CN114657107B - Lactobacillus plantarum with acne treatment effect and application thereof - Google Patents

Abstract

The invention belongs to the technical field of screening and application of probiotics, and particularly relates to lactobacillus plantarum with an acne treatment effect and application thereof. The lactobacillus plantarum is separated from fresh infant feces, and the preservation number of the lactobacillus plantarum is CCTCC NO: m2021683 has remarkable inhibiting effect on the growth of Propionibacterium acnes, and can effectively improve skin health condition, and prevent and relieve acne symptoms. The acne-removing essence cream prepared by taking the lactobacillus plantarum lysate as the only functional component can effectively improve light and medium acne, reduce acne area and skin grease content, reduce percutaneous water diversion and increase skin moisture content, enable skin color to be normal, make pores thin and balance skin pH value through human body test verification. The lactobacillus plantarum has no toxic or harmful effect on organisms, can be added into skin care products, is used for preventing and treating acne, and has wide application prospect.

Description

A strain of Lactobacillus plantarum with acne treatment effect and its application

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

Acne is a chronic inflammatory disfiguring skin disease that often occurs in adolescents. After acne occurs, pigmentation, enlarged pores and even scars often occur, which puts great pressure on the patient's appearance and psychology. Studies have found that disorders in sex hormone secretion, the proliferation of Propionibacterium acnes in the sebaceous glands, abnormal keratinization of the sebaceous gland ducts and excessive secretion of sebum can all lead to acne. Current acne treatments include phototherapy, topical antibiotics or hormone ointments and oral Chinese medicine. Some existing treatments have limited effects, some have long treatment cycles, and the patients themselves do not pay attention to them, which makes acne worse and worse, seriously affecting the quality of life of patients and bringing great mental pressure to patients. Therefore, finding a simple, economical and effective acne treatment plan is of great significance to acne patients.

The skin is the largest organ of the human body and the first barrier separating the internal and external environments of the human body. There are about 10 ⁴ to 10 ⁹ microorganisms per cm ² on human skin, which can be divided into resident flora and transient flora according to the length of their colonization time. The resident flora can be regarded as the core flora of the skin. It has been colonized in the skin of an individual for a long time, has fully adapted to the skin environment, and has become dependent on the skin environment. This type of flora has a more direct impact on the skin. Hair follicles and sebaceous glands are the main habitats of skin microorganisms. Studies have also found that the colonization sites of skin microorganisms are not only the surface of the skin, but also traces of resident flora have been found in the dermis.

The World Health Organization/FAO defines probiotics as live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. Probiotics and their metabolites have been shown to interact with gut-associated lymphoid tissue, an interaction that is critical in helping the immune system respond to pathogens or allergens or commensal bacteria.

Probiotics represented by Lactobacillus and Bifidobacterium not only have the ability to inhibit the growth and reproduction of pathogens, but also have the ability to improve the host's own immunity and enhance the host's resistance to pathogenic bacteria. Therefore, as a new generation of antibacterial biological agents for skin diseases, probiotics have the potential to replace antibiotic therapy, making up for the shortcomings of increased drug resistance and high recurrence rate caused by antibiotic and hormone drug treatment. It is reported that probiotics can inhibit the cytokine IL-8 in epithelial cells and keratinocytes, reduce the inflammatory mediators produced by Propionibacterium acnes, and reduce vasodilation, edema, mast cell degranulation and TNF-α release. For adult acne, a phase 2 clinical trial report showed that the use of topical probiotic spray can significantly reduce the severity of acne and inflammatory damage. Other studies have also reported skin-related improvements brought about by the use of topical probiotic spray in acne patients. Therefore, the use of probiotic preparations as active ingredients to prevent and treat acne has become a research hotspot in recent years, and the development of probiotic strains with acne prevention and treatment effects has important social benefits and economic value.

Summary of the invention

The purpose of the present invention is to provide a new strain of Lactobacillus plantarum and its application. The provided Lactobacillus plantarum is separated from fresh feces of infants, can reduce skin inflammation, improve skin health, and prevent and alleviate acne symptoms.

The plant lactobacillus provided by the present invention is Lactobacillus plantarum VHProbi V22 (Lactiplantibacillus plantarum VHProbi V22), which was deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) on June 7, 2021, and its deposit number is CCTCC NO: M2021683.

The 16s rDNA sequence of the Lactobacillus plantarum VHProbi V22 strain is SEQ ID NO:1.

The Lactobacillus plantarum VHProbi V22 strain provided by the present invention has a MALDI-TOF ribosomal protein molecular weight map as shown in FIG3 ; a Riboprinter fingerprint map as shown in FIG4 ; a RAPD fingerprint map as shown in FIG5 ; and a rep-PCR fingerprint map as shown in FIG6 .

The Lactobacillus plantarum 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 present invention can be used to prepare products with antioxidant function;

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

The product is a skin care product or a functional food, medicine or health care product.

The present invention also provides a product for preventing or treating acne, which contains live bacteria of Lactobacillus plantarum VHProbi V22 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 V22 strain.

The plant lactobacillus VHProbi V22 screened and obtained by the present invention has strong tolerance to artificial gastrointestinal fluid and is sensitive to common antibiotics such as erythromycin and ampicillin; the strain can grow normally at 15°C, does not grow at 45°C, has a maximum tolerance salt concentration of 7%, and can ferment glucose to produce acid without producing gas. The plant lactobacillus VHProbi V22 has strong antioxidant capacity, and its fermentation supernatant has an anti-lipid peroxidation inhibition rate of 52.94%, and a DPPH clearance rate of 37.98%. The strain can effectively degrade cholesterol, and the degradation rate reaches 29.61%. The strain has a strong adhesion to skin cells, which is 2.75.

The plant lactobacillus VHProbi V22 of the present invention has a significant inhibitory effect on the growth of Propionibacterium acnes, and the average diameter of the inhibition zone is 26.33±0.58mm. The acne-clearing cream prepared by the applicant with the lysate of plant lactobacillus VHProbi V22 as the only effective ingredient has been confirmed by human trials to be able to effectively improve mild to moderate acne, reduce the acne area and skin oil content, reduce transepidermal water flow and increase skin moisture content, make the skin color tend to normal, the pores become finer, and balance the skin pH. After using the acne-clearing cream for 4 weeks, 70% of the subjects with mild to moderate acne had a reduced Visia-CR acne area, of which 78.6% of the subjects had a reduced Visia-CR acne area of 50%; 85% of the subjects had a reduced transepidermal water loss rate, of which 35.3% of the subjects had a reduced transepidermal water loss rate of more than 40%; 80% of the subjects had a decreased skin oil content. This shows that the lysate of Lactobacillus plantarum VHProbi V22 is significantly effective in improving the skin of patients with mild to moderate acne, and can achieve the effect of controlling oil and removing acne.

The Lactobacillus plantarum VHProbi V22 provided by the invention has no toxic effect on the body, can be added into skin care products, is used for preventing and treating acne, and has broad application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a photo of a single colony of the V22 strain;

Fig. 2 is a graph showing the test results of the API 50CH of the V22 strain;

FIG3 is a MALDI-TOF ribosomal protein fingerprint of the V22 strain;

Fig. 4 is the Riboprinter fingerprint of V22 strain;

Fig. 5 is the RAPD fingerprint of strain V22;

Fig. 6 is the rep-PCR fingerprint of strain V22;

FIG7 is a graph showing the results of a coaggregation test between the V22 strain and Propionibacterium acnes;

FIG8 is a graph showing the results of a V22 strain antibacterial test against Propionibacterium acnes;

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

DETAILED DESCRIPTION

The Lactobacillus plantarum VHProbi V22 provided by the present invention meets regulatory requirements, and is identified as a newly discovered strain by polyphasic taxonomy. The Lactobacillus plantarum VHProbi V22 provided by the present invention can effectively prevent and alleviate mild to moderate acne, and can alleviate acne by using the strain alone without being compounded with prebiotics and/or other probiotics; and has important application value.

On June 7, 2021, the applicant deposited the plant lactobacillus VHProbi V22 in the Chinese Center for Type Culture Collection of Wuhan University, and its preservation number is CCTCC NO: M2021683.

The screening method of the present invention is not limited to the examples, and any known method that can achieve the screening purpose can be used. The screening description of the examples 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 V22

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 1g of fresh infant feces, and the sampling process follows the ethical standards of biological sampling. After diluting with sterile saline, put it into a sterile sample bag and beat it with a homogenizer to mix it; take 100μL of the mixed solution for gradient dilution, spread it on MRS agar medium, and 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 25 potential lactobacilli, named V01, V02,..., V20, V21, V22, V23, V24, and V25.

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 25 strains of lactobacillus V01, V02, ..., V20, V21, V22, V23, V24 and V25 obtained by screening were inoculated into the above acid-resistant culture medium at an inoculum rate of 6%, and cultured anaerobically at 37°C for 48 hours. The fermentation liquid was taken for bacterial count.

The results showed that the V22 strain had the highest number of viable bacteria after rescreening with acid-resistant culture medium, with a logarithmic value of up to 8.22Log CFU/mL, indicating that the V22 strain had the highest acid resistance.

Example 2 Strain Identification

1. Colony morphology identification

The V22 strain was inoculated on MRS agar medium and cultured anaerobically at 37°C for 24 hours. The colony morphology is shown in Figure 1. The V22 single colony is white, with a colony diameter of about 1-2 mm, and a moist, smooth and round surface.

2. Identification of physiological and biochemical characteristics

The inoculation solution in this example was prepared as follows: Under sterile conditions, an appropriate amount of fresh V22 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 to serve 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 V22 strain multiplied rapidly at 15°C but did not grow at 45°C.

2.2 Carbon source metabolism test

The API 50CH reagent strip was used to perform a carbon source metabolism experiment on the V22 strain. The experimental method and result interpretation are detailed in the API 50CH kit manual. The identification results of the V22 strain were: %ID=99 and T value=0.97. The API result was Lactobacillus plantarum, and the identification comment was good. The carbon source metabolism results are shown in Figure 2.

2.3 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. Do 3 parallels for each salt concentration, 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 V22 was 7%.

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 V22 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 operation 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 V22 strain was obtained by sequencing, and the sequence was compared with the NCBI database, and the V22 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 V22 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 V22 strain were: m/z 5204.361, 7891.650, 3946.735, 5373.481, 7875.818, 3938.509, 2603.241, 2595.273, 4748.037, 7859.458. 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, and DL2000 DNA Marker was used as the result control. The electrophoresis was performed at a constant voltage of 100 V for 80 min, and finally the electrophoresis pattern was detected using a gel imaging system. The RAPD fingerprint of the V22 strain is shown in Figure 5.

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 the result control. The voltage was 100 V and the electrophoresis time was 80 min to detect the amplification results. The rep-PCR fingerprint of the V22 strain is shown in Figure 6.

3.5 Whole genome sequencing

Take fresh V22 bacterial liquid and inoculate it into 500mL MRS broth medium at a volume ratio of 1%, culture it at 37℃ for 20h, centrifuge it 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 CP095386-CP095389.

In summary, the colony morphology and physiological and biochemical characteristics of the V22 strain were uploaded to the website http://www.tgw1916.net/bacteria_logare_desktop.htmL, and compared with the results published in the literature De Clerck E, et al. Systematic and applied microbiology, 2004, 27 (1) 50. Based on the molecular biological identification results, it can be concluded that the V22 strain is a new plant lactobacillus, which is named plant lactobacillus VHProbi V22.

On June 7, 2021, the applicant deposited the Lactobacillus plantarum VHProbi V22 (Lactiplantibacillus plantarum VHProbi V22) in the Chinese Center for Type Culture Collection of Wuhan University, and its preservation number is CCTCC NO: M2021683.

Example 3 Antibiotic tolerance experiment of Lactobacillus plantarum VHProbi V22

1. Preparation of antibiotics: Ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline, and vancomycin were prepared into 2048μg/mL stock solutions and stored at -20℃ 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 1024μg/mL.

2. Preparation of inoculum: Preparation of inoculum: Take an appropriate amount of fresh bacterial culture (cultured for 24-48 hours at 40°C), 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 V22 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 the 2nd to 12th columns, 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 3.

Table 3: Antibiotic MIC values of Lactobacillus plantarum VHProbi V22

MIC unit: μg/mL

It can be seen from the results in Table 3 that the Lactobacillus plantarum VHProbi V22 provided by the present invention is sensitive to common antibiotics such as erythromycin and ampicillin, and has good biosafety.

Example 4 Tolerance test of Lactobacillus plantarum VHProbi V22 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 4.

Table 4: Table of live bacteria after digestion of artificial gastrointestinal fluid

As can be seen from Table 4, after the Lactobacillus plantarum VHProbi V22 screened by the present invention was digested with artificial gastrointestinal fluid, the amount of live bacteria increased slightly, indicating that the strain can tolerate artificial gastrointestinal fluid and can germinate.

Example 5 Determination of the antioxidant function of Lactobacillus plantarum VHProbi V22

1. The strain removes DPPH (1,1-diphenyl-2-trinitrophenylhydrazine)

Take 1mL of PBS bacterial suspension of the strain to be tested, add 1mL of 0.4mM DPPH free radical solution, mix well and then place it at room temperature for 30min under light shielding, then measure the absorbance of the sample at a wavelength of 517nm A sample, and measure 3 times in parallel. The control group sample is adjusted to zero with an equal volume of PBS solution and DPPH·ethanol mixture, and an equal volume of PBS bacterial suspension and ethanol mixture blank. The clearance rate is calculated according to the following formula: Clearance rate % = [1-(A _sample -A _blank )/A _control ] × 100%. The results are shown in Table 5.

Table 5: DPPH free radical scavenging rate table

2. Identification of strains against lipid peroxidation

Preparation of linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water. Add 1mL linoleic acid emulsion, 1mL FeSO ₄ (1%) to 0.5mL PBS solution (pH 7.4), then add 0.5mL sample, incubate at 37℃ for 1.5h, add 0.2mL TCA (4%), 2mL TBA (0.8%) to the mixture, incubate at 100℃ for 30min, cool rapidly, centrifuge at 4000rpm/min for 15min, collect the supernatant and measure the absorbance at 532nm as A; the control group uses 0.5mL distilled water instead of sample as A ₀ . Inhibition rate/% = (A ₀ -A)/A ₀ ×100%

Note: A is the absorbance of the sample group; _A0 is the absorbance of the control group. The results are shown in Table 6.

Table 6: Anti-lipid peroxidation inhibition rate table

Example 6 In vitro cholesterol degradation experiment with Lactobacillus plantarum VHProbi V22

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 potassium dihydrogen 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 at 0.1% inoculum, 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. 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 V22 provided by the present invention reached 29.61% (this is the data without bile salt).

Example 7 Caco-2 cell adhesion test of Lactobacillus plantarum VHProbi V22

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 to digest for 3 minutes, then add 1.5mL of cell culture medium to stop digestion, repeatedly blow and collect the resulting solution into a sterile EP tube, and dilute the collected solution 10 times, 100 times, 1000 times, 10000 times, and count the plates. 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 bacteria adhered to each culture well/total number of cells in each culture well.

The results showed that the cell adhesion of Lactobacillus plantarum VHProbi V22 provided by the present invention was 2.75, and the standard deviation was 0.32.

Example 8 Test on the inhibition of pathogenic bacteria by Lactobacillus plantarum VHProbi V22

1. Agglutination test

Take the frozen Lactobacillus plantarum VHProbi V22 and aseptically streak it onto the MRS plate and culture it at 37℃ for 48-72h. Inoculate Propionibacterium acnes ATCC6919 and Propionibacterium acnes ATCC11827 into BHI broth medium and culture it anaerobically at 37℃ for 3-4d.

Take appropriate amount of the activated fresh bacterial liquid of Lactobacillus plantarum and Propionibacterium acnes, 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 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 showed that after co-incubation of Lactobacillus plantarum VHProbi V22 and Propionibacterium acnes for 18 hours, cross-agglutination occurred, as shown in Figure 7. The results showed that Lactobacillus plantarum VHProbi V22 could effectively inhibit the growth of Propionibacterium acnes, with significant antagonistic effect.

2. Inhibition zone test

Take the frozen lactic acid bacteria glycerol tube, aseptically streak it onto the MRS plate, and culture it at 37℃ for 48-72h. After a single colony grows on the plate, aseptically pick it into MRS broth and culture it at 37℃ for 24h. Take 1mL of bacterial solution and centrifuge it at 5000rpm for 5min to obtain bacterial slurry, rinse the bacterial slurry twice with sterile water, and then re-dissolve it with 2mL of sterile 1% peptone solution for standby use. Inoculate Propionibacterium acnes ATCC11827 and Propionibacterium acnes ATCC6919 into the enhanced Clostridium culture medium and culture it anaerobically at 37℃ for 48h.

Lay the bottom layer of culture medium, prepare 1.5% agar solution, sterilize and pour into the plate, 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 top 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 showed that inhibition zones appeared on all plates, as shown in Figure 8, and the average diameter of the inhibition zones was 26.33±0.58 mm. The results showed that Lactobacillus plantarum VHProbi V22 could significantly inhibit the growth of Propionibacterium acnes, with significant effects.

Example 9 Anti-acne efficacy test of Lactobacillus plantarum VHProbi V22 lysate

1.1 Preparation of Lactobacillus plantarum VHProbi V22 lysate

Lactobacillus plantarum VHProbi V22 was cultured in MRS broth to the logarithmic phase; the activated strain was added to a culture medium (2% brown sugar, 3% collagen peptide, 0.3% yeast powder, and 0.25% diammonium hydrogen phosphate) at an inoculum rate of 1%, and fermented at 37°C for 24 hours; the bacteria were collected in an ice bath, the cell walls were broken by ultrasound 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 7, deionized water was used to prepare the lactic acid bacteria anti-acne cream. The lysate of Lactobacillus plantarum VHProbiV22 is the only effective component in the lactic acid bacteria anti-acne cream, and its content is 8%.

Table 7: Formula of lactic acid bacteria anti-acne 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:

3.1.1 Healthy people, aged 18 to 45 years old

3.1.2 Mild or moderate acne on both sides of the face

3.1.3 Can cooperate well with the experimenter and maintain a regular life during the study

3.1.4 Be able to read and understand all the contents of the informed consent form and voluntarily sign the informed consent form

3.1.5 During the trial, you agree not to use any cosmetics, drugs or health products that may affect the results.

3.2 Exclusion criteria: Patients with any of the following conditions must be excluded from this study:

3.2.1 Those with facial skin diseases that may affect the judgment of the test results

3.2.2 People with severe allergies

3.2.3 Women who are pregnant, breastfeeding, or planning to become pregnant during the trial

3.2.4 Those with severe heart, liver, kidney damage and severe immunodeficiency

3.2.5 People with mental illness, severe endocrine disease, or taking oral contraceptives

3.2.6 Participants in drug clinical trials or other trials within the past 30 days, or those who have systematically used drugs that may affect the trial results within the past week

3.2.7 Have taken orally or applied externally beauty products that may affect the test results within 14 days

3.2.8 Those who cannot cooperate with the experiment

3.2.9 Those deemed by the researcher to be unsuitable for participation in this study

3.2.10 Other relevant exclusion criteria

Table 8: Subject information table

Serial number gender age Is the test completed? Is it included in statistics? S001 male twenty two yes yes S002 male 20 yes yes S003 female 18 yes yes S004 female 19 yes yes S005 female twenty three yes yes S006 female 18 yes yes S007 male 20 yes yes S008 female 19 yes yes S009 female twenty three yes yes S010 male 19 yes yes S011 female 20 yes yes S012 male twenty four yes yes S013 male twenty two yes yes S014 female 20 yes yes S015 male twenty three yes yes S016 male 19 yes yes S017 male 19 yes yes S018 male twenty one yes yes S019 female twenty one yes yes S020 female twenty two yes yes

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 test level of 0.05.

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 9: Acne area detection results

Table 10: 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 percentage, the less obvious the redness symptom. The acne area percentage of the subjects decreased significantly compared with the baseline value after using the product for 4 weeks (P<0.01). 70.0% of the subjects had a decrease in the Visia-CR acne area percentage, of which 78.6% of the subjects had a 50% decrease in the Visia-CR acne area. The changes in the acne area percentage of the subjects within 4 weeks are shown in Figure 9.

6.2 Skin color test results

Table 11: Skin color detection results

Table 12: 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, there was no significant difference in the subject's skin color compared to the baseline value.

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

Table 13: Pore area/AOI area (mm2) test results

Table 14: Statistical analysis results of pore area/AOI area (mm ² )

*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, 40.0% of the subjects had a decrease in pore area/AOI area (mm ^{2 )} compared to the baseline value, but there was no significant difference.

6.4 Skin stratum corneum moisture content test results

Table 15: Skin stratum corneum moisture content test results

Table 16: Statistical analysis results of skin stratum corneum moisture content

*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, 35.0% 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 17 Transepidermal water loss test results

Table 18 Statistical analysis 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<0.001). Among them, 85.0% of the subjects had a reduced transepidermal water loss rate, and 35.3% of the subjects had a reduced transepidermal water loss rate of more than 40%.

6.6 Oil content test results

Table 19 Oil content test results

Table 20 Statistical analysis results of oil content

*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 80% of the subjects had a decrease in skin oil content.

6.7 Skin pH Balance

Table 21 pH test results

Table 22 Statistical analysis results of pH

Normal skin is slightly acidic, with a pH between 4.0 and 5.5. After using the product for four weeks, 55.0% of the subjects showed a decrease in skin pH compared to the baseline value, but the difference was not significant.

In summary, after using the lactic acid bacteria acne essence cream for 4 weeks, the proportion of acne area of subjects with mild to moderate acne was significantly reduced, and the proportion of Visia-CR acne area of 70% of the subjects was reduced, of which 78.6% of the subjects had a 50% reduction in Visia-CR acne area; the subjects' skin color and pH were not significantly different from the baseline values; the transepidermal water loss rate of the subjects was significantly reduced, of which 85% of the subjects had a reduced transepidermal water loss rate, and 35.3% of the subjects had a reduced transepidermal water loss rate of more than 40%; the subjects' skin oil content was significantly reduced compared with the baseline value, of which 80% of the subjects had a reduced skin oil content. In addition, the subjects' pore area/AOI area ( ^mm2 ), skin stratum corneum water content, and skin pH were also improved compared with the baseline values. This shows that lactobacillus acne-clearing cream can effectively improve mild to moderate acne, reduce the area of acne and the oil content of the skin, reduce transepidermal water flow and increase the moisture content of the skin, normalize the skin color, narrow the pores, and balance the pH of the skin. It also further shows that the only active ingredient in the acne-clearing cream, Lactobacillus plantarum VHProbi V22 lysate, has a significant effect on improving the skin of patients with mild to moderate acne, and can achieve the effect of controlling oil and removing acne.

Sequence Listing

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

<120> A strain of Lactobacillus plantarum with acne treatment effect and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1475

<212> DNA

<213> Artificial Sequence

<400> 1

agacaggtta acacgtgcta tacatgcagt cgaacgaact ctggtattga ttggtgcttg 60

catcatgatt tacatttgag tgagtggcga actggtgagt aacacgtggg aaacctgccc 120

agaagcgggg gataacacct ggaaacagat gctaataccg cataacaact tggaccgcat 180

ggtccgagtt tgaaagatgg cttcggctat cacttttgga tggtcccgcg gcgtattagc 240

tagatggtgg ggtaacggct caccatggca atgatacgta gccgacctga gagggtaatc 300

ggccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt agggaatctt 360

ccacaatgga cgaaagtctg atggagcaac gccgcgtgag tgaagaaggg tttcggctcg 420

taaaactctg ttgttaaaga agaacatatc tgagagtaac tgttcaggta ttgacggtat 480

ttaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 540

cgttgtccgg atttattggg cgtaaagcga gcgcaggcgg ttttttaagt ctgatgtgaa 600

agccttcggc tcaaccgaag aagtgcatcg gaaactggga aacttgagtg cagaagagga 660

cagtggaact ccatgtgtag cggtgaaatg cgtagatata tggaagaaca ccagtggcga 720

aggcggctgt ctggtctgta actgacgctg aggctcgaaa gtatgggtag caaacaggat 780

tagataccct ggtagtccat accgtaaacg atgaatgcta agtgttggag ggtttccgcc 840

cttcagtgct gcagctaacg cattaagcat tccgcctggg gagtacggcc gcaaggctga 900

aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 960

tacgcgaaga accttaccag gtcttgacat actatgcaaa tctaagagat tagacgttcc 1020

cttcggggac atggatacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg 1080

ggttaagtcc cgcaacgagc gcaaccctta ttatcagttg ccagcattaa gttgggcact 1140

ctggtgagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc 1200

ccttatgacc tgggctacac acgtgctaca atggatggta caacgagttg cgaactcgcg 1260

agagtaagct aatctcttaa agccattctc agttcggatt gtaggctgca actcgcctac 1320

atgaagtcgg aatcgctagt aatcgcggat cagcatgccg cggtgaatac gttcccgggc 1380

cttgtacaca ccgcccgtca caccatgaga gtttgtaaca cccaaagtcg gtggggtaac 1440

cttttaggaa ccagccgcct aagtagactt tccgg 1475

Claims (3)

1. A Lactobacillus plantarum, characterized in that the deposit number of Lactiplantibacillus plantarum is CCTCC NO: M2021683. 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.