CN114657106A - A Lactobacillus plantarum and its application in the prevention and treatment of acne - Google Patents

Abstract

The present invention provides a new strain of Lactobacillus plantarum, the preservation number of which is CCTCC NO: M2021594. The provided Lactobacillus plantarum is isolated from kimchi fermentation liquid, which can reduce skin inflammation, improve skin health, and prevent and relieve acne symptoms. The Lactobacillus plantarum VHProbi E15 strain provided by the invention has strong tolerance to artificial gastrointestinal juice; the strain is sensitive to common antibiotics such as erythromycin and ampicillin; it can ferment glucose to produce acid without gas production. It was confirmed by coagglutination test and inhibition zone test that Lactobacillus plantarum VHProbi E15 could significantly inhibit P. acnes. The acne-clearing essence cream prepared with Lactobacillus plantarum VHProbi E15 lysate as the only functional ingredient has been confirmed by human trials to effectively reduce the severity of mild to moderate acne. The Lactobacillus plantarum VHProbi E15 provided by the invention has no toxic effect on the body, can be added to skin care products for treating acne, and has broad application prospects.

Description

A Lactobacillus plantarum and its application in the prevention and treatment of acne

technical field

The invention belongs to the technical field of screening and application of probiotics, in particular to a Lactobacillus plantarum with the effect of treating acne and its application.

Background technique

The skin is the body's epithelial surface where microorganisms can interact. This surface is a dynamic interface rather than an impermeable barrier, and the skin's microbiome can extend to the dermis and dermal fat. Human skin is a unique environment with a microbiome different from that of other primates. The skin microbiome is characterized by high diversity, site-specificity, and stability; specifically, some species are dominant, while the overall degree of diversity is relatively low. low characteristic. 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 bacteria in the skin area with developed sebaceous glands. In the skin of different environments, such as In dry skin areas, Staphylococcus and Streptococcus predominated; the common characteristics of skin microbes between different sites reflected commonalities between skin physiology.

After studying the pathogenesis of acne, researchers generally believe that the pathogenesis mainly includes: endocrine dysfunction, increased secretion of androgen leads to increased sebaceous gland sebum secretion; abnormal keratinization of pilosebaceous ducts leads to narrowing of the diameter of the ducts, and the excretion of sebum and normal exfoliated cells Obstructed; local pathogen infection. Current acne treatment drugs include retinoic acid, antibiotics, corticosteroids, anti-androgens, anti-seborrhea, and physical therapy including visible light and laser therapy. However, most of the existing treatments have the problems of high treatment cost, long treatment period and serious drug toxicity and side effects. Therefore, it is imperative to find a new acne relief solution.

Probiotics are live microorganisms that, when administered in sufficient quantities, can confer health benefits to the host. As a new generation of antibacterial biologics for skin diseases, probiotics have the potential to replace antibiotic therapy, and can make up for the shortcomings of increased drug resistance and high recurrence rate caused by antibiotics and hormone therapy. According to reports, Fabbrocini et al. found that the anti-inflammatory cytokine IL-10 level in serum was significantly increased in patients with acne vulgaris after oral administration of the probiotic mixture for one month, indicating that oral probiotics can be used as an adjuvant therapy for acne vulgaris by regulating the inflammatory response. Rahmayani et al found that patients who received Lactobacillus rhamnosus SP1-containing fluids restored expression of genes associated with insulin signaling and improved acne appearance compared with patients who received fluids without the strain. Although the World Health Organization stipulates that only live microorganisms can be considered probiotics, there is evidence that heat-killed probiotics can also exert immunomodulatory effects. Therefore, the use of probiotic preparations as active ingredients to prevent and treat acne has become a research hotspot in recent years. Postbiotics prepared by 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 and economic value.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a new Lactiplantibacillus plantarum. The provided Lactiplantibacillus plantarum is isolated from kimchi fermentation broth, which can reduce skin inflammation, improve skin health, and prevent and relieve acne symptoms.

The Lactobacillus plantarum provided by the present invention is the Lactiplantibacillus plantarum 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 plant lactobacillus 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 Figure 3; its MALDI-TOF ribosomal protein molecular weight map is shown in Figure 4; its RAPD fingerprint is shown in Figure 5; The PCR fingerprints are shown in Figure 6.

The Lactobacillus plantarum provided by the present invention can be used for preparing functional food, health care product, medicine or skin care product.

The Lactobacillus plantarum provided by the invention can be used to prepare a product with antioxidant function.

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

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

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

The Lactobacillus plantarum VHProbi E15 strain provided by the 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. No growth, the maximum tolerated salt concentration is 7%, it can ferment glucose to produce acid without gas; cholesterol degradation rate is 16.24%; skin cell adhesion is 2.65. It was confirmed by coagglutination test and inhibition zone test that Lactobacillus plantarum VHProbi E15 could significantly inhibit P. acnes.

The acne-clearing essence cream prepared with Lactobacillus plantarum VHProbi E15 lysate as the only functional ingredient has been confirmed by human trials to effectively reduce the severity of mild to moderate acne, and can significantly reduce the acne area of patients after 4 weeks of use. The person's skin pales. The rate of transepidermal water loss is significantly reduced, and skin oil is significantly reduced. In addition, the subject's pore area/AOI area (mm ² ), skin stratum corneum water content, and skin pH were also improved compared to baseline values. After 4 weeks of use, the anti-acne essence cream can effectively control oil and 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 to skin care products for treating acne, and has broad application prospects.

Description of drawings

Fig. 1 is a photograph of a single colony of E15 strain;

Fig. 2 is E15 strain API 50CH identification result map;

Fig. 3 is the fingerprint map of E15 strain Riboprinter;

Fig. 4 is E15 strain MALDI-TOF ribosomal protein fingerprint map;

Fig. 5 is the RAPD fingerprint map of E15 strain;

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

Figure 7 is a graph showing the results of a co-agglutination test between E15 strain and P. acnes;

FIG. 8 is a graph showing the results of the Oxford Cup test for the inhibition of Propionibacterium acnes by strain E15;

Fig. 9 is a graph showing an example of improvement in facial acne of a subject.

Detailed ways

The Lactobacillus plantarum VHProbi E15 provided by the present invention complies with the requirements of laws and regulations, and is identified as a newly discovered strain by the multiphase taxonomy identification. 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 compounding with prebiotics and/or other probiotics; it has important application value .

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

The screening method of the present invention is not limited to those described in the examples, and any known methods that can achieve the purpose of screening can be used. The screening descriptions in the examples are only for the description of the present invention, and do not limit the protection scope of the present invention. Modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all belong to the scope of the present invention.

The present invention will be described in detail below with reference to specific embodiments.

Example 1 Isolation and screening of Lactobacillus plantarum VHProbi E15

1. Preliminary screening

MRS agar medium was prepared, adjusted to pH 6.2-6.5, and autoclaved at 121 °C for 15 min.

Take 1 mL of kimchi fermentation broth, dilute it with sterile saline, put it in a sterile sample bag, beat and mix it with a homogenizer; take 100 μL of the mixed solution for gradient dilution, spread it on MRS agar medium, and anaerobicize it at 37°C Incubate for 48h, when the plate grows a single colony for microscopic examination. According to the microscopic examination results, the applicant screened out a total of 18 potential Lactobacillus strains, named E1, E2, ..., E13, E14, E15, E16, E17, E18, respectively.

2. Re-screening

Prepare 1 L of MRS liquid medium and sterilize it by autoclaving at 121°C for 15min. After the medium is cooled, add 3.2g of porcine mucosal pepsin, shake well to dissolve, and place it in a water bath at 37°C for 1 h to make an acid-resistant medium.

The 18 strains of Lactobacillus E1, E2,..., E13, E14, E15, E16, E17, and E18 obtained by screening were inoculated into the above acid-resistant medium at 6% inoculum, and anaerobic static culture at 37 °C. 48h, take the fermentation broth for bacterial count.

The results showed that the E15 strain had the largest amount of viable bacteria after re-screening in the acid-resistant medium, and the logarithmic value was as high as 7.28Log CFU/mL. This shows that the E15 strain has the highest acid resistance.

Example 2 Identification of strains

1. Identification of colony morphology

The E15 strain was inoculated on the MRS agar medium, and after anaerobic culture at 37°C for 24 hours, the single colony of E15 was milky white, the colony diameter was about 2-3mm, and the surface was moist and smooth and rounded. The colony photo is shown in Figure 1.

2. Identification of physiological and biochemical characteristics

The preparation of the inoculum in this example is as follows: under aseptic conditions, take an appropriate amount of fresh E15 bacterial solution, centrifuge at 5000 rpm/min for 5 min, wash twice with PBS buffer, resuspend the bacterial cells with the same volume of PBS buffer, and dilute 50 times as the inoculum.

2.1. Temperature tolerance test

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

The inoculum was inoculated into 10 mL of MRS liquid medium at 10% of the inoculum amount, and 5 mL of MRS liquid medium without inoculation was used as a control, and were placed in a 15°C constant temperature incubator for 7 days and a 45°C constant temperature incubator for 2 days. , and observe whether the bacterial solution becomes cloudy.

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 medium with a salt concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8% to a 96-well plate, respectively, for each salt concentration Do 3 parallels, and then add 10 μL of the inoculum, and the wells without inoculation are used as controls. Add 50 μL of autoclaved paraffin oil to each well to prevent evaporation of water during incubation. Incubate at a constant temperature of 37°C and observe whether the medium becomes turbid.

The results showed that the maximum tolerant salt concentration of E15 strain was 7%.

2.3. Carbon source metabolism test

The carbon source metabolism experiment was carried out on the E15 strain using the API 50CH reagent strip. For details of the experimental method and the interpretation of the results, please refer to the API 50CH kit instructions. The identification results of the E15 strain are: %ID=99 and T value=1, the API result is Lactobacillus plantarum, and the identification comment is excellent identification. The metabolic test results are shown in FIG. 2 .

2.4. Glucose acid production and gas production test

The medium formula used in the present embodiment is as follows:

Peptone 0.5g; Yeast Extract 0.3g; Tween 80 0.1mL; Salt Solution A 0.5mL; Salt Solution B 0.5mL; Sodium Acetate 0.5g; Glucose 2.5g; 2% Bromocresol Green (w/v) 0.05mL ; Distilled water 100mL; pH6.8～7.0.

Dispense the prepared culture medium into large test tubes containing inverted small test tubes, 3mL/tube, 121°C, and autoclave for 15min.

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

Salt solution B component: MgSO ₄ ·7H ₂ O 11.5g, MnSO ₄ ·2H ₂ O 2.4g, FeSO ₄ ·7H ₂ O 0.68g, dissolve in distilled water, and make up to 100mL.

Under sterile conditions, inoculate the medium with 10% of the inoculum, and use the medium without inoculation as a control, then seal the top with 2 mL of sterile liquid paraffin, and incubate at 37°C for 24 hours, observe the color of the medium with or without change.

The results showed that after culturing at 37°C for 24 hours, the 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 biological identification

3.1 16s rDNA gene sequence analysis

3.1.1. Genomic DNA extraction

The procedure was carried out with reference to the Genomex DNA Extraction Kit (Cat. No.: DP302).

3.1.2. 16s rDNA gene amplification

Primer sequence:

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 in the NCBI database, and the E15 strain was preliminarily determined to be Lactobacillus plantarum.

3.2 Riboprinter Fingerprint

Dip the purified single colony from the agar medium plate with a stick, put it into a sample tube with buffer, stir it with a hand stirrer to suspend it in the buffer, and then place the sample holder. After being inactivated in a heater, it was put into the Riboprinter system. After DNA preparation, membrane transfer, imaging detection and data processing, the bacterial identification results were obtained. The identification results showed that the E15 strain was Lactobacillus plantarum, and its Riboprinter fingerprint was shown in Figure 3.

3.3 MALDI-TOF-MS detection of strain ribosomal protein expression

According to the inoculation amount of 0.1%, fresh bacterial liquid was inoculated into MRS liquid medium, and after culturing at 37° C. and 150 rpm for 48 h, the bacterial cells were collected, washed with sterile water for 4 times, and dried on the surface. Then take a small amount of fresh cells and spread them evenly on the target plate in the form of a thin film, add 1 μL of lysis solution to cover the sample, and after drying, add 1 μL of matrix solution to cover the sample, after drying, put the sample target into the mass spectrometer for identification . The co-crystal film formed by the sample and the matrix is irradiated with a laser to ionize the protein in the sample. The ions are accelerated to fly through the flight pipeline under the action of an electric field of 10-20KV, and the molecular weight of the protein is detected according to the flight time to the detector. Using Autofms 1000 analysis software Autof Analyzer v1.0 to obtain protein fingerprints, the ion peaks of the main ribosomal proteins of E15 strain are: 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 a result control, electrophoresis was performed at 100V for 80 min, and finally the electrophoresis was detected by a gel imaging system. The RAPD fingerprint of E15 strain is shown in Figure 5.

3.4.2. Fingerprint of rep-PCR

1) Primer sequence: CTACGGCAAGGCGACGCTGACG.

2) The reaction system of rep-PCR

Table 2: Reaction system table of rep-PCR

3) Electrophoresis

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

3.5 Whole genome sequencing

Take fresh E15 bacterial solution and inoculate it into 500 mL MRS broth medium according to the volume ratio of 1%, cultivate at 37° C. for 20 h, centrifuge at 8000 rpm for 10 min, and collect the bacterial cells. The bacteria were sent to the sequencing center to obtain the whole genome sequence of the bacteria. The whole genome sequence was uploaded to NCBI gene database, GenBank accession number was CP094961-CP094962.

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

Embodiment 3 Tolerance test of plant lactobacillus VHProbi E15 to artificial gastric juice and artificial intestinal juice

1. Preparation of artificial gastric juice

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

2. Preparation of artificial intestinal juice

Weigh 5 g of peptone, 2.5 g of yeast extract, 1 g of glucose, 6.8 g of KH ₂ PO ₄ and 3.0 g of bovine bile salts, respectively, add 77 mL of 0.2 mol/L NaOH solution, make up to 1000 mL, and add dilute hydrochloric acid or sodium hydroxide. The solution was adjusted to pH 6.8±0.1 and sterilized at 115°C for 20min. Then add 1g of pancreatin before use, shake well to dissolve, and put it in a warm water bath for 1 hour in a 37°C water bath shaker to simulate human body temperature.

3. Test method

Take 2 mL of fresh bacterial liquid, centrifuge at 5000 rpm/min for 5 min to collect the bacterial cells, wash the bacterial cells three times with physiological saline, and resuspend with 2 mL of physiological saline as the inoculum. Take 1 mL of inoculum, add it to 24 mL of artificial intestinal juice, place it on a water bath shaker (200 rpm/min) at 37°C for 3 h, and sample 1 mL to detect the amount of viable bacteria.

The viable count method was used to measure the bacterial count according to the national standard "GB4789.35-2016 - Food Microbiological Inspection Lactic Acid Bacteria Inspection", and the viable bacterial count (Log CFU/mL) of this strain after being digested by artificial intestinal juice is shown in Table 3.

Table 3: Scale of viable bacteria after artificial gastrointestinal juice digestion

As can be seen from Table 3, the Lactobacillus plantarum VHProbi E15 screened by the present invention has only a small decrease in the amount of viable bacteria after digestion by artificial gastric juice, and the amount of viable bacteria only decreases by about 1.2Log after continuing to be digested by artificial intestinal juice, indicating that this bacterial strain is effective for artificial Gastrointestinal juices are well tolerated.

Example 4 Antibiotic tolerance test of Lactobacillus plantarum VHProbi E15

1. Antibiotic preparation: Ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline, and vancomycin were all prepared into a stock solution of 2048 μg/mL, and stored at -20°C for later use. When using, the stock solution was serially diluted 2-fold with BMS liquid medium to obtain the use solution, and the concentration of the gradient dilution was 1-1024 μg/mL in a total of 11 gradients.

2. Preparation of inoculum: Preparation of inoculum: Take an appropriate amount of fresh bacterial solution (24-48h, 40°C culture), centrifuge at 5000rpm for 5min, wash once with sterile saline, resuspend the bacteria with 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 micro-broth dilution method.

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

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

c. Take out the 96-well plate after shaking culture at 40°C for 48 hours, measure the OD600 value, and use the results of 48 hours to calculate the MIC value of antibiotics against the strain. The specific results are shown in Table 4.

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

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

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

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

2. Weigh peptone 10.0g beef extract 10.0g yeast extract 5.0g diammonium hydrogen citrate 2.0g glucose 20.0g, Tween 80 1.0mL, sodium acetate 5.0g magnesium sulfate 0.1g manganese sulfate 0.05g, dipotassium hydrogen phosphate 2.0 g, 1 g of bile salts, 1000 mL of distilled water to adjust the pH value to 7.3, sterilized at 115° C. for 30 min, and then added cholesterol solution to make the final concentration of cholesterol 0.1%. Inoculate the fresh bacterial liquid of Lactobacillus plantarum VHProbi E15 according to the inoculation amount of 0.1%, and incubate at 37°C for 48 hours, then take 0.2 mL of bacterial liquid, add 1.8 mL of absolute ethanol, mix well, stand still for 10 minutes, and centrifuge at 3000 rpm for 5 minutes. The supernatant was taken for the determination of cholesterol content. The cholesterol determination method is in accordance with GB/T5009.128-2003 <Determination of Cholesterol in Food>.

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

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

Caco-2 cells were seeded in a six-well plate at an inoculation amount of 2×10 ⁶ cells/well, and cultured in a carbon dioxide incubator for 24 hours for cell adhesion experiments;

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

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

Repeated washing with PBS 3 times to remove unadhered bacteria;

Add 500ul trypsin to digest for 3 minutes, then add 1.5mL cell culture solution to stop digestion, pipetting repeatedly, and collect the resulting solution into a sterile EP tube, and carry out 10 times, 100 times, 1000 times, and 10000 times of the collected solution. Serial dilution, plate count. At the same time, the cells in the blank group were counted. The adhesion ability of the tested strains was calculated according to the following formula:

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

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

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

1. Agglutination test

The cryopreserved Lactobacillus plantarum VHProbi E15 was taken, aseptically operated, streaked to MRS plate, and cultured at 37°C for 48-72h. P. acnes ATCC6919 and P. acnes ATCC11827 were inoculated into BHI broth medium and cultured anaerobic at 37°C for 3-4 days.

Take an 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 salt buffer, and then resuspend with the same buffer.

Take 300 μL of Lactobacillus plantarum suspension and add it to a 24-well microtiter plate, and then add 300 μL mixed suspension of P. Two controls and samples were set in parallel. The 24-well plate was placed on a microplate constant temperature shaker, 400 rpm, and incubated at room temperature for at least 24 h. During this period, the machine was stopped for 30 minutes every 30 minutes of vibration, and the initial orifice plate state and the orifice plate state at each shutdown were recorded by taking pictures to observe whether there was agglutination.

The results are shown in Figure 7: The phenomenon of mutual agglutination occurred after the co-incubation of Lactobacillus plantarum VHProbi E15 and Propionibacterium acnes for 30h10min. Thus, it was shown that Lactobacillus plantarum VHProbi E15 can effectively antagonize P. acnes and significantly inhibit its growth.

2. Inhibition zone test

The cryopreserved Lactobacillus plantarum VHProbi E15 was taken, aseptically operated, streaked to MRS plate, and cultured at 37°C for 48-72h. After a single colony was grown on the plate, it was aseptically picked into MRS broth medium, and cultured at 37°C for 24h. Take 1 mL of bacterial liquid, centrifuge at 5000 rpm for 5 min to obtain bacterial slurry, rinse the bacterial slurry twice with sterile water, and then reconstitute it with 2 mL of sterile 1% peptone solution for use.

P. acnes ATCC11827 and P. acnes ATCC6919 were inoculated into fortified Clostridium medium, 37 ° C, anaerobic culture for 48 h.

Lay the lower medium, configure 1.5% agar solution, pour it into the plate after sterilization, and cover the plate. After the agar has solidified, place an appropriate number of sterile Oxford cups evenly. Overlay medium, upper semi-solid medium: fortified Clostridium medium with 0.7% agar and 1% Tween-80, followed by slight heating to dissolve Tween-80. Sterilize at 121°C for 15 minutes, and then cool down until it is not hot to the touch (about 45°C).

Two strains of P. acnes cultured for 48 hours were mixed in equal amounts, and 0.4% (v/v) was added to the upper semi-solid medium after thorough mixing. After mixing, 14 mL was poured onto the lower medium. After solidification, remove the Oxford cup and mark the added sample on the bottom of the plate. 100uL of bacterial cells + peptone solution was added to the well, and 1% peptone solution was added as a control. Antibacterial results were observed. After culturing for 72 h, the plate was taken out to observe whether there was a bacteriostatic zone, take pictures and measure the size of the bacteriostatic zone with a ruler.

The results are shown in Figure 8: obvious inhibition zones appeared on the plates, and the average diameter of the inhibition zones was 29.00±1.00 mm. This shows that Lactobacillus plantarum VHProbi E15 can significantly inhibit P. 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 to log phase using MRS broth;

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

The activated strains were added to the fermentation medium according to the inoculum amount of 1%, and left to ferment at 37°C for 24 hours; after the fermentation, the bacteria were collected in an ice bath, the walls were broken by ultrasonic for 30 minutes, and the supernatant was obtained by centrifugation; the supernatant was 75 Heating at ℃ for 15min, thermal inactivation was performed to obtain a lysate.

1.2 Preparation of Lactobacillus Acne Cleansing Essence Cream:

Referring to the formula described in Table 5, use deionized water to prepare the lactobacillus acne-clearing cream. Among them, Lactobacillus plantarum VHProbi E15 lysate with a content of 8% is the only functional component in the lactic acid bacteria cleansing cream.

Table 5. Formula of lactic acid bacteria clearing acne cream

2. Population trial scheme

Twenty healthy subjects aged 18-25 with mild or moderate acne on the face were selected. The subjects applied the Lactobacillus Acne Cleansing Cream every morning and evening after cleansing their face for a total of 4 weeks. And before using the product, and continuously using the product for 1 week, 2 weeks, 3 weeks, and 4 weeks, the circle beam photos were taken. Water loss rate, skin oil content and skin pH. The ambient temperature of this test was 20.4°C to 21.5°C, and the relative humidity was 47.7% to 51.9%.

3. Sample completion

3.1 Inclusion criteria:

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

(2) mild or moderate acne on both the left and right face;

(3) Be able to cooperate well with the experimenter and maintain the regularity of life during the study period;

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

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

(1) Those who have skin diseases on the face that may affect the judgment of the test results;

(2) Those with high allergies;

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

(4) Those with severe heart, liver and kidney function damage and severe immune dysfunction;

(5) People with mental illness, severe endocrine disease and oral contraceptives;

(6) Those who have participated in drug clinical trials or other trials within 30 days, or those who have used drugs that have an impact on the test results within the past 1 week;

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

(8) Those who cannot cooperate with the test;

(9) The researcher believes that it is not suitable to participate in the researcher;

(10) Other corresponding exclusion criteria.

Table 6 Subject Information Sheet

4. Test process

1) After the subject's face is cleansed and the water is absorbed with a paper towel, the subject sits for 20 minutes in the efficacy evaluation room at 21±1°C and relative humidity of 50±10%;

2) The technicians operate two instruments, CK-MPA and Visia-CR, to measure the basic value index of the subject's test site.

5. Statistical methods

Statistical analysis software was SPSS. The measured values at different time points were compared with the baseline values, and the level of α=0.05 was tested by repeated measures analysis of variance.

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

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

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

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

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

The improvement rate of using the product for 1 week = △1/W0*100%;

The improvement rate of using the product for 2 weeks = △2/W0*100%;

The improvement rate of using the product for 3 weeks = △3/W0*100%;

The improvement rate of using the product for 4 weeks = Δ4/W0*100%.

Among them, W0——the basic value of skin parameters before using the product in the test area;

W1——The skin parameter value of the product used in the test area for 1 week;

W2——The skin parameter value of the product used in the test area for 2 weeks;

W3——The skin parameter value of the product used in the test area for 3 weeks;

W4——The skin parameter value of the product used in the test area for 4 weeks.

6. Test results

6.1 The detection result of acne area

Table 7 The results of the detection of the area of acne

Table 8 Statistical analysis results of the proportion of acne areas in Visia-CR

*Significance labeling method: "n.s." means no statistical difference, P≥0.05; P<0.05 means significant difference ("*" means 0.01≤P<0.05; "**" means 0.001≤P<0.01; " ***” means P<0.001).

The smaller the acne area, the less obvious the redness. The percentage of acne area in subjects decreased significantly compared with the baseline value after 4 weeks of using the product, 59.1% of the subjects had a reduction in the percentage of acne areas in Visia-CR, of which 61.5% of subjects had Visia -CR pimple area reduced by 50%. The change in the proportion of acne in the subjects within 4 weeks is shown in Figure 9.

6.2 Skin color test results

Table 9 Skin color test result table

Table 10 Statistical analysis results of skin color

*Significance labeling method: "n.s." means no statistical difference, P≥0.05; P<0.05 means significant difference ("*" means 0.01≤P<0.05; "**" means 0.001≤P<0.01; " ***” means P<0.001).

The smaller the skin color value, the less noticeable the redness. After 4 weeks of product use, 22.7% of the subjects had lighter skin, but there was no significant difference.

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

Table 11 Pore area/AOI area (mm2) test result table

Table 12 Pore area/AOI area (mm2) statistical result table

*Significance labeling method: "n.s." means no statistical difference, P≥0.05; P<0.05 means significant difference ("*" means 0.01≤P<0.05; "**" means 0.001≤P<0.01; " ***” means P<0.001).

The smaller the pore area/AOI area (mm ² ), the smaller the number of pores. The subject's pore area/AOI area ( ^mm2 ) decreased in 31.8% of the subjects pore area/AOI area ( ^mm2 ) after 4 weeks of product use compared to basal, but there was no significant difference.

6.4 Test results of moisture content of skin stratum corneum

Table 13 Test result table of skin stratum corneum moisture content

Table 14 Statistical result table of moisture content of skin stratum corneum

*Significance labeling method: "n.s." means no statistical difference, P≥0.05; P<0.05 means significant difference ("*" means 0.01≤P<0.05; "**" means 0.001≤P<0.01; " ***” means P<0.001).

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

6.5 Transdermal Water Loss Test Test

Table 15 Test Table for Transdermal Water Loss Test

Table 16 Statistical result table of percutaneous water loss test

*Significance labeling method: "n.s." means no statistical difference, P≥0.05; P<0.05 means significant difference ("*" means 0.01≤P<0.05; "**" means 0.001≤P<0.01; " ***” means P<0.001).

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

6.6 Test results of oil and fat content

Table 17 Test result table of oil and fat content

Table 18 Statistical result table of oil content test

*Significance labeling method: "n.s." means no statistical difference, P≥0.05; P<0.05 means significant difference ("*" means 0.01≤P<0.05; "**" means 0.001≤P<0.01; " ***” means P<0.001).

The lower the oil content value, the less oily the skin is. After four weeks of using the product, the skin oil content of the subjects decreased significantly compared with the basic value (P<0.05), and 72.7% of the subjects showed a decrease in the skin oil content.

6.7 Skin pH balance

Table 19 pH test result table

Table 20 Statistical result table of pH detection

*Significance labeling method: "n.s." means no statistical difference, P≥0.05; P<0.05 means significant difference ("*" means 0.01≤P<0.05; "**" means 0.001≤P<0.01; " ***” means P<0.001).

Normal skin is weakly acidic, between 4.0-5.5. 22.7% of subjects experienced a decrease in skin pH after four weeks of product use compared to baseline, but the difference was not significant.

After 4 weeks of using lactobacillus acne-clearing cream in subjects with moderate to mild acne, the area of acne was significantly reduced (P<0.01), among which 59.1% of the subjects had a reduction in the area of Visia-CR acne , 61.5% of the subjects had more than 50% reduction in acne area. The transepidermal water loss rate of the subjects decreased significantly (P<0.05), 68.2% of the subjects had a reduced transepidermal water loss rate, and 66.7% of the subjects had a reduction of more than 20% in the transepidermal water loss rate. After four weeks of using the product, the skin oil content of the subjects decreased significantly compared with the basic value (P<0.05), and 72.7% of the subjects showed a decrease in the skin oil content. In addition, the subject's skin color, pore area/AOI area (mm ² ), skin stratum corneum water content, and skin pH were also improved compared to baseline values. This shows that the Lactobacillus plantarum VHProbiE15 lysate has an obvious effect on 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 Weilan Biological Co., Ltd. Qingdao Weilan Biological Group Co., Ltd.

<120> A Lactobacillus plantarum and its application in the prevention and treatment of 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

gtcaattcct ttgagtttca 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 acgtgttact caccagttcg ccactcactc 1380

aaatgtaaat catgatgcaa gcaccaatca ataccagagt tcgttcgact gc 1432

Claims (10)

1. The lactobacillus plantarum is characterized in that the preservation number of the lactobacillus plantarum is CCTCC NO: m2021594.

2. The lactobacillus plantarum of claim 1, wherein the 16s rDNA sequence of lactobacillus plantarum is SEQ ID No. 1.

3. The Lactobacillus plantarum of claim 1, wherein the Riboprinter fingerprint of Lactobacillus plantarum is shown in figure 3.

4. The lactobacillus plantarum of claim 1, wherein the MALDI-TOF ribosomal protein molecular weight profile of the lactobacillus plantarum is shown in fig. 4.

5. The lactobacillus plantarum of claim 1, wherein the RAPD fingerprint of the lactobacillus plantarum is as shown in figure 5; the rep-PCR fingerprint is shown in FIG. 6.

6. Use of the lactobacillus plantarum of claim 1 for the preparation of functional food, health care product, pharmaceutical product or skin care product.

7. Use of lactobacillus plantarum as defined in claim 1 for the preparation of a product with antioxidant function.

8. Use of lactobacillus plantarum as defined in claim 1 for the preparation of a product for the prevention or treatment of acne.

9. A preparation for preventing or treating acne, characterized in that it comprises the Lactobacillus plantarum of claim 1 and/or its fermentation product.

10. A preparation for preventing or treating acne, characterized in that it comprises a lysate of lactobacillus plantarum according to claim 1.

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