CN114657106B - A strain of Lactobacillus plantarum and its application in the prevention and treatment of acne - Google Patents

Abstract

The invention provides a novel lactobacillus plantarum, the preservation number of which is CCTCC NO: m2021594. The provided Lactobacillus plantarum is separated from pickle fermentation liquor, and can reduce skin inflammation, improve skin health condition, and prevent and relieve acne symptoms. The lactobacillus plantarum VHProbi E15 strain provided by the invention has strong tolerance to artificial intestinal juice; the strain is sensitive to common antibiotics such as erythromycin and ampicillin; can ferment glucose to produce acid without producing gas. The coagglutination test and the inhibition zone test prove that the lactobacillus plantarum VHProbi E15 can produce obvious inhibition effect on propionibacterium acnes. The acne-removing essence cream prepared by taking the lactobacillus plantarum VHProbi E15 lysate as the only effective component can effectively reduce the severity of moderate acne through human body test verification. The lactobacillus plantarum VHProbi E15 provided by the invention has no toxic or harmful effect on organisms, can be added into skin care products, is used for treating acne, and has wide application prospect.

Description

A strain of Lactobacillus plantarum and its application in preventing and treating acne

Technical Field

The invention belongs to the technical field of probiotic screening and application, and specifically relates to a Lactobacillus plantarum strain capable of treating acne and application thereof.

Background Art

The skin is an epithelial surface where the human body can interact with microorganisms. This surface is a dynamic interface rather than an impermeable barrier. The skin's microbiome can extend to the dermis and dermal fat. Human skin is a unique environment with a different microbiome from other primates. The skin microbiome is characterized by high diversity, site specificity and stability; specifically, some species are dominant, while the overall diversity is low. The microorganisms of healthy skin belong to 19 different phyla and 200 genera, and the microbial composition of different parts of the body's skin is different. For example, Propionibacterium acnes is a common bacterium in skin areas with developed sebaceous glands. In skin in different environments, such as dry skin areas, Staphylococcus and Streptococcus are dominant. The common characteristics of skin microorganisms between different sites reflect the commonalities between skin physiology.

After studying the pathogenesis of acne, researchers generally believe that the pathogenesis mainly includes: endocrine dysfunction, increased secretion of androgen causing increased secretion of sebum from sebaceous glands; abnormal keratinization of the sebaceous gland ducts of the hair follicles, resulting in narrowing of the duct diameter, obstruction of the excretion of sebum and normal exfoliated cells; local pathogen infection. Current acne treatment drugs include retinoic acid, antibiotics, corticosteroids, anti-androgen drugs, anti-seborrheic drugs, and physical therapy includes visible light and laser therapy. However, most existing therapies have the problems of high treatment costs, long treatment cycles, and large drug toxicity and side effects. Therefore, it is imperative to find a new acne relief solution.

Probiotics are living microorganisms that can bring health benefits to the host when administered in sufficient quantities. As a new generation of antimicrobial biological agents for skin diseases, probiotics have the potential to replace antibiotic therapy and can make up for the shortcomings of increased resistance and high recurrence rate caused by antibiotic and hormone drug treatment. It is reported that Fabbrocini et al. found that after one month of oral administration of a probiotic mixture, the level of anti-inflammatory cytokine IL-10 in the serum of patients with acne vulgaris was significantly increased, indicating that oral probiotics can be used as an adjuvant treatment for acne vulgaris by regulating inflammatory responses. Rahmayani et al. found that patients who took a liquid containing Lactobacillus rhamnosus SP1 restored the expression of genes related to insulin signaling and improved the appearance of acne compared with patients who took a liquid without the strain. Although the World Health Organization stipulates that only living microorganisms can be considered probiotics, there is evidence that heat-killed probiotics can also play an immunomodulatory role. Therefore, the use of probiotic preparations as active ingredients to prevent and treat acne has become a research hotspot in recent years. Postbiotics prepared from probiotics can be used as a potential treatment strategy for skin diseases, and the development of probiotic strains with acne prevention and treatment effects has important social benefits and economic value.

Summary of the invention

The invention aims to provide a new Lactobacillus plantarum. The provided Lactobacillus plantarum is separated from kimchi fermentation liquid and can reduce skin inflammation, improve skin health, and prevent and alleviate acne symptoms.

The plant lactobacillus provided by the present invention is the plant lactobacillus VHProbi E15 strain (Lactiplantibacillus plantarum VHProbi E15), which has been deposited in the China Center for Type Culture Collection on May 24, 2021, and its preservation number is CCTCC NO: M2021594.

The 16s rDNA sequence of the Lactobacillus plantarum VHProbi E15 strain is SEQ ID NO:1.

The Riboprinter fingerprint of the Lactobacillus plantarum VHProbi E15 strain provided by the present invention is shown in FIG3 ; its MALDI-TOF ribosomal protein molecular weight map is shown in FIG4 ; its RAPD fingerprint is shown in FIG5 ; and its rep-PCR fingerprint is shown in FIG6 .

The plant lactobacillus provided by the present invention can be used to prepare functional foods, health products, medicines or skin care products.

The plant lactobacillus provided by the invention can be used to prepare products with antioxidant function.

The Lactobacillus plantarum provided by the invention can be used to prepare products for preventing or treating acne.

The present invention also provides a product for preventing or treating acne, which contains live bacteria of Lactobacillus plantarum VHProbi E15 strain and/or its fermentation product.

The present invention also provides a product for preventing or treating acne, which contains the lysate of Lactobacillus plantarum VHProbi E15 strain.

The plant lactobacillus VHProbi E15 strain provided by the present invention has strong tolerance to artificial gastrointestinal fluid; the strain is sensitive to common antibiotics such as erythromycin and ampicillin; the strain can grow at 15°C, does not grow at 45°C, has a maximum tolerance salt concentration of 7%, can ferment glucose to produce acid without producing gas, has a cholesterol degradation rate of 16.24%, and has a skin cell adhesion of 2.65. Coaggregation test and inhibition zone test confirm that plant lactobacillus VHProbi E15 can produce a significant inhibitory effect on Propionibacterium acnes.

The acne-clearing cream, which is made with the lysate of Lactobacillus plantarum VHProbi E15 as the only active ingredient, has been proven to effectively reduce the severity of mild to moderate acne through human trials. After 4 weeks of use, it can significantly reduce the acne area of patients, and the skin color of the subjects becomes lighter. The transepidermal water loss rate is significantly reduced, and the skin oil is significantly reduced. In addition, the pore area/AOI area ( ^mm2 ), water content of the stratum corneum, and skin pH of the subjects are also improved compared with the baseline value. After 4 weeks of use, the acne-clearing cream can effectively control oil and remove acne, and repair the skin barrier.

The Lactobacillus plantarum VHProbi E15 provided by the invention has no toxic effect on the body, can be added into skin care products, is used for treating acne, and has broad application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a photo of a single colony of the E15 strain;

FIG2 is a diagram showing the identification results of the API 50CH of the E15 strain;

Fig. 3 is a Riboprinter fingerprint spectrum of E15 strain;

FIG4 is a MALDI-TOF ribosomal protein fingerprint spectrum of E15 strain;

Fig. 5 is a RAPD fingerprint of strain E15;

Fig. 6 is a rep-PCR fingerprint of strain E15;

FIG7 is a graph showing the results of a coaggregation test between strain E15 and Propionibacterium acnes;

FIG8 is a graph showing the results of the Oxford Cup test of E15 strain inhibiting Propionibacterium acnes;

FIG. 9 is an example of improvement in facial acne in subjects.

DETAILED DESCRIPTION

The Lactobacillus plantarum VHProbi E15 provided by the present invention meets regulatory requirements, and is identified as a newly discovered strain by polyphasic taxonomy. The Lactobacillus plantarum VHProbi E15 provided by the present invention can effectively prevent and relieve mild to moderate acne, and can relieve acne by using the strain alone without being compounded with prebiotics and/or other probiotics; and has important application value.

On May 24, 2021, the applicant deposited the Lactobacillus plantarum VHProbi E15 in the China Type Culture Collection of Wuhan University, and its preservation number is CCTCC NO: M2021594.

The screening method of the present invention is not limited to the examples described in the embodiments, and any known method that can achieve the screening purpose can be used. The screening description of the embodiments is only an explanation of the present invention, and is not a limitation on the protection scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all belong to the scope of the present invention.

The present invention is described in detail below in conjunction with specific embodiments.

Example 1 Isolation and screening of Lactobacillus plantarum VHProbi E15

1. Initial screening

Prepare MRS agar medium and adjust the pH to 6.2-6.5, and sterilize by high pressure at 121°C for 15 min.

Take 1mL of kimchi fermentation liquid, dilute it with sterile saline, put it into a sterile sample bag, and beat it with a homogenizer to mix it; take 100μL of the mixed liquid for gradient dilution, spread it on MRS agar medium, and then culture it anaerobically at 37℃ for 48h, and wait for single colonies to grow on the plate for microscopic examination. According to the results of microscopic examination, the applicant screened out a total of 18 potential lactobacilli, named E1, E2, ..., E13, E14, E15, E16, E17, and E18.

2. Rescreening

Prepare 1L of MRS liquid culture medium and sterilize it under high pressure at 121℃ for 15min. After the culture medium has cooled, add 3.2g of porcine mucosal pepsin, shake well to dissolve, and place in a 37℃ water bath shaker for 1h to make an acid-resistant culture medium.

The 18 strains of lactobacillus E1, E2, ..., E13, E14, E15, E16, E17, and E18 obtained by screening were inoculated into the above-mentioned acid-resistant culture medium at an inoculation rate of 6%, and cultured anaerobically at 37°C for 48 hours, and the fermentation broth was taken for bacterial count.

The results showed that the E15 strain had the highest number of viable bacteria after rescreening with acid-resistant culture medium, with a logarithmic value of up to 7.28Log CFU/mL, indicating that the E15 strain had the highest acid resistance.

Example 2 Strain Identification

1. Colony morphology identification

The E15 strain was inoculated on MRS agar medium and cultured anaerobically at 37°C for 24 hours. Single E15 colonies were observed to be milky white with a diameter of about 2-3 mm. The surface was moist, smooth and round. The colony photo is shown in Figure 1.

2. Identification of physiological and biochemical characteristics

The inoculation solution in this embodiment was prepared as follows: under sterile conditions, an appropriate amount of fresh E15 bacterial solution was taken, centrifuged at 5000 rpm/min for 5 min, washed twice with PBS buffer, and then resuspended with the same volume of PBS buffer and diluted 50 times as the inoculation solution.

2.1 Temperature tolerance test

Take an appropriate amount of fresh bacterial liquid (24h, 37℃), centrifuge at 5000rpm for 5min, wash once with PSP solution, resuspend with the same volume of PSP solution and dilute 50 times as inoculum.

The inoculum was inoculated into 10 mL of MRS liquid culture medium at a 10% inoculum volume, and 5 mL of MRS liquid culture medium without bacteria was used as a control. The cultures were cultured in a 15°C constant temperature incubator for 7 days and a 45°C constant temperature incubator for 2 days, and the bacterial solution was observed to see whether it became turbid.

The results showed that the E15 strain grew normally at 15°C but did not grow at 45°C.

2.2 Salinity tolerance test

Under sterile conditions, add 190 μL of BSM liquid culture medium with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8% to a 96-well plate. Make three parallels for each salt concentration, and then add 10 μL of inoculation solution. The wells without bacteria are used as controls. Add 50 μL of autoclaved paraffin oil to each well to prevent water evaporation during the culture process. Incubate at 37°C and observe whether the culture medium becomes turbid.

The results showed that the maximum salt concentration tolerated by strain E15 was 7%.

2.3 Carbon source metabolism test

The API 50CH reagent strip was used to perform a carbon source metabolism experiment on the E15 strain. The experimental method and result interpretation are detailed in the API 50CH kit manual. The identification result of the E15 strain was: %ID=99 and T value=1. The API result was Lactobacillus plantarum, and the identification comment was excellent. The metabolic test results are shown in Figure 2.

2.4 Glucose acid and gas production test

The culture medium formula used in this example is as follows:

Peptone 0.5 g; yeast extract 0.3 g; Tween 80 0.1 mL; saline solution A 0.5 mL; saline solution B 0.5 mL; sodium acetate 0.5 g; glucose 2.5 g; 2% bromocresol green (w/v) 0.05 mL; distilled water 100 mL; pH 6.8-7.0.

The prepared culture medium was dispensed into a large test tube containing an inverted small test tube, 3 mL/tube, and sterilized under high pressure at 121°C for 15 min.

Salt solution A ingredients: KH ₂ PO ₄ 10g, K ₂ HPO ₄ 1.0g, dissolved in distilled water, and the volume is adjusted to 100mL.

Salt solution B components: MgSO ₄ ·7H ₂ O 11.5 g, MnSO ₄ ·2H ₂ O 2.4 g, FeSO ₄ ·7H ₂ O 0.68 g, dissolved in distilled water and fixed to 100 mL.

Under sterile conditions, inoculate the culture medium with 10% of the inoculum and use the uninoculated culture medium as a control. Then seal the top with 2 mL of sterile liquid paraffin and culture at 37°C for 24 hours to observe whether the color of the culture medium changes.

The results showed that after culturing at 37°C for 24 hours, the culture medium changed from green to yellow and there was no gas in the small inverted tube, indicating that the E15 strain fermented glucose to produce acid but not gas.

3. Molecular Biology Identification

3.1 16S rDNA gene sequence analysis

3.1.1 Genomic DNA extraction

The procedure was performed according to the Tiangen Bacteria Genomic DNA Extraction Kit (Catalog Number: DP302).

3.1.2. 16S rDNA gene amplification

Primer sequences:

27F:AGAGTTTGATCCTGGCTCA;

1492R: GGTTACCTTGTTACGACTT.

The 16s rDNA sequence SEQ ID NO: 1 of the E15 strain was obtained by sequencing, and the sequence was compared with the NCBI database, and the E15 strain was preliminarily determined to be Lactobacillus plantarum.

3.2 Riboprinter fingerprint

Use a bacterial stick to pick up a purified single colony from the agar medium plate, put it into a sample tube with buffer, stir it with a handheld stirrer to suspend it in the buffer, then put the sample rack into the heater to inactivate it and put it into the Riboprinter system. After DNA preparation, membrane transfer, imaging detection and data processing, the bacterial identification results are obtained. The identification results show that the E15 strain is Lactobacillus plantarum, and its Riboprinter fingerprint results are shown in Figure 3.

3.3 MALDI-TOF-MS detection of ribosomal protein expression

Inoculate fresh bacterial liquid in MRS liquid culture medium at a 0.1% inoculation rate, culture at 37°C, 150rpm for 48h, collect the bacteria, wash with sterile water 4 times, and dry the surface moisture. Then take a small amount of fresh bacteria and evenly apply it on the target plate in the form of a film, add 1μL of lysis solution to cover the sample, dry it, add 1μL of matrix solution to cover the sample, and after drying, put the sample target into the mass spectrometer for identification. Use laser to irradiate the co-crystallized film formed by the sample and the matrix to ionize the protein in the sample. The ions are accelerated to fly through the flight pipe under the action of 10-20KV electric field, and the molecular weight of the protein is detected according to the different flight time to the detector. The protein fingerprint was obtained using Autofms 1000 analysis software Autof Analyzer v1.0. The ion peaks of the main ribosomal proteins of the E15 strain were: m/z 5201.363, 7887.113, 5734.785, 7303.595, 5185.929, 5421.786, 7878.099, 3943.720. The identification results are shown in Figure 4.

3.4 RAPD and rep-PCR fingerprint identification

3.4.1. RAPD fingerprint identification

1) Primer sequence: GAGGGTGGCGGTTCT.

2) RAPD reaction system

Table 1: RAPD reaction system table

3) Electrophoresis

A 1.5% agarose gel plate was prepared, DL2000 DNA Marker was used as the result control, and the electrophoresis was carried out at a constant voltage of 100 V for 80 min. Finally, the electrophoresis pattern was detected using a gel imaging system. The RAPD fingerprint of the E15 strain is shown in FIG5 .

3.4.2 rep-PCR fingerprint

1) Primer sequence: CTACGGCAAGGCGACGCTGACG.

2) Rep-PCR reaction system

Table 2: Rep-PCR reaction system

3) Electrophoresis

DL2000 DNA Marker was used as a control. The voltage was 100 V and the electrophoresis time was 80 min to detect the amplification results. The rep-PCR fingerprint of strain E15 is shown in Figure 6.

3.5 Whole genome sequencing

Take fresh E15 bacterial liquid and inoculate it into 500mL MRS broth medium at a volume ratio of 1%, culture at 37℃ for 20h, centrifuge at 8000rpm for 10min, and collect the bacteria. The bacteria are sent to the sequencing center to obtain the whole genome sequence of the bacteria. The whole genome sequence is uploaded to the NCBI gene database, and the GenBank accession number is CP094961-CP094962.

The colony morphology and physiological and biochemical characteristics of strain E15 were uploaded to http://www.tgw1916.net/bacteria_logare_desktop.htmL, and compared with the results published in De Clerck E, et al. Systematic and applied microbiology, 2004, 27 (1) 50. Based on the molecular biological identification results, strain E15 was determined to be a new strain of Lactobacillus plantarum, which was named Lactiplantibacillus plantarum VHProbi E15.

Example 3 Tolerance test of Lactobacillus plantarum VHProbi E15 to artificial gastric juice and artificial intestinal juice

1. Preparation of artificial gastric juice

Weigh 5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl respectively, add 1000mL of distilled water, adjust pH to 3.0 with dilute hydrochloric acid, and then sterilize at 115℃ for 20min. Then add 3.2g of porcine mucosal pepsin before use, shake well to dissolve, and place in a 37℃ water bath shaker for 1h to simulate human body temperature.

2. Preparation of artificial intestinal fluid

Weigh 5g of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH ₂ PO ₄ and 3.0g of ox bile salt, add 77mL of 0.2mol/L NaOH solution, dilute to 1000mL, adjust pH to 6.8±0.1 with dilute hydrochloric acid or sodium hydroxide solution, sterilize at 115℃ for 20min. Then add 1g of pancreatic enzyme before use, shake well to dissolve, and place in a 37℃ water bath shaker for 1h to simulate human body temperature.

3. Test methods

Take 2mL of fresh bacterial solution, centrifuge at 5000rpm/min for 5min to collect the bacteria, wash the bacteria 3 times with saline, and resuspend with 2mL of saline as the inoculum. Take 1mL of the inoculum, add it to 24mL of artificial intestinal fluid, place it in a 37℃ water bath shaker (200rpm/min) for 3h, take 1mL of the sample, and detect the amount of live bacteria.

The live bacteria count method was carried out in accordance with the national standard GB4789.35-2016-Food Microbiology Inspection Lactic Acid Bacteria Inspection to determine the bacterial count. The live bacteria count (Log CFU/mL) of the strain after digestion with artificial intestinal fluid is shown in Table 3.

Table 3: Table of live bacteria after digestion of artificial gastrointestinal fluid

As shown in Table 3, the live bacteria amount of Lactobacillus plantarum VHProbi E15 screened by the present invention decreased only slightly after digestion with artificial gastric juice, and decreased only by about 1.2Log value after further digestion with artificial intestinal juice, indicating that the strain has strong tolerance to artificial gastrointestinal juice.

Example 4 Antibiotic tolerance experiment of Lactobacillus plantarum VHProbi E15

1. Preparation of antibiotics: Ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline, and vancomycin were prepared into 2048 μg/mL stock solutions and stored at -20°C for later use. When used, the stock solution was diluted 2-fold with BMS liquid culture medium to form a working solution, with a total of 11 gradients of gradient dilution concentrations ranging from 1 to 1024 μg/mL.

2. Preparation of inoculum: Preparation of inoculum: Take an appropriate amount of fresh bacterial culture (cultured at 40°C for 24-48 hours), centrifuge at 5000rpm for 5 minutes, wash once with sterile saline, resuspend the bacteria in the same volume of saline and dilute 50 times as the inoculum.

3. The minimum inhibitory concentration (MIC) of antibiotics against Lactobacillus plantarum VHProbi E15 was determined by the microbroth dilution method.

a. Add BMS liquid culture medium without antibiotics to the first column of the 96-well plate as a negative control, and add 190 μL of BMS liquid culture medium containing different concentrations of antibiotics to columns 2 to 12, and then inoculate 10 μL of the above inoculation solution to make 3 parallel wells, and use 1 well without bacterial solution as a blank.

b. Add 50 μL paraffin oil to cover to prevent water evaporation.

c. The 96-well plate was taken out after shaking culture at 40°C for 48 hours, and the OD600 value was measured. The MIC value of the antibiotic to the strain was calculated using the result of 48 hours. The specific results are shown in Table 4.

Table 4: Antibiotic MIC values (μg/mL) of Lactobacillus plantarum VHProbi E15

It can be seen from the results in Table 4 that the Lactobacillus plantarum VHProbi E15 provided by the present invention is sensitive to common antibiotics such as erythromycin and ampicillin, and has good biosafety.

Example 5 In vitro cholesterol degradation experiment with Lactobacillus plantarum VHProbi E15

1. Preparation of cholesterol micelle solution: Accurately weigh 1g of cholesterol, dissolve it in anhydrous ethanol, and make up to 100mL. Filter and sterilize it with a 0.22μm microporous filter membrane under sterile conditions.

2. Weigh 10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 2.0g diammonium citrate, 20.0g glucose, 1.0mL Tween 80, 5.0g sodium acetate, 0.1g magnesium sulfate, 0.05g manganese sulfate, 2.0g dipotassium hydrogen phosphate, 1g bile salt, 1000mL distilled water, adjust pH to 7.3, sterilize at 115℃ for 30min, then add cholesterol solution to make the final cholesterol concentration 0.1%. Inoculate fresh bacterial solution of Lactobacillus plantarum VHProbi E15 at a 0.1% inoculum volume, culture at 37℃ for 48h, then take 0.2mL bacterial solution, add 1.8mL anhydrous ethanol, mix, let stand for 10 minutes, centrifuge at 3000 rpm for 5 minutes, and take the supernatant for cholesterol content determination. The cholesterol determination method is in accordance with GB/T5009.128-2003 <Determination of cholesterol in food>.

The results showed that the degradation rate of cholesterol by Lactobacillus plantarum VHProbi E15 provided by the present invention reached 16.24% (this is the data without bile salt).

Example 6 Caco-2 cell adhesion test of Lactobacillus plantarum VHProbi E15

Caco-2 cells were seeded in six-well plates at a seeding density of 2 × 10 ⁶ cells/well and cultured in a CO incubator for 24 h for cell adhesion experiments;

The strains in the stable phase were resuspended to 5 × 10 ⁷ CFU/mL in MRS medium;

Take 1 mL of the above strain and add it to the six-well plate with attached cells, and culture it in a carbon dioxide incubator for 2 h;

The cells were washed three times with PBS to remove the nonadherent bacteria;

Add 500ul of trypsin for digestion for 3 minutes, then add 1.5mL of cell culture medium to terminate digestion, blow repeatedly, collect the resulting solution into a sterile EP tube, and dilute the collected solution 10 times, 100 times, 1000 times, 10000 times, and count on a plate. At the same time, count the cells in the blank group. Calculate the adhesion ability of the test strain according to the following formula:

Adhesion capacity (CFU/cells) = total number of adhered bacteria in each culture well/total number of cells in each culture well.

The results showed that the cell adhesion of Lactobacillus plantarum VHProbi E15 provided by the present invention was 2.65, and the standard deviation was 0.44.

Example 7 Inhibition test of Lactobacillus plantarum VHProbi E15 on Propionibacterium acnes

1. Agglutination test

Take the frozen Lactobacillus plantarum VHProbi E15, streak it onto the MRS plate aseptically, and culture it at 37°C for 48-72 hours. Inoculate Propionibacterium acnes ATCC6919 and Propionibacterium acnes ATCC11827 into BHI broth medium and culture it anaerobically at 37°C for 3-4 days.

Take appropriate amount of the above activated fresh bacterial solution, centrifuge at 6000 rpm for 4 min, concentrate to a suitable multiple, wash twice with tris saline buffer, and then resuspend with the same buffer.

Take 300 μL of plant lactobacillus suspension and add it to a 24-well ELISA plate, then add 300 μL of Propionibacterium acnes mixed suspension as the experimental group, and take an equal amount of plant lactobacillus suspension and buffer solution as the control group, and set up 2 parallels for each control and sample. Place the 24-well plate in a microplate constant temperature oscillator at 400 rpm and incubate at room temperature for at least 24 hours. During the period, stop the machine for 30 minutes every 30 minutes of oscillation, take pictures to record the initial plate state and the plate state at each stop, and observe whether agglutination occurs.

The results are shown in Figure 7: after co-incubation with Propionibacterium acnes for 30h10min, cross-agglutination occurred, indicating that Lactobacillus plantarum VHProbi E15 can effectively antagonize Propionibacterium acnes and significantly inhibit its growth.

2. Inhibition zone test

Take the frozen Lactobacillus plantarum VHProbi E15, aseptically streak it onto an MRS plate, and culture it at 37°C for 48-72 hours. After a single colony grows on the plate, aseptically pick it into an MRS broth medium and culture it at 37°C for 24 hours. Take 1 mL of the bacterial solution, centrifuge it at 5000 rpm for 5 minutes to obtain bacterial sludge, rinse the bacterial sludge twice with sterile water, and then re-dissolve it with 2 mL of sterile 1% peptone solution for use.

Propionibacterium acnes ATCC11827 and Propionibacterium acnes ATCC6919 were inoculated into reinforced Clostridium medium and cultured anaerobically at 37°C for 48 hours.

Lay the bottom layer of culture medium, prepare 1.5% agar solution, pour it into the plate after sterilization, and cover the plate. After the agar solidifies, evenly place an appropriate number of sterile Oxford cups. Lay the top layer of culture medium, the upper layer of semi-solid culture medium: add 0.7% agar and 1% Tween-80 to the fortified Clostridium culture medium, and then heat it slightly to dissolve the Tween-80. Sterilize at 121℃ for 15min, and wait until it is not hot to the touch (about 45℃) for use.

Mix two strains of Propionibacterium acnes cultured for 48 hours in equal amounts, mix thoroughly, and add 0.4% (v/v) to the upper semi-solid culture medium. After mixing, pour 14 mL onto the lower culture medium. After solidification, take out the Oxford cup and mark the added sample at the bottom of the plate. Take 100uL of bacteria + peptone solution and add it to the well, and add 1% peptone solution as a control. Observe the antibacterial results. After 72 hours of culture, take out the plate, observe whether there is an inhibition zone, take a picture and measure the size of the inhibition zone with a ruler.

The results are shown in Figure 8: obvious inhibition zones appeared on all plates, and the average diameter of the inhibition zones was 29.00±1.00 mm, indicating that Lactobacillus plantarum VHProbi E15 can significantly inhibit Propionibacterium acnes.

Example 8 Anti-acne efficacy test of Lactobacillus plantarum VHProbi E15 lysate

1.1 Preparation of Lactobacillus plantarum VHProbi E15 lysate

Lactobacillus plantarum VHProbi E15 was cultured in MRS broth to the logarithmic phase;

Fermentation medium: brown sugar 2%, collagen peptide 3%, yeast powder 0.3%, diammonium hydrogen phosphate 0.25%;

The activated strain was added to the fermentation medium at an inoculation rate of 1%, and fermented at 37°C for 24 hours. After the fermentation, the bacteria were collected in an ice bath, ultrasonically broken for 30 minutes, and the supernatant was obtained by centrifugation. The supernatant was heated at 75°C for 15 minutes for heat inactivation to obtain a lysate.

1.2 Preparation of lactic acid bacteria acne-clearing cream:

Referring to the formula described in Table 5, deionized water was used to prepare the lactic acid bacteria anti-acne cream. Among them, the lysate of Lactobacillus plantarum VHProbi E15 with a content of 8% was the only effective component in the lactic acid bacteria anti-acne cream.

Table 5 Formula of Lactic Acid Bacteria Anti-Acne Essence Cream

2. Population trial plan

Twenty healthy subjects aged 18 to 25 years with mild or moderate acne on their faces were selected. The subjects used lactic acid bacteria anti-acne essence cream after cleansing their faces every morning and evening for a total of 4 weeks. Photos of the ring beam were taken before using the product, and after using the product for 1 week, 2 weeks, 3 weeks, and 4 weeks. The moisture content of the stratum corneum, the rate of transepidermal water loss, the skin oil content, and the skin pH were tested before using the product, and after using the product for 2 weeks and 4 weeks. The ambient temperature of this test was 20.4℃～21.5℃, and the relative humidity was 47.7%～51.9%.

3. Sample completion status

3.1 Inclusion criteria:

(1) Healthy people, aged 18 to 45 years;

(2) Mild or moderate acne on both sides of the face;

(3) Be able to cooperate well with the experimenters and maintain a regular life during the study;

(4) Be able to read and understand all the contents of the informed consent form and voluntarily sign the informed consent form;

(5) Agree not to use any cosmetics, drugs or health products that may affect the results during the trial. 3.2 Exclusion criteria: Patients with any of the following conditions must be excluded from this study:

(1) Those with facial skin diseases that may affect the judgment of the test results;

(2) Those with highly allergic constitution;

(3) Women who are pregnant, breastfeeding, or planning to become pregnant during the trial;

(4) Those with severe heart, liver, or kidney damage and severe immunodeficiency;

(5) People with mental illness, severe endocrine diseases, or those taking oral contraceptives;

(6) Those who have participated in drug clinical trials or other trials within 30 days, or those who have used drugs that may affect the trial results within the past week;

(7) Those who have taken oral or external cosmetic products that may affect the test results within 14 days;

(8) Those who are unable to cooperate with the experiment;

(9) Those deemed by the researcher to be unsuitable for participation in this study;

(10) Other corresponding exclusion criteria.

Table 6 Subject information

4. Test process

1) After cleaning the face and drying it with a tissue, the subject should sit quietly in an efficacy evaluation room at 21±1℃ and relative humidity of 50±10% for 20 minutes;

2) The technicians operate the CK-MPA and Visia-CR instruments to measure the basal value indicators of the test parts of the subjects.

5. Statistical methods

SPSS was used for statistical analysis. The measured values at different time points were compared with the baseline values using repeated measures analysis of variance with an α=0.05 test level.

The improvement rate is the rate of change relative to that before use, and the calculation formula is as follows:

△1 after using the product for 1 week = W1-W0;

△2 after using the product for 2 weeks = W2-W0;

△3 after using the product for 3 weeks = W3-W0;

△4 after using the product for 4 weeks = W4-W0;

Improvement rate after using the product for one week = △1/W0*100%;

Improvement rate after using the product for 2 weeks = △2/W0*100%;

Improvement rate after using the product for 3 weeks = △3/W0*100%;

The improvement rate after using the product for 4 weeks = △4/W0*100%.

Wherein, W0 is the basic value of skin parameters in the test area before using the product;

W1——skin parameter values of the test area after using the product for 1 week;

W2——skin parameter values of the test area after using the product for 2 weeks;

W3——skin parameter values of the test area after using the product for 3 weeks;

W4——Skin parameter values of the test area after using the product for 4 weeks.

6. Test results

6.1 Results of acne area measurement

Table 7 Acne area detection results

Table 8 Statistical analysis results of Visia-CR acne area ratio

*Significance marking method: “n.s.” indicates no statistical difference, P ≥ 0.05; P < 0.05 indicates a significant difference (“*” indicates 0.01 ≤ P < 0.05; “**” indicates 0.001 ≤ P < 0.01; “***” indicates P < 0.001).

The smaller the acne area, the less obvious the redness. The acne area of the subjects decreased significantly compared with the baseline value after using the product for 4 weeks. 59.1% of the subjects had a decrease in the Visia-CR acne area, and 61.5% of the subjects had a 50% decrease in the Visia-CR acne area. The changes in the acne area of the subjects within 4 weeks are shown in Figure 9.

6.2 Skin color test results

Table 9 Skin color test results

Table 10 Skin color statistical analysis results

*Significance marking method: “n.s.” indicates no statistical difference, P ≥ 0.05; P < 0.05 indicates a significant difference (“*” indicates 0.01 ≤ P < 0.05; “**” indicates 0.001 ≤ P < 0.01; “***” indicates P < 0.001).

The smaller the skin color value, the less obvious the redness symptom. After using the product for 4 weeks, 22.7% of the subjects had their skin color lightened, but there was no significant difference.

6.3 Pore area/AOI area (mm ² ) test results

Table 11 Pore area/AOI area (mm2) test results

Table 12 Statistical results of pore area/AOI area (mm2)

*Significance marking method: “n.s.” indicates no statistical difference, P ≥ 0.05; P < 0.05 indicates a significant difference (“*” indicates 0.01 ≤ P < 0.05; “**” indicates 0.001 ≤ P < 0.01; “***” indicates P < 0.001).

The smaller the pore area/AOI area (mm ² ), the fewer the pores. After using the product for 4 weeks, 31.8% of the subjects had a decrease in pore area/AOI area (mm ^{2 )} compared with the baseline value, but there was no significant difference.

6.4 Skin stratum corneum moisture content test results

Table 13 Skin stratum corneum moisture content test results

Table 14 Statistical results of moisture content in the stratum corneum of the skin

*Significance marking method: “n.s.” indicates no statistical difference, P ≥ 0.05; P < 0.05 indicates a significant difference (“*” indicates 0.01 ≤ P < 0.05; “**” indicates 0.001 ≤ P < 0.01; “***” indicates P < 0.001).

The higher the moisture content of the stratum corneum, the more hydration the skin contains. After using the product for 4 weeks, 59.1% of the subjects had an increase in the moisture content of the stratum corneum compared to the baseline value, but there was no significant difference.

6.5 Transepidermal water loss test

Table 15 Transepidermal water loss test table

Table 16 Statistical results of transepidermal water loss test

*Significance marking method: “n.s.” indicates no statistical difference, P ≥ 0.05; P < 0.05 indicates a significant difference (“*” indicates 0.01 ≤ P < 0.05; “**” indicates 0.001 ≤ P < 0.01; “***” indicates P < 0.001).

The lower the value of transepidermal water loss, the better the skin barrier function. The transepidermal water loss of the subjects decreased significantly after using the product for 4 weeks compared with the baseline value (P<0.05). Among them, 68.2% of the subjects had a reduced transepidermal water loss rate, and 66.7% of the subjects had a reduced transepidermal water loss rate of more than 20%.

6.6 Oil content test results

Table 17 Oil content test results

Table 18 Statistical results of oil content test

*Significance marking method: “n.s.” indicates no statistical difference, P ≥ 0.05; P < 0.05 indicates a significant difference (“*” indicates 0.01 ≤ P < 0.05; “**” indicates 0.001 ≤ P < 0.01; “***” indicates P < 0.001).

The smaller the oil content value, the less oil content in the skin. After using the product for four weeks, the skin oil content of the subjects decreased significantly compared with the baseline value (P<0.05), and 72.7% of the subjects had a decrease in skin oil content.

6.7 Skin pH Balance

Table 19 pH test results

Table 20 Statistical results of pH test

*Significance marking method: “n.s.” indicates no statistical difference, P ≥ 0.05; P < 0.05 indicates a significant difference (“*” indicates 0.01 ≤ P < 0.05; “**” indicates 0.001 ≤ P < 0.01; “***” indicates P < 0.001).

Normal skin is slightly acidic, between 4.0-5.5. After using the product for four weeks, 22.7% of the subjects showed a decrease in skin pH compared to the baseline value, but the difference was not significant.

After using the lactobacillus acne cream for 4 weeks, the proportion of acne in the subjects with mild to moderate acne was significantly reduced (P<0.01), and the proportion of Visia-CR acne in 59.1% of the subjects was reduced, and the area of acne in 61.5% of the subjects was reduced by more than 50%. The transepidermal water loss rate of the subjects was significantly reduced (P<0.05), and the transepidermal water loss rate of 68.2% of the subjects was reduced, and the transepidermal water loss rate of 66.7% of the subjects was reduced by more than 20%. After using the product for four weeks, the skin oil content of the subjects decreased significantly compared with the baseline value (P<0.05), and the skin oil content of 72.7% of the subjects decreased. In addition, the skin color, pore area/AOI area (mm ² ), skin stratum corneum water content, and skin pH of the subjects were also improved compared with the baseline value. This shows that the plant lactobacillus VHProbiE15 lysate is effective in improving the skin of patients with mild to moderate acne, and can achieve the effect of oil control and acne removal.

Sequence Listing

<110> Qingdao Blue Biotechnology Co., Ltd. Qingdao Blue Biotechnology Group Co., Ltd.

<120> A strain of Lactobacillus plantarum and its application in preventing and treating acne

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1432

<212> DNA

<213> Artificial Sequence

<400> 1

cggctggttc ctaaaaggtt accccaccga ctttgggtgt tacaaactct catggtgtga 60

cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg catgctgatc cgcgattact 120

agcgattccg acttcatgta ggcgagttgc agcctacaat ccgaactgag aatggcttta 180

agagattagc ttactctcgc gagttcgcaa ctcgttgtac catccattgt agcacgtgtg 240

tagcccaggt cataaggggc atgatgattt gacgtcatcc ccaccttcct ccggtttgtc 300

accggcagtc tcaccagagt gcccaactta atgctggcaa ctgataataa gggttgcgct 360

cgttgcggga cttaacccaa catctcacga cacgagctga cgacaaccat gcaccacctg 420

tatccatgtc cccgaaggga acgtctaatc tcttagattt gcatagtatg tcaagacctg 480

gtaaggttct tcgcgtagct tcgaattaaa ccacatgctc caccgcttgt gcgggccccc 540

gtcaattcctttgagtttca gccttgcggc cgtactcccc aggcggaatg cttaatgcgt 600

tagctgcagc actgaagggc ggaaaccctc caacacttag cattcatcgt ttacggtatg 660

gactaccagg gtatctaatc ctgtttgcta cccatacttt cgagcctcag cgtcagttac 720

agaccagaca gccgccttcg ccactggtgt tcttccatat atctacgcat ttcaccgcta 780

cacatggagt tccactgtcc tcttctgcac tcaagtttcc cagtttccga tgcacttctt 840

cggttgagcc gaaggctttc acatcagact taaaaaaccg cctgcgctcg ctttacgccc 900

aataaatccg gacaacgctt gccacctacg tattaccgcg gctgctggca cgtagttagc 960

cgtggctttc tggttaaata ccgtcaatac ctgaacagtt actctcagat atgttcttct 1020

ttaacaacag agttttacga gccgaaaccc ttcttcactc acgcggcgtt gctccatcag 1080

actttcgtcc attgtggaag attccctact gctgcctccc gtaggagttt gggccgtgtc 1140

tcagtcccaa tgtggccgat taccctctca ggtcggctac gtatcattgc catggtgagc 1200

cgttacccca ccatctagct aatacgccgc gggaccatcc aaaagtgata gccgaagcca 1260

tctttcaagc tcggaccatg cggtccaagt tgttatgcgg tattagcatc tgtttccagg 1320

tgttatcccc cgcttctggg caggtttccc acgtgttat caccagttcg ccactcactc 1380

aaatgtaaat catgatgcaa gcaccaatca ataccagagt tcgttcgact gc 1432

Claims (3)

1. Lactiplantibacillus plantarum, characterized in that the deposit number of Lactiplantibacillus plantarum is CCTCC NO: M2021594. 2. Application of the lysate of Lactobacillus plantarum according to claim 1 in the preparation of skin care products for preventing or treating acne. 3. A skin care product for preventing or treating acne, characterized in that the skin care product contains the lysate of Lactobacillus plantarum according to claim 1.