Use of lactobacillus casei lc-12 fermentation lysate in oil control efficacy cosmetics

Abstract

The application discloses application of a lactobacillus casei LC-12 fermentation lysate product in oil control functional cosmetics, and confirms the oil control effect of the lactobacillus casei LC-12 fermentation lysate product through 5-alpha reductase inhibition experiments and human body experiments; and the experimental results show that the lactobacillus casei LC-12 fermentation lysate product not only has the oil control effect, but also can achieve long-acting oil control.

Description

Technical Field

[0001] This invention relates to a novel application of a fermentation lysate of Lactobacillus casei LC-12, particularly the application of a fermentation lysate of Lactobacillus casei LC-12 in oil-controlling cosmetics. Background Technology

[0002] Oily skin, also known as "oily skin," refers to a skin type characterized by excessive sebum secretion, large pores, thick, rough, and dull skin with a yellowish appearance, and a slightly alkaline pH. A study indicates that the prevalence of oily skin in the total population of China is 25.6%. People with oily skin are prone to clogged pores, shine, and acne due to excessive sebum production. Therefore, they need to use cosmetic ingredients or skincare products specifically formulated for oily skin to regulate sebum secretion and control shine.

[0003] Lactobacillus ferment lysate is a lysate produced during the fermentation of lactic acid bacteria. It contains the bacterial cells and all the active ingredients synthesized during fermentation, such as peptidoglycans, teichoic acid, proteins, phospholipids, sterols, fatty acids, various enzymes, peptides and amino acids, nucleotides, and extracellular polysaccharides. It possesses antibacterial, repairing, anti-allergic, and moisturizing effects, and has therefore received widespread attention in the cosmetics industry.

[0004] Invention patent CN202211362271.2 provides a *Lactobacillus plantarum* fermentation broth, fermentation lysate, and their preparation method and application. It uses *Lactobacillus plantarum* to ferment pomegranate peel, and the resulting fermentation broth and fermentation lysate exhibit superior antioxidant and whitening effects. This invention uses plant raw materials as a fermentation base to prepare the fermentation broth and its lysate; its effective components are not only the strain's own metabolites but also the active ingredients in the plant. Invention patent CN202210207122.2 relates to a method for preparing *Lactobacillus casei* LC-12 fermentation lysate and its application. Research has found that it has anti-inflammatory effects on the skin and can be used in cosmetics for anti-inflammatory purposes. Invention patent CN202310343078.2 uses *Lactobacillus casei* to ferment white willow bark extract to prepare a *Lactobacillus* fermentation filtrate, and an acne-repairing gel containing this fermentation filtrate has excellent oil-control and acne-removing effects. However, current research rarely reports that lactic acid bacteria lysates have oil-controlling effects. Summary of the Invention

[0005] The purpose of this invention is to provide an application of Lactobacillus casei LC-12 fermentation lysate in oil-controlling cosmetics. This invention features excellent oil-controlling effects.

[0006] The technical solution of this invention: the application of Lactobacillus casei LC-12 fermentation lysate in oil-controlling cosmetics.

[0007] In the application of the aforementioned Lactobacillus casei LC-12 fermentation lysate in oil-controlling cosmetics, the amount of Lactobacillus casei LC-12 fermentation lysate is less than or equal to 25%.

[0008] In the application of the aforementioned Lactobacillus casei LC-12 fermentation lysate in oil-controlling cosmetics, the dosage forms of the oil-controlling cosmetics include, but are not limited to, water, lotion, serum, or cream.

[0009] The aforementioned application of Lactobacillus casei LC-12 fermentation lysate in oil-controlling cosmetics involves the following steps in its preparation:

[0010] S1. Preparation of seed culture: Lactobacillus casei LC-12 was inoculated into MRS medium and cultured to obtain seed culture;

[0011] S2. Preparation of fermentation broth: Inoculate the seed culture into MRS medium and culture it in a shaker at 30-42℃ and 80-180 rpm / min for 15-20 hours to obtain the fermentation broth;

[0012] S3. Isolation of bacterial cells: Centrifuge the fermentation broth at 3500-8000 rpm and 4℃ for 20 min, and filter to obtain Lactobacillus casei cells;

[0013] S4. Cleaning of bacterial cells: Wash the Lactobacillus casei cells with phosphate buffer, centrifuge at 3500-8000 rpm and 4℃ for 20 min, and repeat the washing 2-5 times to obtain clean Lactobacillus casei cells.

[0014] S5. Preparation of Lactobacillus casei lysate: Clean Lactobacillus casei cells and phosphate buffer were mixed at a mass ratio of 1:1 and then sonicated for 20-40 min to obtain a cell wall-broken mixture. The cell wall-broken mixture was centrifuged at 3500-8000 rpm and 4℃ for 20 min to obtain the supernatant, which is the fermentation lysate of Lactobacillus casei LC-12.

[0015] In the application of the aforementioned Lactobacillus casei LC-12 fermentation lysate in oil-controlling cosmetics, the culture conditions in step S1 are: 30-42℃, 80-180rpm / min, shaker culture for 20-80 hours.

[0016] In the application of the aforementioned Lactobacillus casei LC-12 fermentation lysate in oil-controlling cosmetics, the MRS culture medium used comprises the following components in parts by weight: casein digest, 10.0 parts; triammonium citrate, 2.0 parts; beef extract powder, 10.0 parts; magnesium sulfate, 0.2 parts; yeast extract powder, 4.0 parts; manganese sulfate, 0.05 parts; sodium acetate, 5.0 parts; dipotassium hydrogen phosphate, 2.0 parts; glucose, 20.0 parts; Tween 80, 1.08 parts.

[0017] The pH value of the MRS medium is 5.5-5.9.

[0018] In the application of the aforementioned Lactobacillus casei LC-12 fermentation lysate in oil-controlling cosmetics, in step S4, the molar concentration of the phosphate buffer is 0.01-0.05 mol / L, and the pH is 6.5-7.5.

[0019] Compared with the prior art, the present invention applies the lysate of Lactobacillus casei LC-12 to cosmetics with oil-controlling effects, and confirms its oil-controlling effect through 5-α reductase inhibition experiment and human experiment. The experimental results show that the lysate of Lactobacillus casei LC-12 not only has an oil-controlling effect, but also has a long-lasting oil-controlling effect. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the Sebumeter test area;

[0021] Figure 2 This is a schematic diagram illustrating the rate of change in skin lipid content in volunteers over 4 weeks. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0023] The MRS medium used below consists of: 10.0 g casein digest, 2.0 g triammonium citrate, 10.0 g beef extract, 0.2 g magnesium sulfate (MgSO4·7H2O), 4.0 g yeast extract, 0.05 g manganese sulfate (MnSO4·4H2O), 5.0 g sodium acetate, 2.0 g dipotassium hydrogen phosphate, 20.0 g glucose, and 1.08 g Tween 80; the pH of the MRS medium is 5.5-5.9.

[0024] The following Lactobacillus casei LC-12 is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC NO.21372.

[0025] The preparation process of fermentation lysate of Lactobacillus casei LC-12 is as follows:

[0026] S1. Preparation of seed culture: Lactobacillus casei LC-12 was inoculated into MRS medium and cultured under the following conditions: 30-42℃, 80-180rpm / min, and shaker culture for 20-80 hours to obtain seed culture.

[0027] S2. Preparation of fermentation broth: The seed culture obtained in step S1 is inoculated into MRS medium and cultured on a shaker at 30-42℃ and 80-180 rpm / min for 15-20 hours to obtain the fermentation broth.

[0028] S3. Isolation of bacterial cells: The fermentation broth obtained in step S2 is centrifuged at 3500-8000 rpm and 4℃ for 20 min, and then filtered to obtain Lactobacillus casei cells.

[0029] S4. Cleaning of bacterial cells: The Lactobacillus casei cells obtained in step S3 are washed with phosphate buffer, centrifuged at 3500-8000 rpm at 4°C for 20 min, and the washing step is repeated 2-5 times to obtain clean Lactobacillus casei cells; the molar concentration of the phosphate buffer is 0.01-0.05 mol / L and the pH is 6.5-7.5.

[0030] S5. Preparation of Lactobacillus casei lysate: The clean Lactobacillus casei cells obtained in step 4 are mixed with phosphate buffer at a mass ratio of 1:1 and then sonicated for 20-40 min to obtain a cell wall-broken mixture; the cell wall-broken mixture is centrifuged at 3500-8000 rpm and 4℃ for 20 min to obtain the supernatant, which is the Lactobacillus casei LC-12 fermentation lysate.

[0031] The fermentation lysate of Lactobacillus casei LC-12 obtained has good oil-controlling effect when used in oil-controlling cosmetics, and the amount of Lactobacillus casei LC-12 fermentation lysate used is less than or equal to 25%.

[0032] As a preferred option, the dosage forms of oil-controlling cosmetics include, but are not limited to, water, lotion, serum, or cream.

[0033] Example 1

[0034] A method for preparing Lactobacillus casei LC-12 lysate, comprising the following steps:

[0035] 1. Inoculate Lactobacillus casei LC-12 into MRS medium and culture under the following conditions: 30℃, 80 rpm / min, shaker culture for 28 hours to obtain seed culture.

[0036] 2. Inoculate the seed culture obtained in step 1 into MRS medium and culture it in a shaker at 30°C and 80 rpm / min for 18 hours to obtain the fermentation broth.

[0037] 3. Centrifuge the fermentation broth obtained in step 2 at 3500 rpm and 4℃ for 20 min, and filter to obtain Lactobacillus casei cells.

[0038] 4. Wash the Lactobacillus casei cells obtained in step 3 with phosphate buffer, centrifuge at 3500 rpm and 4°C for 20 min, and repeat washing steps 2-5 times to obtain clean Lactobacillus casei cells; the phosphate buffer has a molar concentration of 0.01 mol / L and a pH of 6.5.

[0039] 5. Mix the clean Lactobacillus casei cells obtained in step 4 with phosphate buffer at a mass ratio of 1:1, and sonicate for 20 min to obtain a cell wall-broken mixture; centrifuge the cell wall-broken mixture at 3500-8000 rpm and 4℃ for 20 min to obtain the supernatant, which is the fermentation lysate of Lactobacillus casei LC-12.

[0040] Example 2

[0041] A method for preparing Lactobacillus casei LC-12 lysate, comprising the following steps:

[0042] 1. Inoculate Lactobacillus casei LC-12 into MRS medium and culture under the following conditions: 37℃, 100 rpm / min, shaker culture for 20 hours to obtain seed culture.

[0043] 2. Inoculate the seed culture obtained in step 1 into MRS medium and culture it in a shaker at 37°C and 100 rpm / min for 15 hours to obtain the fermentation broth.

[0044] 3. Centrifuge the fermentation broth obtained in step 2 at 3500 rpm and 4℃ for 20 min, and filter to obtain Lactobacillus casei cells.

[0045] 4. Wash the Lactobacillus casei cells obtained in step 3 with phosphate buffer, centrifuge at 3500 rpm and 4°C for 20 min, and repeat washing steps 2-5 times to obtain clean Lactobacillus casei cells; the phosphate buffer has a molar concentration of 0.01 mol / L and a pH of 6.5.

[0046] 5. Mix the clean Lactobacillus casei cells obtained in step 4 with phosphate buffer at a mass ratio of 1:1, and then sonicate for 40 min to obtain a cell wall-broken mixture. Centrifuge the cell wall-broken mixture at 3500 rpm and 4℃ for 20 min to obtain the supernatant, which is the fermentation lysate of Lactobacillus casei LC-12.

[0047] Example 3

[0048] A method for preparing Lactobacillus casei LC-12 lysate, characterized by the following steps:

[0049] 1. Inoculate Lactobacillus casei LC-12 into MRS medium and culture under the following conditions: 42℃, 180 rpm / min, shaker culture for 80 hours to obtain seed culture.

[0050] 2. Inoculate the seed culture obtained in step 1 into MRS medium and culture it in a shaker at 42℃ and 180 rpm / min for 20 hours to obtain the fermentation broth.

[0051] 3. Centrifuge the fermentation broth obtained in step 2 at 8000 rpm and 4℃ for 20 min, and filter to obtain Lactobacillus casei cells.

[0052] 4. Wash the Lactobacillus casei cells obtained in step 3 with phosphate buffer, centrifuge at 8000 rpm and 4°C for 20 min, and repeat washing steps 2-5 times to obtain clean Lactobacillus casei cells; the phosphate buffer has a molar concentration of 0.05 mol / L and a pH of 7.5.

[0053] 5. Mix the clean Lactobacillus casei cells obtained in step 4 with phosphate buffer at a mass ratio of 1:1, and then sonicate for 40 min to obtain a cell wall-broken mixture. Centrifuge the cell wall-broken mixture at 8000 rpm and 4℃ for 20 min to obtain the supernatant, which is the fermentation lysate of Lactobacillus casei LC-12.

[0054] Experimental Example 1

[0055] Lactobacillus casei LC-12 lysate inhibits 5α-reductase activity

[0056] Experimental steps:

[0057] Sample tubes: Add 1 mL of sample working solution (0.625%, 1.25%, 2.5%, 5%, or 10% of the fermentation lysate of *Lactobacillus casei* LC-12 as indicated in Example 1), 1 mL of enzyme solution, 1 mL of NADPH solution, and 1 mL of testosterone solution to a test tube. Set up three parallel tubes, gently shake well, and pipette 200 μL into a 96-well ELISA plate. Place the plate in an ELISA reader and measure the absorbance at 340 nm. This is the first measurement value for sample A (0). After incubating the sample at 37°C for 20 min, place it in an ELISA reader and measure the absorbance at 340 nm. This is the second measurement value for sample A (20).

[0058] Enzyme tubes: Add 1 mL of PBS solution, 1 mL of enzyme solution, 1 mL of NADPH solution, and 1 mL of testosterone solution to three parallel tubes. Gently mix, and pipette 200 μL into a 96-well ELISA plate. Place the plate in a microplate reader and measure the absorbance at 340 nm; this is the first measurement value (A enzyme 0). Incubate the sample at 37°C for 20 minutes, then measure the absorbance at 340 nm again; this is the second measurement value (A enzyme 20). Perform blank and positive control tests simultaneously.

[0059] Calculation formula:

[0060] Inhibition rate (%) = [1 - (A sample 0 - A sample 20) / (A enzyme 0 - A enzyme 20)] * 100%

[0061] Data processing:

[0062] All experiments were conducted in parallel, with at least 2-3 replicates. Experimental data are expressed as mean ± standard deviation (Mean ± SD), and data analysis was performed using Excel statistical software. When using the t-test method for statistical analysis, significance compared to the blank control group is indicated by *, P-value < 0.05 is indicated as *, and P-value < 0.01 is indicated as **.

[0063] Experimental results:

[0064] Table 1. Effects of different concentrations of Lactobacillus casei LC-12 lysate products on the inhibition rate of 5-α reductase.

[0065]

[0066]

[0067] The experimental results showed that, compared with the control group, the lysate of Lactobacillus casei LC-12 at concentrations of 0.625–10% significantly inhibited the activity of 5-α reductase (P<0.01), and the inhibition rate increased with increasing concentration.

[0068] Sebum metabolism is influenced by both external and internal factors. External factors mainly include environmental temperature, humidity, diet, and lifestyle habits; while internal factors mainly refer to age, genes, and endocrine function. Among these, androgen levels have a significant impact on sebum metabolism. 5-α reductase is primarily involved in the regulation of androgen metabolism, converting testosterone into dihydrotestosterone (DHT), a more active androgen. Therefore, by inhibiting 5-α reductase activity and reducing DHT production, sebum secretion can be effectively controlled.

[0069] Experimental Example 2

[0070] Lactobacillus casei LC-12 lysate inhibits lipid secretion in human skin.

[0071] Prior to conducting clinical efficacy testing, the safety of the fermentation lysate of Lactobacillus casei LC-12 at a concentration of 25% in Example 1 was verified by human skin patch testing.

[0072] 1. Test Background and Purpose

[0073] Volunteers used 20% of the fermentation lysate of Lactobacillus casei LC-12 from Example 1 for 4 weeks. The changes in skin oil content before and after product use were tested using non-invasive instruments to verify the oil-controlling effect of the sample.

[0074] 2. Instructions for use of test samples

[0075] Apply two pumps of the sample evenly to the designated half of the face once in the morning and once in the evening, and gently pat until absorbed. Replace your usual oil-controlling skincare sample with the Lactobacillus casei LC-12 fermentation lysate from Example 1, and continue with other skincare routines as usual.

[0076] 3. Criteria for selecting volunteers

[0077] (1) A person who voluntarily participates in the trial, is able to read and understand all the contents of the informed consent form, and signs the informed consent form;

[0078] (2) Healthy volunteers aged 18 to 45 (both men and women are eligible);

[0079] (3) No skincare products were applied, and the face was not cleansed in the morning after leaving the face on overnight. The Sebumeter reading on either side of the forehead was greater than 100 μg / cm. 2 Candidates can be selected.

[0080] (4) A person who can cooperate well with the test subjects and maintain a regular lifestyle during the research period.

[0081] (5) Understand and fill out the project questionnaire and cooperate with the staff.

[0082] Volunteer situation

[0083] The number of participants and their information are shown in Tables 2 and 3.

[0084] Table 2 Number of Participants

[0085] Group Number of participants Number of people who withdrew Number of people who completed Number of people analyzed Example 1 33 people 0 people 33 people 29 people Blank control group 33 people 3 people 30 people 24 people

[0086] Table 3 Volunteer Age

[0087] Group Mean age ± standard deviation Minimum age Maximum age Example 1 37.59±5.53 26 years old 45 years old Blank control group 35.83±6.38 21 years old 45 years old

[0088] Test methods

[0089] The Sebumeter (Courage & Khazaka, Germany) skin oil analyzer was used to test the oil content of the skin in the test area. Figure 1 The test was conducted on symmetrical areas of the forehead, at three points. Each point was tested three times, and the readings were recorded for each test. The first reading for each point was the test value, and the subsequent two readings were the verification values. The mean of the three test values ​​was subtracted from the mean of the three verification values; the result must be less than or equal to 10 μg / cm³. 2 When the test value is greater than 10 μg / cm, the test value is taken; the difference between the test value and the verification value is also taken. 2 At that time, the verification data should be taken. Oil testing should be performed one hour after each face wash. Finally, analysis and calculations are performed.

[0090] Data processing

[0091] (1) Baseline value verification: The Mann-Whitney U test was used, with a significance level of α = 0.05.

[0092] (2) Comparison results between the sample and the blank control: First, a normality test was performed. If the sample conforms to a normal distribution, an independent sample t-test was used.

[0093] The significance level is determined by the Mann-Whitney U test if the distribution does not conform to normality, with a significance level of α = 0.05. Significance compared to the blank control group is indicated by *, with P-value < 0.05 indicating * and P-value < 0.01 indicating **.

[0094] Experimental results

[0095] Table 44: Skin lipid content and changes in volunteers over 4 weeks

[0096]

[0097] Note: Change rate (%) = Change in lipid content / Baseline value * 100%.

[0098] As shown in the above chart, after continuous use of the Lactobacillus casei LC-12 fermentation lysate from Example 1, the changes in skin oil content in weeks 2 and 4 (compared to baseline) were significantly reduced by 27.21% and 18.93%, respectively; while the blank control group only changed by 10.08% (week 2) and 2.48% (week 4), indicating that the Lactobacillus casei LC-12 lysate has an oil-controlling effect and can control oil for a long time.

[0099] Application Example 1: #1 Essence Water

[0100] The manufacturing process is as follows: (Note: Ensure all equipment and containers are clean and dry).

[0101] A. Add the A phase raw material (A01-A11 phase raw materials in the table below) into the preparation tank, heat to 50-60℃, and stir until dissolved evenly;

[0102] B. Cool down to about 40℃ and add phase B raw materials (raw materials B01-B02 in the table below), stir and disperse evenly;

[0103] C. Add phase C (raw material C01 in the table below), stir and disperse evenly, and discharge through a 300-mesh filter cloth.

[0104]

[0105] Application Example 2: #2 Essence Lotion

[0106] The manufacturing process is as follows (Note: Ensure all equipment and containers are clean and dry).

[0107] A. Add the A phase raw material (A01-A12 phase raw materials in the table below) into the preparation tank, heat to 70-80℃, and stir until dissolved evenly;

[0108] B. Add phase B (phases B01-B09 in the table below) to the oil phase tank, heat to 70-80℃, and stir until dissolved.

[0109] C. After the B phase raw material is stirred evenly, it is pumped into the preparation tank, homogenized at 1500-2000 rpm for 5-10 mins, then the C phase (C01 raw material in the table below) is added, homogenized at 1000-1500 rpm for 1-2 mins, and then stirred and cooled.

[0110] D. Cool to about 45℃ and add phase D (raw material D01 in the table below), stir and disperse evenly; cool to about 38℃ and discharge through a 200-mesh filter cloth.

[0111]

[0112]

Claims

1. Application of Lactobacillus casei LC-12 fermentation lysate in the preparation of oil-controlling cosmetics.

2. The application of the Lactobacillus casei LC-12 fermentation lysate according to claim 1 in the preparation of oil-controlling cosmetics, characterized in that, The amount of fermentation lysate of Lactobacillus casei LC-12 should be less than or equal to 25%.

3. The application of the Lactobacillus casei LC-12 fermentation lysate according to claim 1 in the preparation of oil-controlling cosmetics, characterized in that, The dosage forms of the oil-controlling cosmetics include water, lotion, serum, and cream.

4. The application of the Lactobacillus casei LC-12 fermentation lysate according to claim 1 in the preparation of oil-controlling cosmetics, characterized in that, The preparation of the Lactobacillus casei LC-12 fermentation lysate includes the following steps: S1. Preparation of seed culture: Lactobacillus casei LC-12 was inoculated into MRS medium and cultured to obtain seed culture; S2. Preparation of fermentation broth: Inoculate the seed culture into MRS medium and culture it in a shaker at 30-42℃ and 80-180 rpm / min for 15-20 hours to obtain the fermentation broth; S3. Isolation of bacterial cells: Centrifuge the fermentation broth at 3500-8000 rpm at 4℃ for 20 min, and filter to obtain Lactobacillus casei cells; S4. Cleaning of bacterial cells: Wash the Lactobacillus casei cells with phosphate buffer, centrifuge at 3500-8000 rpm and 4℃ for 20 min, and repeat the washing 2-5 times to obtain clean Lactobacillus casei cells. S5. Preparation of Lactobacillus casei lysate: Clean Lactobacillus casei cells and phosphate buffer were mixed at a mass ratio of 1:1 and then sonicated for 20-40 min to obtain a cell wall-broken mixture. The cell wall-broken mixture was centrifuged at 3500-8000 rpm and 4℃ for 20 min to obtain the supernatant, which is the fermentation lysate of Lactobacillus casei LC-12.

5. The application of the Lactobacillus casei LC-12 fermentation lysate according to claim 4 in the preparation of oil-controlling cosmetics, characterized in that, The culture conditions in step S1 are: 30-42℃, 80-180 rpm / min, and cultured on a shaker for 20-80 hours.

6. The application of the Lactobacillus casei LC-12 fermentation lysate according to claim 4 in the preparation of oil-controlling cosmetics, characterized in that, The MRS medium used consisted of the following components in parts by weight: casein digest, 10.0 parts; Triammonium citrate, 2.0 parts; Beef extract powder, 10.0 parts; magnesium sulfate, 0.2 parts; Yeast extract powder, 4.0 parts; manganese sulfate, 0.05 parts; sodium acetate, 5.0 parts; dipotassium hydrogen phosphate, 2.0 parts; glucose, 20.0 parts; Tween 80, 1.08 parts; The pH value of the MRS medium is 5.5-5.

9.

7. The application of the Lactobacillus casei LC-12 fermentation lysate according to claim 4 in the preparation of oil-controlling cosmetics, characterized in that, In step S4, the molar concentration of the phosphate buffer is 0.01-0.05 mol / L, and the pH is 6.5-7.5.

Images