Probiotic liquid cultures and their use in improving skin-related conditions

Abstract

The present invention relates to a probiotic liquid culture and its use in improving skin-related conditions. The present invention discloses a probiotic liquid culture, which is prepared by the following method: a probiotic composition containing Lactobacillus acidophilus TYCA06 (CGMCC NO.15210), Lactobacillus salivarius AP-32 (CCTCC M 2011127) and Bifidobacterium animalis subsp. lactis CP-9 (CCTCC M 2014588) with a bacterial count ratio of 1:0.125:0.125 to 1:8:8 is co-cultured in a collagen solution. The present invention also discloses that the probiotic liquid culture can be used to improve skin conditions and inhibit pathogenic bacteria infection.

Description

Technical Field

[0001] The present invention relates to a probiotic liquid culture obtained by culturing probiotics using a collagen solution. The present invention also relates to a probiotic liquid culture that can be used to improve skin conditions and inhibit pathogen infection. Background Art

[0002] The skin is the largest barrier to protect the human body. It can prevent water loss, invasion of skin pathogens (such as Staphylococcus aureus and Propionibacterium acnes), and damage from various harmful environments. When the skin is damaged, it may lead to a variety of skin conditions, including eczema, acne, melanogenesis, and delay of wound healing.

[0003] In recent years, the demand for skin care has been increasing day by day. Therefore, researchers in the medical field and the cosmetics industry are committed to developing products that can effectively improve various skin conditions to meet the needs of the vast market.

[0004] Probiotics are a group of microorganisms that can improve intestinal microflora and intestinal immunity, and have been widely used in food and health products. Commonly used probiotics include: Lactobacillus, Lactococcus, Pediococcus, Enterococcus, Streptococcus, Bifidobacterium, Bacillus and Leuconostoc, etc.

[0005] At present, there are studies on the application of probiotic strains in skin care. For example, TW 202108125A discloses a composition containing lactic acid bacteria fermentation products for whitening effect, which comprises a fermentation product with whitening effect produced by fermentation of lactic acid bacteria strains, wherein the lactic acid bacteria strain is selected from at least one of the following: Bifidobacterium breve Bv-889, Bifidobacterium longum subsp. infantis BLI-02, Bifidobacterium animalis subsp. lactis CP-9, Bifidobacterium bifidum Bf-688, Lactobacillus salivarius subsp. salicinius AP-32 and Lactobacillus paracasei GL-156. The fermented products of these lactic acid bacteria strains have been experimentally confirmed to have the effect of inhibiting melanin production when used alone. However, these strains have not been found to improve other skin conditions.

[0006] Although there are the above literature reports, there is still a need in the art to develop products that can improve multiple skin conditions at the same time for use by the industry. Summary of the invention

[0007] In the present invention, the applicant has found that a probiotic liquid culture obtained by co-cultivating strains of Lactobacillus acidophilus TYCA06 (CGMCC NO.15210), Lactobacillus salivarius subsp.salicinius AP-32 (CCTCC M 2011127) and Bifidobacterium animalis subsp.lactis CP-9 (CCTCC M 2014588) at a bacterial count ratio of 1:0.125:0.125 to 1:8:8 in a collagen solution can effectively promote wound healing, inhibit melanogenesis, treat acne, reduce skin inflammation and reduce skin brown spots, and is therefore expected to be applied to improve various skin conditions. In addition, the applicant has also found that the probiotic liquid culture has an effect against Staphylococcus aureus and Propionibacterium acnes, and is therefore expected to be applicable to inhibiting pathogen infection.

[0008] Therefore, in a first aspect, the present invention provides a probiotic liquid culture, which is prepared by the following method: a probiotic composition comprising Lactobacillus acidophilus TYCA06, Lactobacillus salivarius AP-32 and Bifidobacterium animalis subsp. lactis CP-9 at a bacterial count ratio of 1:0.125:0.125 to 1:8:8 is co-cultured in a collagen solution.

[0009] In a second aspect, the present invention provides a method for preparing a probiotic liquid culture, comprising a method as described above.

[0010] In a third aspect, the present invention provides a use of the probiotic liquid culture as described above for preparing a composition for improving skin conditions.

[0011] In a fourth aspect, the present invention provides a method for improving skin conditions, comprising administering a probiotic liquid culture as described above to a subject in need thereof.

[0012] In a fifth aspect, the present invention provides a use of the probiotic liquid culture as described above for preparing a composition for inhibiting pathogen infection.

[0013] In a sixth aspect, the present invention provides a method for inhibiting pathogen infection, comprising administering a probiotic liquid culture as described above to an individual in need thereof.

[0014] Preferably, in the probiotic composition, the bacterial count ratio of Lactobacillus acidophilus TYCA06, Lactobacillus salivarius AP-32 and Bifidobacterium animalis subsp. lactis CP-9 is 1:1:1.

[0015] Preferably, the probiotic liquid culture contains substantially no bacterial cells.

[0016] Preferably, the skin condition comprises at least one of the following: wounds, melanin production, acne, skin inflammation, skin brown spots, and skin pathogen infection.

[0017] Preferably, the pathogenic bacteria infection is caused by a pathogen selected from the group consisting of Staphylococcus aureus, Propionibacterium acnes, Escherichia coli, Gardnerella vaginalis, Epidermophyton floccosum, Trichophyton rubrum, and combinations thereof.

[0018] Preferably, the composition is a pharmaceutical composition.

[0019] More preferably, the pharmaceutical composition is in a dosage form for oral administration.

[0020] Preferably, the composition is a cosmeceutical composition.

[0021] Biomaterial Deposit Information

[0022] The Lactobacillus acidophilus TYCA06 of the present invention was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on January 15, 2018, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, the deposit number is: CGMCC No.15210, the culture name is TYCA06, and the classification name is Lactobacillus acidophilus.

[0023] The animal Bifidobacterium lactis subsp. CP-9 of the present invention was deposited in China Center for Type Culture Collection (CCTCC) on November 24, 2014, with the deposit address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, China, postal code 430072, deposit number: CCTCC NO: M2014588, and the culture name is Bifidobacterium animalis subsp. lactis CP-9.

[0024] The salivarius Lactobacillus AP-32 of the present invention was deposited in China Center for Type Culture Collection (CCTCC) on April 10, 2011, with the deposit address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, China, postal code 430072, the deposit number is CCTCCNO: M2011127, and the culture name is Bifidobacterium animalis subsp.salicinius AP-32. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The wound healing percentage (%) measured in each group in Example 2 below is shown, wherein "**" indicates: when compared with the control group, p < 0.01;

[0026] Figure 2 The melanin content (%) of each group measured in Example 3 below is shown, wherein "***" indicates: when compared with the normal control group, p < 0.001; and "##" and "###" respectively indicate: when compared with the pathological control group, p < 0.01 and p < 0.001;

[0027] Figure 3 The following table shows the growth inhibition rate (%) of each group on Staphylococcus aureus measured in Example 4, wherein "**" and "***" respectively indicate: when compared with the normal control group, p < 0.01 and p < 0.001;

[0028] Figure 4 The following table shows the growth inhibition rate (%) of Propionibacterium acnes measured in each group in Example 4, where "*" and "***" respectively indicate: when compared with the normal control group, p < 0.05 and p < 0.001;

[0029] Figure 5 The morphology of skin acne in the experimental area and the control area in Example 5 below is shown;

[0030] Figure 6The relative amount (%) of the skin inflammation index measured in the experimental area and the control area in Example 5 below is shown, wherein "***" means: when compared with before application of the gel (i.e., day 0), p <0.001; and "##" means: when compared with the control area, p <0.01; and

[0031] Figure 7 The relative amounts (%) of skin brown spots measured in the experimental area and the control area in Example 5 below are shown, wherein "***" indicates: p < 0.001 when compared with before gel application (ie, day 0). DETAILED DESCRIPTION

[0032] For the purposes of this specification, it will be expressly understood that the word "comprising" means "including, but not limited to," and that the word "comprises" has a corresponding meaning.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used to implement the present invention. Of course, the present invention is in no way limited to the methods and materials described.

[0034] The present invention provides a probiotic liquid culture, which is prepared by the following method: a probiotic composition comprising Lactobacillus acidophilus TYCA06 (CGMCC NO.15210), Lactobacillus salivarius subsp.salicinius AP-32 (CCTCC M 2011127) and Bifidobacterium animalis subsp.lactis CP-9 (CCTCC M 2014588) with a bacterial count ratio of 1:0.125:0.125 to 1:8:8 is co-cultivated in a collagen solution.

[0035] Preferably, in the probiotic composition, the bacterial count ratio of Lactobacillus acidophilus TYCA06, Lactobacillus salivarius AP-32 and Bifidobacterium animalis subsp. lactis CP-9 is 1:1:1 to 1:4:4.

[0036] In a preferred embodiment of the present invention, in the probiotic composition, the bacterial count ratio of Lactobacillus acidophilus TYCA06, Lactobacillus salivarius AP-32 and Bifidobacterium animalis subsp. lactis CP-9 is 1:1:1.

[0037] According to the present invention, the probiotic composition may have a range of 10 7 Up to 10 12 In a preferred embodiment of the present invention, the bacterial concentration of the probiotic composition is 10 9 -10 10 CFU / mL.

[0038] According to the present invention, the collagen solution can be a commercially available product, or a product prepared by techniques well known and commonly used by those skilled in the art, such as a natural product isolated from animal tissues or a recombinant protein obtained through genetic engineering.

[0039] According to the present invention, the collagen solution may have a concentration ranging from 10% (w / v, g / mL) to 50% (w / v, g / mL). In a preferred embodiment of the present invention, the concentration of the collagen solution is 20% (w / v, g / mL).

[0040] As used herein, the terms "co-cultivation" and "co-fermentation" are used interchangeably.

[0041] The operation process and parameter conditions of co-cultivation are within the professional qualities and routine skills of people familiar with this technology.

[0042] According to the present invention, the co-cultivation may be performed at a temperature ranging from 35° C. to 45° C. In a preferred embodiment of the present invention, the co-cultivation is performed at 37° C.

[0043] According to the present invention, the co-cultivation is carried out for a period ranging from 42 to 72 hours. In a preferred embodiment of the present invention, the co-cultivation is carried out for 48 hours.

[0044] According to the present invention, the probiotic liquid culture can be substantially free of cells by solid-liquid separation.

[0045] According to the present invention, the solid-liquid separation treatment is selected from the group consisting of: centrifugation treatment, filtration treatment, concentration treatment, and combinations thereof. In a preferred embodiment of the present invention, the solid-liquid separation treatment is centrifugation treatment.

[0046] According to the present invention, the probiotic liquid culture may be sterilized to be substantially free of viable cells.

[0047] According to the present invention, the sterilization treatment can be performed by heating at 90° C. to 180° C. for 10 to 30 minutes. In a preferred embodiment of the present invention, the sterilization treatment is performed by heating at 100° C. for 30 minutes.

[0048] As used herein, the term "substantially free of" means that a specified component lacks a meaningful content. Preferably, the content of the component has no measurable effect on the properties of the probiotic liquid culture. More preferably, the probiotic liquid culture does not contain the component at all.

[0049] According to the present invention, the probiotic liquid culture can be further subjected to a drying process selected from the following to become a powder: freeze-drying, spray-drying, and fluid bed drying.

[0050] The present invention also provides a method for preparing a probiotic liquid culture, comprising: co-culturing a probiotic composition comprising Lactobacillus acidophilus TYCA06, Lactobacillus salivarius AP-32 and Bifidobacterium animalis subsp. lactis CP-9 at a bacterial ratio of 1:0.125:0.125 to 1:8:8 in a collagen solution.

[0051] According to the present invention, the probiotic composition, collagen solution and co-culture conditions used in the method are as described above.

[0052] The present invention also provides a use of the probiotic liquid culture as described above for preparing a composition for improving skin condition.

[0053] According to the present invention, the skin condition comprises at least one of the following: wound, melanogenesis, acne, skin inflammation, skin brown spot, and skin pathogen infection.

[0054] The present invention also provides a use of the probiotic liquid culture as described above for preparing a composition for inhibiting pathogen infection. Preferably, the pathogen infection is a skin pathogen infection.

[0055] As used herein, the term "inhibition" or "inhibiting" means preventing or slowing the growth of pathogenic bacteria in an individual.

[0056] According to the present invention, the pathogenic bacteria infection is caused by a pathogen selected from the group consisting of: Staphylococcus aureus, Propionibacterium acnes, Escherichia coli, Gardnerella vaginalis, Epidermophyton floccosum, Trichophyton rubrum, and combinations thereof.

[0057] In a preferred embodiment of the present invention, the pathogen is Staphylococcus aureus. In another preferred embodiment of the present invention, the pathogen is Propionibacterium acnes.

[0058] According to the present invention, the composition may be a pharmaceutical composition.

[0059] According to the present invention, the pharmaceutical composition may further include a pharmaceutically acceptable carrier widely used in drug manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the following: solvent, buffer, decomposer, excipient, stabilizer, diluent, wetting agent, lubricant and the like. The selection and amount of these agents fall within the professional qualities and routine technical scope of those skilled in the art.

[0060] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for oral administration using techniques known to those skilled in the art, including, but not limited to, sterile powders, tablets, troches, lozenges, pellets, capsules, dispersible powders or granules, solutions, emulsions, syrups, elixirs, slurries and the like. Preferably, the pharmaceutical composition is manufactured into a dosage form suitable for administration in capsules.

[0061] In addition, the present invention also provides a method for improving skin conditions, which comprises administering a probiotic liquid culture as described above to an individual in need thereof. The present invention also provides a method for inhibiting pathogenic bacteria infection, which comprises administering a probiotic liquid culture as described above to an individual in need thereof.

[0062] As used herein, the terms "administration" and "administration" are used interchangeably and refer to introducing, providing or delivering a predetermined active ingredient to a subject by any appropriate route to perform its intended effect.

[0063] As used herein, the term "subject" means any mammal of interest, such as humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, and rats.

[0064] According to the present invention, the dosage and frequency of administration of the probiotic liquid culture will vary depending on the severity of the condition to be improved, the route of administration, and the age, physical condition and response of the individual to be improved. Generally speaking, the probiotic liquid culture can be administered orally in the form of a single dose or divided into several doses.

[0065] According to the present invention, the composition may be a cosmeceutical composition.

[0066] According to the present invention, the cosmeceutical composition may further include a pharmaceutically acceptable carrier as described above and / or a cosmetically acceptable adjuvant widely used in cosmetic manufacturing technology. For example, the cosmetically acceptable adjuvant may include one or more agents selected from the following: solvents, gelling agents, active agents, antioxidants, screening agents, surfactants, coloring agents, thickening agents, fillers, fragrances and odor absorbers. The selection and amount of these agents fall within the professional qualities and routine technical scope of those skilled in the art.

[0067] According to the present invention, the cosmeceutical composition can be manufactured into a form suitable for skincare or makeup using techniques well known to those skilled in the art, including, but not limited to: aqueous solution, aqueous-alcohol solution or oily solution, emulsion of oil-in-water type, water-in-oil type or combination type, gel, ointment, cream, mask, patch, pack, bandage, liniment, powder, aerosol, spray, lotion, serum, paste, foam, dispersion, suspension, drop, mousse, salve, sunblock, tonic water. water), foundation, eye shadow, makeup remover products, soap and other body cleansing products, etc. In a preferred embodiment of the present invention, the cosmeceutical composition is prepared into a dosage form suitable for application as a gel.

[0068] The present invention will be further described with reference to the following examples. However, it should be understood that these examples are only for illustration and should not be construed as limitations on the implementation of the present invention.

[0069] General experimental materials:

[0070] 1. Probiotic strains:

[0071] The probiotic strains used for efficacy evaluation in the following examples have been deposited in the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) of Taiwan, China (300 No. 331, Food Road, Hsinchu City, Taiwan, China), and are publicly available. In addition, the probiotic strains are also deposited in the China Center for Type Culture Collection (CCTCC) and the China General Microbiological Culture Collection Center (CGMCC) of the China Microbiological Culture Collection Committee according to the provisions of the Budapest Treaty. For clarity, the relevant information of each probiotic strain (including: scientific name, relevant source, deposit number and deposit date, etc.) has been integrated in the following Table 1.

[0072] Table 1 Relevant information of each probiotic strain

[0073]

[0074] 2. Staphylococcus aureus (BCRC12154) and Propionibacterium acnes (BCRC16146) used in the following examples were purchased from the Bioresource Conservation and Research Center (BCRC) of the Food Industry Development Institute (FIRDI) in Taiwan, China.

[0075] 3. The collagen solution used in the following examples was purchased from Hangzhou Nutrition Biotech Co., Ltd. under the trade name of collagen peptide.

[0076] 4. Source and culture of cell lines:

[0077] The human epidermal keratinocyte cell line (Adult) HaCaT used in the following examples was provided by Professor Zhiyao Wang of the College of Medicine, National Cheng Kung University, Taiwan; and the mouse skin melanoma cell line B16F10 (BCRC 60031) was purchased from the Bioresource Conservation and Research Center (BCRC) of the Food Industry Development Research Institute (FIRDI), Taiwan, China.

[0078] Both cells were cultured in 10 cm petri dishes containing Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (EMDMillipore Corp.) and 1% penicillin-streptomycin (Sigma-Aldrich) at 37°C and 5% CO. 2 The cells were cultured in an incubator at 4 °C. After that, fresh culture medium was replaced approximately every 2-3 days. When the cell density reached about 80-90% confluence, the cells were subcultured.

[0079] General experimental methods:

[0080] 1. Statistical analysis:

[0081] In the following examples, the experiments of each group were repeated 3 times, and the experimental data were expressed as "mean ± standard error of the mean (SEM). All data were analyzed by Student's t-test to evaluate the differences between the groups. If the statistical analysis results obtained were p < 0.05, this indicated statistical significance.

[0082] Example 1 Preparation of probiotic liquid culture

[0083] Experimental methods:

[0084] First, the three probiotic strains described in item 1 of the above "General Experimental Materials" were inoculated into MRS broth (Difco) supplemented with 0.05% cysteine ​​and cultured at 37°C for 24 hours to activate the strains. Then, the activated probiotic strain cultures were adjusted to have a concentration of 10 9 -10 10 CFU / mL of bacterial concentration (counted by plate count medium), and then mixed according to the bacterial count ratio shown in Table 2 below to obtain 7 probiotic compositions (i.e., probiotic compositions 1 to 7), and inoculated into 1L of 20% (w / v, g / mL) collagen solution at an inoculum of 3% (v / v), and cultured at 37°C for 48 hours to obtain cultures of probiotic compositions 1 to 7. Thereafter, the cultures were centrifuged at 3,000 rpm at 4°C for 10 minutes to precipitate the bacteria, and the supernatant was collected and sterilized at 100°C for 30 minutes to obtain the culture supernatant of each probiotic composition (hereinafter referred to as probiotic liquid culture C1 to C7).

[0085] Table 2 Ratio of the number of bacteria used in each probiotic composition

[0086] Probiotic composition TYCA06:AP-32:CP-9 1 1：0.125：0.125 2 1：1：1 3 1：1：8 4 1：8：1 5 1：0.071：0.071 6 1：1：14 7 1：14：1

[0087] In addition, the probiotic composition 2 was inoculated into 5 mL of MRS broth medium at an inoculum volume of 3% (v / v), and cultured at 37° C. for 48 hours. The obtained culture of the probiotic composition 2 was then centrifuged at 3,000 rpm at 4° C. for 10 minutes to precipitate the bacteria, and the supernatant was collected and sterilized at 100° C. for 30 minutes to obtain the culture supernatant of the probiotic composition 2 (hereinafter referred to as the probiotic liquid culture M).

[0088] Example 2 Evaluation of the efficacy of probiotic liquid culture in promoting wound healing

[0089] Experimental methods:

[0090] First, the HaCaT cells that were subcultured according to the fourth item "Source and culture of cell lines" in the "General experimental materials" above were divided into 10 groups, including 1 control group, 1 comparative experimental group, and 8 experimental groups (i.e., experimental group M and experimental groups C1 to C7). 6The number of cells / well was cultured in a 6-well culture plate containing 3 mL of DMEM (supplemented with 10% FBS and 1% penicillin-streptomycin) and kept in an incubator (37°C, 5% CO 2 ) and cultured for 24 hours.

[0091] Next, a pipette tip was used to scrape the HaCaT cells along the diameter of each well to produce a wound area with no cell attachment and a width of about 5 μm along the diameter of the culture plate. After that, the culture medium was removed and each well was washed with phosphate buffered saline (PBS). After replacing with fresh DMEM (supplemented with 10% FBS and 1% penicillin-streptomycin), an appropriate amount of the probiotic liquid culture M obtained in Example 1 above and the probiotic liquid cultures C1 to C7 were added to the corresponding experimental groups, respectively, so that each experimental group had a final concentration of 6.3 g / L of probiotic liquid culture. In addition, an appropriate amount of collagen solution was added to the comparative experimental group, so that the comparative experimental group had a final concentration of 6.3 g / L of collagen. As for the control group, no treatment was performed.

[0092] The cells of each group were cultured in an incubator (37°C, 5% CO 2 ) for 24 hours. Before culturing and at 24 hours after the start of culturing, the wound area of ​​each group was observed using an Olympus CKX41 microscope at a magnification of 100 times, and photographed using a digital camera (Canon brand), and then the photographs were analyzed using Image J software to calculate the area of ​​the wound area. The wound healing percentage (%) was calculated by substituting the calculated area of ​​the wound area into the following formula (1):

[0093] Formula (1): A = [1-(B / C)] × 100

[0094] Where: A = wound healing percentage (%)

[0095] B = area of ​​wound area measured 24 hours after the start of culture

[0096] C = area of ​​wound measured before incubation

[0097] Afterwards, the obtained experimental data were analyzed according to the method described in item 1 of the above “General Experimental Methods”.

[0098] result:

[0099] Figure 1 The figure shows the percentage of wound healing measured after HaCaT cells were treated with different probiotic liquid cultures. Figure 1 It can be seen that compared with the control group, there is no significant difference in the wound healing percentage of the comparative experimental group and the experimental group M. In contrast, the wound healing percentage of the experimental group C2 has a significant increase, which means that the probiotic liquid culture obtained by co-cultivating the probiotic composition 2 (Lactobacillus acidophilus TYCA06, Lactobacillus salivarius AP-32 and Bifidobacterium animalis subsp. lactis CP-9 at a bacterial ratio of 1:1:1) in the collagen solution has an excellent effect on promoting wound healing, and this effect is significantly better than that of the probiotic liquid culture obtained by co-cultivating the probiotic composition 2 in the MRS culture medium and that of the collagen itself. In addition, the excellent effect similar to that of the experimental group C2 can also be observed in the experimental groups C1, C3 and C4, while there is no such effect in the experimental groups C5 to C7. The experimental results show that the probiotic liquid cultures obtained by co-culturing probiotic compositions 1, 3 and 4 (the bacterial count ratios of Lactobacillus acidophilus TYCA06, Lactobacillus salivarius AP-32 and Bifidobacterium animalis subspecies lactis CP-9 are 1:0.125:0.125, 1:1:8 and 1:8:1) in collagen solution can all exhibit excellent effects in promoting wound healing.

[0100] Example 3 Evaluation of the effectiveness of the probiotic liquid culture of the present invention in inhibiting melanogenesis

[0101] Experimental methods:

[0102] First, the B16F10 cells that were subcultured according to the fourth item "Source and culture of cell lines" in the "General experimental materials" above were divided into 11 groups, including 1 normal control group, 1 pathological control group, 1 comparative experimental group, and 8 experimental groups (i.e., experimental group M and experimental groups C1 to C7). Each group of cells was divided into 2×10 6 The number of cells / well was cultured in a 6-well culture plate containing 3 mL of DMEM (supplemented with 10% FBS and 1% penicillin-streptomycin) and kept in an incubator (37°C, 5% CO 2 ) and cultured for 24 hours.

[0103] Then, the cell cultures of the pathological control group, comparative experimental group and each experimental group were replaced with fresh DMEM [supplemented with 10% FBS, 1% penicillin-streptomycin and 100 nM α-melanocyte stimulating hormone (α-MSH)] and placed in an incubator (37°C, 5% CO 2 ) for 24 hours, so that B16F10 cells were induced to produce melanin. In addition, the cell culture of the normal control group was replaced with fresh DMEM (supplemented with 10% FBS and 1% penicillin-streptomycin) without α-MSH and placed in an incubator (37°C, 5% CO 2 ) and cultured for 24 hours.

[0104] Next, the cell culture of each group was replaced with fresh DMEM (supplemented with 10% FBS and 1% penicillin-streptomycin) without α-MSH, and then an appropriate amount of the probiotic liquid culture M and probiotic liquid cultures C1 to C7 obtained in Example 1 above were added to the corresponding experimental groups, so that each experimental group had a final concentration of 6.3 g / L of probiotic liquid culture. In addition, an appropriate amount of collagen solution was added to the comparative experimental group, so that the comparative experimental group had a final concentration of 6.3 g / L of collagen. No treatment was performed on the normal control group and the pathological control group.

[0105] The cells in each group were incubated in a 37°C, 5% CO 2 ) for 24 hours, the cell culture was collected and washed with PBS, and then 1 mL of 1N NaOH (dissolved in RO water) was added and mixed thoroughly, and then heated at 80°C for 1 hour. After that, the resulting mixture was centrifuged at 3,000 rpm for 3 minutes, and 100 μL of the supernatant was collected and the absorbance value (OD) of each well was measured at a wavelength of 405 nm using an ELISA reader (BioTek). 405 ).

[0106] Melanin content (%) was calculated by measuring the absorbance value (OD 405 ) is substituted into the following formula (2) and calculated:

[0107] Formula (2): D = (E / F) × 100

[0108] Where: D = melanin content (%)

[0109] E = OD measured in each group 405Absorbance

[0110] F = OD measured in the normal control group 405 Absorbance

[0111] Afterwards, the obtained experimental data were analyzed according to the method described in item 1 of the above “General Experimental Methods”.

[0112] result:

[0113] Figure 2 The melanin content of B16F10 cells induced to produce melanin after being treated with different probiotic liquid cultures. Figure 2 It can be seen that the melanin content of the normal control group is significantly lower than that of the pathological control group, which indicates that α-MSH can successfully induce the melanin production of B16F10 cells. Compared with the pathological control group, there is no significant difference in the melanin content of the comparative experimental group and the experimental group M. In contrast, the melanin content of the experimental group C2 is significantly reduced, which indicates that the probiotic liquid culture obtained by co-culturing the probiotic composition 2 (the bacterial count ratio of Lactobacillus acidophilus TYCA06, Lactobacillus salivarius AP-32 and Bifidobacterium animalis subsp. lactis CP-9 is 1:1:1) in the collagen solution has an excellent inhibitory effect on the melanin production of B16F10 cells treated with α-MSH, and this effect is significantly better than that of the probiotic liquid culture obtained by co-culturing the probiotic composition 2 in the MRS culture medium and that of the collagen itself. In addition, similar excellent effects as those of experimental group C2 were observed in experimental groups C1, C3 and C4, but no such effects were observed in experimental groups C5 to C7. This experimental result shows that the probiotic liquid culture obtained by co-culturing probiotic compositions 1, 3 and 4 (Lactobacillus acidophilus TYCA06, Lactobacillus salivarius AP-32 and Bifidobacterium animalis subsp. lactis CP-9 at a bacterial ratio of 1:0.125:0.125, 1:1:8 and 1:8:1) in a collagen solution can all exhibit excellent melanin production inhibition effects, and are therefore expected to achieve skin whitening effects.

[0114] Example 4 Evaluation of the antibacterial efficacy of the probiotic liquid culture of the present invention

[0115] Experimental methods:

[0116] A. Anti-Staphylococcus aureus test:

[0117] Staphylococcus aureus was inoculated into a nutrient broth (HIMEDIA) and cultured at 37°C for 20 hours. Then, the proliferated Staphylococcus aureus was divided into 4 groups, including 1 normal control group, 1 comparative experimental group and 2 experimental groups (i.e., experimental group M and experimental group C). The volume of each group was 100 μL and had a 1×10 9 CFU / mL of bacterial concentration (counting the number of bacteria was performed using a plate counting medium). Next, 4.9 mL of the probiotic liquid culture M and the probiotic liquid culture C2 obtained in Example 1 above were added to the experimental group M and the experimental group C, respectively, and 4.9 mL of the collagen solution was added to the comparative experimental group. No treatment was performed on the normal control group.

[0118] After each group was cultured at 37°C for 6 hours, the cultured samples were serially diluted 10-fold to prepare different dilution factors (10 1 -10 3 Next, 100 μL of each dilution was evenly spread on a nutrient agar plate, and then cultured at 37°C for 48 hours. After that, the number of colonies generated on the nutrient agar plate was counted, and the number of Staphylococcus aureus contained in each group of cultures was calculated based on the dilution multiple.

[0119] The growth inhibition rate (%) was calculated by substituting the obtained bacterial count into the following formula (3):

[0120] Formula (3): G = [1-(H / I)] × 100

[0121] Where: G = Growth inhibition rate (%)

[0122] H = the number of bacteria contained in each group of cultures

[0123] I = the number of bacteria contained in the culture of the normal control group

[0124] Afterwards, the obtained experimental data were analyzed according to the method described in item 1 of the above “General Experimental Methods”.

[0125] B. Anti-Propionibacterium acnes test:

[0126] In order to understand the anti-Propionibacterium acnes effect of the probiotic liquid culture of the present invention, Propionibacterium acnes was inoculated into tryptone soy broth (HIMEDIA) supplemented with 5% defibrinated sheep blood and cultured at 37°C for 48 hours. Then, the grouping of Propionibacterium acnes, the treatment of the probiotic liquid culture and the collagen solution, and the counting of the number of colonies were carried out in a manner generally referring to the above method for Staphylococcus aureus, except that Propionibacterium acnes was used instead of Staphylococcus aureus. Then, the obtained number of Propionibacterium acnes bacteria was substituted into the above formula (3) to calculate the growth inhibition rate (%).

[0127] Afterwards, the obtained experimental data were analyzed according to the method described in item 1 of the above “General Experimental Methods”.

[0128] result:

[0129] Figure 3 and Figure 4 The growth inhibition rates of probiotic liquid culture on Staphylococcus aureus and Propionibacterium acnes are shown respectively. Figure 3 and Figure 4 It can be seen that the growth inhibition rate of both Staphylococcus aureus and Propionibacterium acnes in the experimental group C was significantly higher than that in the experimental group M. As for the growth inhibition rate of the comparative experimental group, there was no significant difference with that of the normal control group.

[0130] These experimental results show that collagen itself has no antibacterial activity, but when strains of Lactobacillus acidophilus TYCA06, Lactobacillus salivarius AP-32 and Bifidobacterium animalis subspecies lactis CP-9 are co-cultured in a collagen solution, the antibacterial effect of the probiotic liquid culture can be significantly enhanced.

[0131] Example 5 Evaluation of the effectiveness of the probiotic liquid culture of the present invention in improving skin conditions

[0132] Experimental subject:

[0133] The subjects participating in this trial were recruited using a procedure approved by the Ethics Committee of Antai Hospital. After a complete description of the experiment was given to all subjects participating in the trial, their informed consent was obtained. A total of 20 subjects aged between 18 and 50 years old, including 8 males and 12 females, were included in this trial.

[0134] Experimental Materials:

[0135] 1. Preparation of gel containing probiotic liquid culture:

[0136] First, the probiotic liquid culture C2 obtained in Example 1 above was formulated into a gel that can be applied to human skin (hereinafter referred to as liquid culture gel). In addition, for comparison, a gel that does not contain a probiotic liquid culture (hereinafter referred to as control gel) was prepared. The formulations of these two gels are shown in Table 3 below.

[0137] Table 3 Gel formulation

[0138]

[0139] The preparation of the liquid culture gel is carried out according to the following steps: first, carbomer, ammonium acryloyldimethyltaurate / VP copolymer and sodium hyaluronate powder are added to water for swelling, and then placed in a blender for full stirring until it becomes a gel, then the probiotic liquid culture C2 is added, and the pH value is adjusted to 5.0-5.5 with sodium hydroxide aqueous solution. In addition, the control gel is generally prepared according to the above steps, except that the probiotic liquid culture C2 is not added.

[0140] Experimental methods:

[0141] A. Application of gel:

[0142] First, the right face skin of each subject was used as the experimental area, and the left face skin was used as the control area. Then, the liquid culture gel and control gel obtained according to item 1 of the above "experimental materials" were applied to the experimental area and the control area, respectively. The application dosage of the experimental area and the control area was: 3-5 mg of gel was applied per 1 square centimeter area, twice a day, and the test period lasted a total of 28 days.

[0143] Before applying the gel (i.e., day 0) and on the 14th and 28th days after applying the gel, 10 subjects with similar acne symptoms on the left and right faces were selected from the 20 subjects, and the skin of the experimental area and the control area of ​​each subject was observed for acne according to the method described in the following item B. In addition, before applying the gel (i.e., day 0) and on the 28th day after applying the gel, the skin inflammation index and the number of skin brown spots were measured according to the methods described in the following items C and D on the skin of the experimental area and the control area of ​​each subject.

[0144] B. Observation of acne:

[0145] The skin of the experimental area and the control area of ​​each subject was observed and photographed using an intelligent skin analysis system (model ES3100, purchased from Yilimei Technology Co., Ltd.) to observe and photograph the acne areas of the skin.

[0146] C. Determination of skin inflammation index:

[0147] The skin of the experimental and control areas of each subject was measured using a VISIA TM Skin Analyzer (VISIA TM Complexion Analysis, USA) was used to determine the skin inflammation index.

[0148] The relative amount (%) of the skin inflammation index was calculated by substituting the number of skin redness calculated for each area before application of the gel (i.e., day 0) and on day 28 after application of the gel into the following formula (4):

[0149] Formula (4): J = (K / L) × 100

[0150] Where: J = relative amount of skin inflammation index (%)

[0151] K = number of skin redness measured in each area on the 28th day after gel application

[0152] L = number of skin redness measured in each area before applying the gel

[0153] Afterwards, the obtained experimental data were analyzed according to the method described in item 1 of the above “General Experimental Methods”.

[0154] D. Determination of the number of brown spots on the skin:

[0155] The skin of the experimental and control areas of each subject was measured using a VISIA TM Skin Analyzer (VISIA TM Complexion Analysis, USA) was used to determine the number of skin brown spots.

[0156] The relative amount (%) of skin brown spots was calculated by substituting the number of skin brown spots calculated for each area before application of the gel (i.e., day 0) and on day 28 after application of the gel into the following formula (5):

[0157] Formula (5): M = (N / O) × 100

[0158] Where: M = relative amount of skin brown spots (%)

[0159] N = the number of brown spots on each area measured on the 28th day after application of the gel

[0160] O = the number of brown spots on the skin measured in each area before applying the gel

[0161] Afterwards, the obtained experimental data were analyzed according to the method described in item 1 of the above “General Experimental Methods”.

[0162] result:

[0163] A. Observation of acne:

[0164] Figure 5 Shows the changes in acne morphology over time in various skin areas after application of liquid culture gel. Figure 5 It can be seen that on the 14th day after applying the gel, the redness and swelling of the acne area of ​​the experimental area were significantly improved, and on the 28th day after applying the gel, the redness and swelling of the area had been completely eliminated and almost no scars remained. In contrast, the redness and swelling of the acne area of ​​the control area did not change significantly over time.

[0165] B. Determination of skin inflammation index:

[0166] Figure 6 The relative amount of skin inflammation index measured in each area of ​​skin before and after application of liquid culture gel is shown. Figure 6 It can be seen that after 28 days of applying the gel, the relative amount of the skin inflammation index measured in the control area did not change significantly, while the relative amount of the skin inflammation index measured in the experimental area decreased significantly and was significantly lower than that in the control area.

[0167] C. Determination of the number of brown spots on the skin:

[0168] Figure 7 The relative amount of skin brown spots measured in each area of ​​skin before and after application of liquid culture gel is shown. Figure 7 It can be seen that after 28 days of applying the gel, the relative amount of skin brown spots measured in the experimental area was significantly reduced, while the relative amount of skin brown spots measured in the control area did not change significantly.

[0169] These experimental results show that the probiotic liquid culture obtained by co-culturing strains of Lactobacillus acidophilus TYCA06, Lactobacillus salivarius AP-32 and Bifidobacterium animalis subsp. lactis CP-9 in a collagen solution can effectively treat acne, reduce skin inflammation and reduce the formation of skin brown spots.

[0170] Based on the above experimental results, it can be seen that the probiotic liquid culture obtained by co-culturing the strains of Lactobacillus acidophilus TYCA06, Lactobacillus salivarius AP-32 and Bifidobacterium animalis subsp. lactis CP-9 in a ratio of 1:0.125:0.125 to 1:8:8 in a collagen solution has excellent antibacterial effect (especially against skin pathogens), and can promote wound healing, inhibit melanin production and improve various skin conditions. Therefore, the applicant believes that the probiotic liquid culture according to the present invention has a high potential for development into skin medicine and skin care products.

[0171] All patents and references cited in this specification are incorporated herein by reference in their entirety. In the event of a conflict, the detailed description (including definitions) of this specification will prevail.

[0172] Although the present invention has been described with reference to the above specific embodiments, it is apparent that many modifications and variations can be made without departing from the scope and spirit of the invention. It is therefore intended that the present invention be limited only as indicated by the appended claims.

Claims

1. A probiotic liquid culture, which is prepared by the following method: a probiotic composition comprising Lactobacillus acidophilus TYCA06, Lactobacillus salivarius subsp.salicinius AP-32 and Bifidobacterium animalis subsp.lactis CP-9 with a bacterial count ratio of 1:(0.125-8):(0.125-8) is co-cultured in a collagen solution, wherein the Lactobacillus acidophilus TYCA06 is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee (CGMCC) with a deposit number of CGMCC 15210; the Lactobacillus salivarius subsp.salicinius AP-32 is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC M 2011127; the animal Bifidobacterium lactis subspecies CP-9 is deposited in China Center for Type Culture Collection (CCTCC) with the deposit number of CCTCC M2014588.

2. The probiotic liquid culture according to claim 1, characterized in that: In the probiotic composition, the bacterial count ratio of Lactobacillus acidophilus TYCA06, Lactobacillus salivarius subspecies AP-32, and Bifidobacterium animalis subspecies lactis CP-9 is 1:1:

1.

3. The probiotic liquid culture according to claim 1, characterized in that: The probiotic liquid culture contains substantially no bacterial cells.

4. A use of the probiotic liquid culture according to any one of claims 1 to 3 for preparing a composition for improving skin conditions, characterized in that: The skin condition comprises at least one of the following: wound, melanin production, acne, skin inflammation, skin brown spots, and skin pathogen infection, wherein the skin pathogen is selected from the group consisting of Staphylococcus aureus, Propionibacterium acnes, and combinations thereof.

5. A use of the probiotic liquid culture according to any one of claims 1 to 3 for preparing a composition for inhibiting pathogen infection, characterized in that: The pathogenic bacteria is selected from the group consisting of Staphylococcus aureus, Propionibacterium acnes, and combinations thereof.

6. The use according to claim 4 or 5, characterized in that: The composition is a pharmaceutical composition.

7. The use according to claim 4 or 5, characterized in that: The composition is a cosmeceutical composition.

Images