysis buffer (Lysis Buffer) was prepared by 300 mM sodium chloride (NaCl; brand: Sigma) and 50 mM sodium phosphate (Sodium Phosphate Monobasic; brand: Choneye).

First, take Lactobacillus salivarius TCI153, Lactobacillus salivarius L208, Lactobacillus gasseri LH37, Lactobacillus gasseri LH49 obtained in Example 1 as target strains and use the methods described in Example 2. The activation procedure activates the target strains separately for subsequent experiments.

The above activated target strains were respectively inoculated into 1L liquid Lactobacillus MRS medium, and the bacterial amount of the target strains in the liquid Lactobacillus MRS medium was adjusted to form an initial bacterial liquid with an absorbance value (OD ₆₀₀ ) of 0.1. Next, the initial bacterial solution was cultured in an anaerobic environment at 37° C. (ie, the oxygen concentration in the culture environment was 1% by volume) for 16 hours to form a test bacterial solution. The test bacterial liquid was centrifuged with a high-speed centrifuge (Thermo Megafuge 16 centrifuge) at 5,000×g for 20 minutes to remove the supernatant and collect the bacteria after the test bacterial liquid was centrifuged. Next, the centrifuged cells were re-dissolved in 50 ml of lysis buffer, and DNase (brand: Cyrubioscience) was added to form a re-dissolved solution, and reacted at room temperature for 10 minutes. The concentration of DNase in the redissolving solution was 1 μg/mL.

The reconstituted solution after the reaction was subjected to a sterilization procedure with different pressure values set by a high pressure sterilizer (Constant System TS series) to form a sterilization solution. Wherein, in the sterilization procedure, the reconstituted solution is sterilized three times under the pressure values of 25 kilopound force per square inch (Kpsi), 30 Kpsi and 32 Kpsi in sequence.

The broken bacteria solution was sent to the external inspection unit (Tendums Technical Consultants Co., Ltd.) for the detection of sialic acid content, as shown in Figure 2.

Please refer to FIG. 2 , wherein the bacteria-breaking liquid of each target strain is marked with the strain number shown in Table 1 respectively. As can be seen from FIG. 2 , 284.6 μg/mL of sialic acid was contained in the bacteria-breaking liquid of Lactobacillus salivarius TCI153. The strain of Lactobacillus salivarius L208 contained 49.69 μg/mL of sialic acid. The sterilized liquid of Lactobacillus gasseri LH37 contained 169.18 μg/mL of sialic acid. The sterilized liquid of Lactobacillus gasseri LH49 contained 155.74 μg/mL of sialic acid. It can be seen that the content of sialic acid in the broken liquid of Lactobacillus salivarius TCI153 is much higher than that of other Lactobacillus salivarius strains.

Based on this, Lactobacillus salivarius TCI153 has better sialic acid production ability. Therefore, when the recipient takes Lactobacillus salivarius TCI153, the Lactobacillus salivarius TCI153 colonized in the recipient can generate sialic acid in the recipient, so as to improve the sialic acid content in the recipient's blood.

Example 4: Detection of sialic acid content of Lactobacillus salivarius TCI153

Here, the sialic acid-producing ability of Lactobacillus salivarius TCI153 obtained in Example 1 was evaluated. Wherein, the medium used is Lactobacillus MRS medium (BD Difco ^™ Lactobacilli MRS Broth). The used lysis buffer (Lysis Buffer) was prepared by 300 mM sodium chloride (NaCl; brand: Sigma) and 50 mM sodium phosphate (Sodium Phosphate Monobasic; brand: Choneye).

First, Lactobacillus salivarius TCI153 obtained in Example 1 was used as the target strain, and the target strain was activated by the activation procedure described in Example 2, so as to facilitate subsequent experiments.

The above activated target strain was inoculated into 2L liquid Lactobacillus MRS medium, and the amount of Lactobacillus salivarius TCI153 in the liquid Lactobacillus MRS medium was adjusted to form an initial bacterial liquid with an absorbance value (OD ₆₀₀ ) of 0.1.

Next, the initial bacterial solution was cultured in an anaerobic environment at 37°C. After culturing for 8 hours, take out a part (about 1L) of the cultured initial bacterial solution (hereinafter referred to as the 8-hour bacterial solution), and the rest of the cultured initial bacterial solution (about 1L) is continuously cultivated for 24 hours to form a 24-hour bacterial solution.

Here, the experimental group was divided into an 8-hour experimental group and a 24-hour experimental group according to the culture time of the bacterial liquid. Among them, the 8-hour bacterial liquid was used as the test sample of the 8-hour experimental group, and the 24-hour bacterial liquid was used as the test sample of the 24-hour experimental group.

Next, each group of test samples was centrifuged at 5,000×g for 20 minutes with a high-speed centrifuge (Thermo Megafuge 16 centrifuge) to remove the supernatant and collect each group of centrifuges later bacteria. Next, the centrifuged cells were re-dissolved in 50 ml of lysis buffer, and then DNase (brand: Cyrubioscience) was added to form each group of back solutions, and reacted at room temperature for 10 minutes. The concentration of DNase in the return solution was 1 μg/mL.

The reacted back solution was subjected to a sterilization procedure with a high pressure sterilizer (Constant System TS series) set with different pressure values to form each group of sterilization solutions. Wherein, in the sterilization procedure, the back solution was sterilized three times under pressure values of 25 kilopound force per square inch (Kpsi), 30 Kpsi and 32 Kpsi in sequence.

The bacteria-broken liquid of the second experimental group was sent to the external inspection unit (Tendums Technical Consultants Co., Ltd.) to detect the sialic acid content, as shown in Figure 3.

See Figure 3, where the 8-hour experimental group is labeled 8 hours, and the 24-hour experimental group is labeled 24 hours. As can be seen from FIG. 3 , 17.56 μg/mL of sialic acid was contained in the strain of Lactobacillus salivarius TCI153 after 8 hours of culture. The 24-hour cultured Lactobacillus salivarius TCI153 strain contained 1040 μg/mL of sialic acid.

Example 5: Preparation of the breaking liquid of Lactobacillus salivarius TCI153

Here, the medium used was Lactobacillus MRS medium (BD Difco ^™ Lactobacilli MRS Broth). The used lysis buffer (Lysis Buffer) was prepared by 300 mM sodium chloride (NaCl; brand: Sigma) and 50 mM sodium phosphate (Sodium Phosphate Monobasic; brand: Choneye).

First, Lactobacillus salivarius TCI153 obtained in Example 1 was used as the target strain, and the target strain was activated by the activation procedure described in Example 2, so as to facilitate subsequent experiments.

The above-mentioned activated target strain was inoculated into 1 L of liquid Lactobacillus MRS medium, and the amount of Lactobacillus salivarius TCI153 in the liquid Lactobacillus MRS medium was adjusted to form an initial bacterial liquid with an absorbance value (OD ₆₀₀ ) of 0.1.

Next, the initial culture solution was cultured in an anaerobic environment at 37° C. for 16 hours to form a Lactobacillus salivarius TCI153 bacterial solution. The Lactobacillus salivarius TCI153 bacterial solution was centrifuged with a high-speed centrifuge (Thermo Megafuge 16 centrifuge) at a set speed of 5,000×g for 20 minutes to remove the supernatant and collect the Lactobacillus salivarius TCI153 bacterial cells after centrifugation. Next, the centrifuged Lactobacillus salivarius TCI153 cells were redissolved in 50 ml of lysis buffer, DNase (brand: Cyrubioscience) was added to form a solution, and the reaction was performed at room temperature for 10 minutes. The concentration of DNase in the return solution was 1 μg/mL. The reacted back solution was subjected to a sterilization procedure with a high pressure sterilizer (Constant System TS series) with different pressure values set to form a sterilization liquid of Lactobacillus salivarius TCI153. Wherein, in the sterilization procedure, the back solution was sterilized three times under pressure values of 25 kilopound force per square inch (Kpsi), 30 Kpsi and 32 Kpsi in sequence.

Here, a lysate of Lactobacillus salivarius TCI153 containing cell fragments of Lactobacillus salivarius TCI153, intracellular protein of Lactobacillus salivarius TCI153, and a lysis buffer can be obtained.

Example 6: Detection of elastin content

Here, the medium used is a minimal essential medium containing 90 vol% (Minimum essential medium; brand Gibco), 10 vol% fetal bovine serum (FBS; brand: Gibco) and 1 mM sodium pyruvate (sodium pyruvate). pyruvate, brand: Gibco) cell culture medium (hereinafter referred to as MEM medium). The cell line used is human skin fibroblasts (CCD-966Sk cell, brand: ATCC ^® , CRL-1881), hereinafter referred to as CCD-966Sk cell.

CCD-966Sk cells were seeded in a 6-well culture dish containing 2 ml of MEM medium per well at 1×10 ⁵ cells per well, and cultured at 37°C for 24 hours, and divided into experimental group, control group and control group. Group. Next, the MEM medium was replaced with the experimental medium, and the experimental group and the control group were cultured at 37° C. for 24 hours. Wherein, the experimental medium of the experimental group was the MEM medium containing 0.25% by volume of the Lactobacillus salivarius TCI153 broken liquid prepared in Example 5. The experimental medium of the control group was the MEM medium containing 0.25% by volume of sterile liquid Lactobacillus MRS medium (that is, without Lactobacillus salivarius TCI153 breaking liquid, only adding sterile liquid Lactobacillus MRS medium). The experimental medium of the control group was a simple MEM medium (ie, a MEM medium without Lactobacillus salivarius TCI153 breaking liquid and sterile liquid Lactobacillus MRS medium).

Next, after treating each group of CCD-966Sk cells with an elastin assay kit (Fastin ^TM Elastin Assay kit, brand: Biocolor), the elastic force of three groups of CCD-966Sk cells was detected with a full-spectrum optical analyzer (brand: BioTek, Epoch). The amount of protein produced is shown in Figure 4. Among them, the elastin content measured in the control group that was not cultured in the experimental medium for 24 hours was regarded as 1 (ie, the amount of elastin production was 100%). It should be noted that the statistically significant differences between the groups were statistically analyzed by student t-test, as shown in FIG. 4 . In Fig. 4, "**" represents that the p-value is less than 0.01 when compared with the control group, and "▲" represents that the p-value is less than 0.05 when compared with the control group.

Referring to Figure 4, compared to the control group, the production of elastin in the experimental group was 116.1%, while the production of elastin in the control group was 103.3%. In other words, there was no significant difference in the production of elastin between the control group and the control group, and compared with the control group, the production of elastin in the experimental group was significantly increased, representing the CCD-966Sk CCD-966Sk treated with Lactobacillus salivarius TCI153 for 24 hours. Cells can make more elastin. From this, it can be seen that the Lactobacillus salivarius TCI153 sterilized liquid has the function of promoting the synthesis of elastin in cells.

Based on this, Lactobacillus salivarius TCI153 and/or its broken liquid have the ability to promote cell synthesis. The function of elastin. When the recipient takes Lactobacillus salivarius TCI153, the Lactobacillus salivarius TCI153 colonized in the recipient can promote the recipient's skin cells to produce elastin, thereby improving the recipient's skin elasticity.

Example 7: Collagen Membrane Shrinkage Test (Skin Firming Test)

Here, the medium used is 90% by volume supplemented with 0.1 mM non-essential amino acids (Non-Essential Amino Acids; brand: Gibco), 1.5 g/L sodium bicarbonate (sodium bicarbonate; brand: Gibco) and 1 mM Sodium pyruvate (Brand: Gibco) in Earle's Minimum essential medium (Eagle) in Earle's BSS; Brand: Gibco, and 10% by volume fetal bovine serum albumin (fetal) bovine serum, FBS; brand: Gibco) Eagle-MEM medium. The cell line used is human skin fibroblasts (CCD-966Sk cell, brand: ATCC ^® , CRL-1881), hereinafter referred to as CCD-966Sk cell.

CCD-966Sk cells were seeded at 2×10 ⁵ per well in a 24-well culture dish containing 0.5 ml of cell culture medium per well, and cultured at 37° C. for 1 hour. Wherein, the above cell culture medium is supplemented with 1 mg/ml type I collagen (Collagen I; brand: Gibco) and adjusted to neutral (pH 7.0) with sodium hydroxide (NaOH; brand: Macron, model 7708-10). ) of Eagle-MEM medium. During the 1-hour incubation period, CCD-966Sk cells reconstituted type 1 collagen and formed collagen membranes.

The CCD-966Sk cells cultured and formed collagen membrane were divided into experimental group, control group and control group. Next, 0.5 ml of experimental medium was added to each well, and the experimental group and the control group were further cultured at 37° C. for 8 hours (the control group was not cultured for 8 hours). Wherein, the experimental medium of the experimental group was the Lactobacillus salivarius TCI153 prepared in Example 5 containing 0.25% by volume Eagle-MEM medium for bacterial breakage. The experimental medium of the control group was MRS medium containing 0.25 vol% sterile liquid Lactobacillus. The experimental medium of the control group was simple Eagle-MEM medium (that is, without Lactobacillus salivarius TCI153 breaking liquid and sterile liquid Lactobacillus MRS medium).

The size of the collagen films in each group was analyzed by image software (imageJ), and the differences in the size of the collagen films in each group were compared. It should be noted that the statistically significant differences between the groups were statistically analyzed by student t-test, as shown in FIG. 5 . In Fig. 5, "*" represents that the p-value is less than 0.05 when compared with the control group, and "▲" represents that the p-value is less than 0.05 when compared with the control group.

Here, the size of the collagen film of the control group was regarded as 100%, and compared with the size of the collagen film of the experimental group and the control group to analyze the shrinkage of the collagen film of the experimental group and the control group. Referring to Figure 5, compared to the control group, the size of the collagen film of the experimental group was 95.71%, while the size of the collagen film of the control group was 99.27%. In other words, there was little difference in the size of the collagen film between the control group and the control group, while the size of the collagen film in the experimental group was smaller than that in the control group. Aggregation of type 1 collagen makes the shrinkage of type 1 collagen membrane more pronounced. From this, it can be seen that the Lactobacillus salivarius TCI153 broken liquid has the function of recombining the aggregated collagen.

Based on this, Lactobacillus salivarius TCI153 and/or its broken solution have the function of recombining collagen aggregation. When the recipient takes Lactobacillus salivarius TCI153, the Lactobacillus salivarius TCI153 colonized in the recipient can promote the reorganization and aggregation of collagen in the recipient's skin cells, thereby improving the skin firmness of the recipient.

Example 8: Human blood test

To further confirm the effect of Lactobacillus salivarius TCI153 on human body. The live capsules of Lactobacillus salivarius TCI153 containing 1×10 ⁹ CFU (ie, 1×10 ⁹ CFU/cap) per capsule were provided to 8 subjects, each taking one capsule per day for 8 weeks. And before taking Lactobacillus salivarius TCI153 capsules (considered the 0th week), after taking Lactobacillus salivarius TCI153 capsules for 4 weeks (considered the 4th week) and taking Lactobacillus salivarius TCI153 capsules for 8 weeks (considered the 8th week) The blood test of the subject is carried out, and the test items include the content of sialic acid in the blood of the subject and the content of epidermal growth factor in the blood of the subject. Among them, the sialic acid content in the blood of the subjects was detected by using the sialic acid detection kit (brand: Cloud-Clone Corp, model: CES099Ge) in the blood of the subjects. The content of growth factor is to detect the content of epidermal growth factor in the blood of the subjects with epidermal growth factor detection kit (brand: biogems, model: BGK01133).

It should be noted that the statistically significant differences between the measurement results at weeks 0, 4 and 8 were statistically analyzed by Student's t-test.

Please refer to Figure 6. Before taking Lactobacillus salivarius TCI153 capsules, the average blood sialic acid content of 8 subjects was 1.47 pg/ml (Figure 6 indicates week 0). The average blood sialic acid content of the subjects rose to 3.19 pg/ml (Figure 6 indicates the 4th week), and then after 8 weeks of taking the 8 subjects, the average blood sialic acid content rose to 4.64 pg /ml (Figure 6 indicates week 8). In other words, compared to week 0, the average blood sialic acid content of the 8 subjects increased by 3.17 pg/ml after taking Lactobacillus salivarius TCI153 capsules for 8 weeks. It can be seen from this that the sialic acid content in the blood can be increased after the subjects took Lactobacillus salivarius TCI153.

Referring to Figure 7, before taking Lactobacillus salivarius TCI153 capsules, the average blood epidermal growth factor level of 8 subjects was 96.85 pg/ml (Figure 7 indicates week 0), and After 4 weeks of taking, the average blood epidermal growth factor content of 8 subjects increased to 130.40 pg/ml (Figure 7 indicates the 4th week), and then after 8 weeks of taking, the average blood level of 8 subjects increased. The epidermal growth factor level rose to 123.94 pg/ml (Figure 7 indicates week 8). In other words, compared to week 0, the average blood sialic acid content of the 8 subjects increased by 27.09 pg/ml after taking Lactobacillus salivarius TCI153 capsules for 8 weeks. It can be seen from this that after taking Lactobacillus salivarius TCI153, the sialic acid content and/or epidermal growth factor content in the blood can be increased, so the anti-aging ability of the subject can be improved.

Example 9: Human skin test

To further confirm the effect of Lactobacillus salivarius TCI153 on human body. The subjects were given one capsule per day of Lactobacillus salivarius TCI153 containing 1×10 ⁹ CFU (ie, 1×10 ⁹ CFU/cap) of live bacteria for 8 weeks. And before taking Lactobacillus salivarius TCI153 capsules (considered the 0th week), after taking Lactobacillus salivarius TCI153 capsules for 4 weeks (considered the 4th week) and taking Lactobacillus salivarius TCI153 capsules for 8 weeks (considered the 8th week) Analysis was performed with a full-face instrument (VISIA Complexion Analysis System (Canfield scientific, USA)). The analysis items include wrinkles, skin pores, skin radiance, skin moisture loss percentage and skin moisture content.

It should be noted that the statistically significant differences between the measurement results at weeks 0, 4 and 8 were statistically analyzed by Student's t-test. In Figure 12, "**" represents that the p-value is less than 0.01 in comparison with the control group.

Referring to Figure 8, the mean value of wrinkles measured at week 0 for 8 subjects was considered as 100%. Compared to week 0, the mean value of wrinkles measured at week 4 was 99.7%, and the mean value of wrinkles measured at week 8 was 86.9%. In other words, taking saliva for 8 consecutive weeks After Lactobacillus TCI153 capsules, the wrinkle reduction of 8 subjects averaged 13.1%. Please refer to FIG. 9 , the subjects measured more wrinkles W0 under the eyes in the 0th week, and after taking Lactobacillus salivarius TCI153 capsules for 8 consecutive weeks, the subjects’ wrinkles W8 under the eyes became less. Thus, it can be seen that Lactobacillus salivarius TCI153 has the ability to improve wrinkles in subjects.

Referring to Figure 10, the mean value of pores measured at week 0 for 8 subjects was considered as 100%. Compared to week 0, the mean value of pores measured at week 4 was 100.2%, and the mean value of pores measured at week 8 was 93.2%. In other words, after taking Lactobacillus salivarius TCI153 capsules for 8 consecutive weeks, the pores of the 8 subjects decreased by an average of 6.8%. Please refer to Figure 11, the facial pores P0 measured by the subjects in the 0th week were denser and more numerous, and after taking Lactobacillus salivarius TCI153 capsules for 8 consecutive weeks, the facial pores P8 measured by the subjects became less. . From this, it can be seen that Lactobacillus salivarius TCI153 has the ability to improve the skin pores of the subjects.

Referring to Figure 12, the average value of skin radiance measured by 8 subjects at week 0 was taken as 100%. Compared to week 0, the average value of skin radiance measured in week 4 was 105.1%, and the average value of skin radiance measured in week 8 was 110.1%. In other words, after taking Lactobacillus salivarius TCI153 capsules for 8 consecutive weeks, the skin radiance of the 8 subjects increased by an average of 10.1%. Please refer to Figure 13, the subject's skin was duller at week 0, and after taking Lactobacillus salivarius TCI153 capsules for 8 consecutive weeks, the subject's skin was more glossy, representing the subject's skin glossiness rise. From this, it can be seen that Lactobacillus salivarius TCI153 has the ability to improve the skin glossiness of the subjects.

Referring to Figure 14, the average value of the percentage of skin moisture loss measured by the 8 subjects at week 0 was regarded as 100%. Compared with the 0th week, the average value of the skin moisture loss percentage measured in the 4th week was 93.9%, and the skin moisture loss measured in the 8th week was 100%. The average value of the scores was 85.8%. In other words, after taking Lactobacillus salivarius TCI153 capsules for 8 consecutive weeks, the skin moisture loss percentage of the 8 subjects decreased by an average of 14.2%, indicating that the skin moisture loss of the subjects decreased. It can be seen that after the subjects took Lactobacillus salivarius TCI153, the loss of water from the skin could be slowed down, thereby improving the skin moisturizing ability of the subjects.

Referring to Figure 15, the average value of skin moisture content measured by 8 subjects at week 0 was regarded as 100%. Compared with week 0, the average value of skin moisture content measured in week 4 was 103.6%, and the average value of skin moisture content measured in week 8 was 109.5%. In other words, after taking Lactobacillus salivarius TCI153 capsules for 8 consecutive weeks, the skin moisture content of the 8 subjects increased by an average of 9.5%, which means that the skin moisture content of the subjects increased. It can be seen that after the subjects take Lactobacillus salivarius TCI153, the water content of the subjects' skin can be increased, thereby improving the skin moisturizing power of the subjects.

Here, through Example 2, it can be known that Lactobacillus salivarius TCI153 can adapt to the human gastrointestinal environment. Through Example 3 and Example 4, it can be seen that Lactobacillus salivarius TCI153 has the ability to generate sialic acid, and Lactobacillus salivarius TCI153 has better ability to generate sialic acid than other Lactobacillus salivarius strains. Through Example 6, it can be seen that the bacteria-breaking liquid of Lactobacillus salivarius TCI153 can promote the production of elastin in cells. From Example 7, it can be seen that the bacteria-breaking liquid of Lactobacillus salivarius TCI153 can promote the cell reorganization and aggregation of collagen. Through Example 8, it can be known that Lactobacillus salivarius TCI153 has the effect of increasing the content of sialic acid and the content of epidermal growth factor in the blood of the recipient. Through Example 9, it can be seen that Lactobacillus salivarius TCI153 has the effect of improving skin condition, wherein improving skin condition includes improving wrinkles, reducing skin pores, improving skin gloss, reducing skin moisture loss percentage, increasing skin moisture content or a combination thereof.

In summary, according to any embodiment of the present invention Lactobacillus salivarius TCI153 and/ Or its bacterial breaking liquid, which can prepare a composition for improving skin condition. Among them, Lactobacillus salivarius TCI153 can produce sialic acid. In addition, Lactobacillus salivarius TCI153 is resistant to gastric acid bile salts and can survive in the gastrointestinal environment. In some embodiments, Lactobacillus salivarius TCI153 has the ability to increase the elasticity of the recipient's skin since Lactobacillus salivarius TCI153 and/or its sterilization fluid can be used to promote elastin production in cells. In some embodiments, Lactobacillus salivarius TCI153 has the ability to increase skin firmness in recipients since Lactobacillus salivarius TCI153 and/or its sterilization fluid can be used to promote cellular reorganization and aggregate collagen. In some embodiments, Lactobacillus salivarius TCI153 can be used to improve recipient skin gloss, increase recipient skin moisture content, reduce recipient skin pores, slow recipient skin water loss, reduce wrinkles, or a combination thereof, thereby improving recipient skin state. In some embodiments, Lactobacillus salivarius TCI153 can be used to increase the content of sialic acid and epidermal growth factor in the blood of the recipient, thereby improving the anti-aging ability of the recipient. Also, the prepared composition can be a pharmaceutical, food, skincare or cosmetic.

Although the technical content of the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person who is familiar with the art, makes some changes and modifications without departing from the spirit of the present invention, should be included in the present invention. Therefore, the protection scope of the present invention should be determined by the scope of the appended patent application.

【Biological Material Deposit】

TW Republic of China Food Industry Development Research Institute 2020/03/27 BCRC 910982

DE Germany German National Culture Collection 2020/04/17 DSM 33503

Claims (10)

A Lactobacillus salivarius TCI153, wherein Lactobacillus salivarius TCI153 (Lactobacillus salivarius TCI153) is deposited in the Food Industry Development Research Institute of a consortium, and the deposit number is BCRC 910982. A use of Lactobacillus salivarius TCI153 and/or its sterilized liquid in the preparation of a composition for improving skin conditions, wherein Lactobacillus salivarius TCI153 (Lactobacillus salivarius TCI153) is deposited in the Food Industry Development Research Institute of a consortium, and the deposit number is BCRC 910982. The use according to claim 2, wherein the Lactobacillus salivarius TCI153 has the function of producing sialic acid. The use according to claim 2, wherein the Lactobacillus salivarius TCI153 has the function of promoting the production of epidermal growth factor by a receptor. The Lactobacillus salivarius TCI153 according to claim 2, wherein the Lactobacillus salivarius TCI153 and/or its sterilized liquid have the function of promoting cell synthesis of elastin. The Lactobacillus salivarius TCI153 according to claim 2, wherein the Lactobacillus salivarius TCI153 and/or its sterilized liquid have the functions of promoting cell reorganization and aggregation of collagen. The use of claim 2, wherein the composition is used to improve skin elasticity, skin firmness, or a combination thereof in a recipient. The use of claim 2, wherein the composition is used to improve wrinkles, skin pores, skin moisture loss, or a combination thereof in a recipient. The use of claim 2, wherein the composition is used to improve skin gloss, skin moisture content, or a combination thereof in a recipient. The use according to claim 2, wherein the composition is a medicine, a food, a skin care product or a cosmetic.

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