CN117448227B - Lactobacillus paracasei and application thereof in preventing and treating acne - Google Patents

Abstract

The invention discloses a strain of cheese bacillus paracasei (Lacticaseibacillusparacasei), which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20231510. Also disclosed is a microbial inoculum, the active ingredient of which comprises Lactobacillus paracasei (Lacticaseibacillusparacasei) LPH01 or a fermentation product thereof, preferably the microbial inoculum is a freeze-dried powder. Also discloses the application of the microbial inoculum in preparing a product for inhibiting propionibacterium acnes and the application in preparing a product for preventing and/or treating acnes.

Description

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

Technical Field

The invention relates to the technical field of microorganisms, and in particular to a strain of Lactobacillus paracasei and application thereof in preventing and treating acne.

Background Art

Acne vulgaris is a chronic inflammatory skin disease of the pilosebaceous unit, which is prone to occur in areas rich in sebaceous glands, such as the face, chest and back. It is often accompanied by various lesions such as papules, pustules, nodules, cysts and scars. It is one of the most common diseases in cosmetic dermatology. The incidence of acne can be as high as 85%, and it is common in adolescents, adults, and even children. Acne seriously affects the quality of life and physical and mental health of patients, leading to low self-esteem, anxiety, depression, and even an increased risk of suicide. Propionibacterium acnes is a Gram-positive anaerobic bacterium that usually resides in the hair follicles and sebaceous glands of the skin. The role of Propionibacterium acnes in the development of acne seems to be related to the production of enzyme activities involved in the degradation of host molecules. Propionibacterium acnes lipase has been considered to be one of the virulence factors involved in the pathogenesis of acne. When a large amount of sebum accumulates in the sebaceous glands, causing the pores to be blocked and forming an anaerobic environment, Propionibacterium acnes will use sebum as the main nutrient to proliferate in large quantities, and then produce lipase to decompose triglycerides in sebum into glycerol and free fatty acids. In addition, the occurrence of acne is related to the strain type of Propionibacterium acnes. Studies have found that the severity of acne is positively correlated with the abundance of Propionibacterium acnes, and the system type IA1 is more strongly associated with acne.

A series of preventive and therapeutic measures targeting the pathological factors of acne vulgaris have emerged in clinical practice. Although the existing strategies have improved the management of acne vulgaris, safety issues such as bacterial resistance and serious adverse reactions have brought great challenges to the use of traditional prevention and control measures. Antibiotics, as a conventional drug for the treatment of acne, can inhibit the reproduction of Propionibacterium acnes and reduce inflammation. However, long-term use of antibiotics can increase the risk of drug resistance and even destroy the skin microbiome and eliminate symbiotic bacteria that are beneficial to the skin. Therefore, in the context of increasing antibiotic resistance, it is necessary to find some safe and effective methods to inhibit Propionibacterium acnes or reduce the inflammatory response induced by Propionibacterium acnes to improve acne.

Summary of the invention

The object of the present invention is to solve the above problems and provide a strain of Lactobacillus paracasei LPH01, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20231510.

Another object of the present invention is to provide a bacterial agent, the active ingredient of which comprises the above-mentioned Lactobacillus paracasei LPH01 or its fermentation product; preferably, the bacterial agent is a freeze-dried powder.

Preferably, the bacterial agent is a fermentation product of Lactobacillus paracasei LPH01, and has a molecular weight > 10 KDa.

Preferably, the bacterial agent is obtained by ultrafiltration and classification of the fermentation broth obtained by fermenting the above-mentioned Lactobacillus paracasei LPH01.

Preferably, the method for preparing the fermentation product is: inoculating the activated bacteria into a liquid culture medium, culturing at 30-37°C to the logarithmic phase, and collecting the fermentation supernatant by centrifugation to obtain the fermentation product; preferably, the culture medium is MRS liquid culture medium.

Another object of the present invention is to provide the use of the above-mentioned Lactobacillus paracasei LPH01 or the above-mentioned bacterial agent in the preparation of a product for inhibiting Propionibacterium acnes.

Another object of the present invention is to provide the use of the above-mentioned Lactobacillus paracasei LPH01 or the above-mentioned bacterial agent in the preparation of a product for preventing and/or treating acne.

In the above-mentioned technical solution of application, the product is a functional food, a health product, a medicine or a skin care product.

In the above-mentioned technical solution of application, the fermentation product of Lactobacillus paracasei LPH01 inhibits the ability of Propionibacterium acnes to degrade triglycerides, and downregulates the expression of extracellular lipase-related genes GehA, PPA1796, PPA2105 and PPA1761.

The last object of the present invention is to provide a product, which is used to inhibit Propionibacterium acnes or to prevent and/or treat acne, and the active ingredient of the product comprises the above-mentioned Lactobacillus paracasei LPH01 or the above-mentioned bacterial agent; the product is a functional food, a health product, a medicine or a skin care product.

The beneficial effects of the present invention are:

The present invention provides a LPH01 strain of Lactobacillus paracasei, whose cell-free metabolites have the effect of effectively inhibiting Propionibacterium acnes, and the average diameter of the inhibition zone is 24.65±0.27mm, and the antibacterial effect is significant. The antibacterial active ingredients of LPH01 fermentation broth are heat-resistant, acid-resistant, and insensitive to proteases. After ultrafiltration and classification treatment of the LPH01 fermentation broth, the antibacterial effect of the molecular weight of ＞10KDa is the best, and the minimum inhibitory concentration for Propionibacterium acnes is 12.5mg/mL. In addition, the antibacterial substances in the LPH01 fermentation broth can also effectively inhibit the ability of Propionibacterium acnes to degrade triglycerides, and downregulate the expression of extracellular lipase-related genes. Therefore, the LPH01 strain provided by the present invention has broad application prospects in the prevention or treatment of common acne, and in the preparation of functional foods, health products, medicines or skin care products.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the inhibitory activity of Lactobacillus paracasei LPH01 against Propionibacterium acnes.

FIG. 2 shows the colony morphology (A) and Gram staining results (B) of strain LPH01.

FIG3 is a phylogenetic tree of strain LPH01.

FIG4 shows the minimum inhibitory concentration of strain LPH01.

Figure 5 shows the effect of heat treatment on the antibacterial effect of metabolites of strain LPH01.

Figure 6 shows the effect of pH treatment on the antibacterial effect of metabolites of strain LPH01.

Figure 7 shows the effect of enzyme treatment on the antibacterial effect of metabolites of strain LPH01.

FIG8 shows the relative molecular weight range of antibacterial substances in the fermentation broth of strain LPH01.

FIG. 9 shows the effect of the metabolites of strain LPH01 on the ability of Propionibacterium acnes to degrade triglycerides.

FIG. 10 is a quantitative analysis of the extracellular lipase gene of Propionibacterium acnes by the metabolites of strain LPH01.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

The experimental methods in the following examples are all conventional methods unless otherwise specified; the biological and chemical reagents used are all conventional reagents in the art and can be obtained commercially.

Example 1. Antibacterial experiment and identification of strain LPH01

1 Experimental Materials

Lactobacillus paracasei strain LPH01, Lactobacillus fermentans strain U22, Lactobacillus crispatus strain h11, and Lactobacillus plantarum strain S22: originated from the strain bank of Suo Huayi Laboratory, School of Food Science, Southwest University.

LPH01 was isolated from human feces of healthy adults.

LPH01 strain deposit information:

The Lactobacillus paracasei LPH01 strain was sent to the China Center for Type Culture Collection (CCTCC) for preservation in August 2023. The address of the preservation unit is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The preservation date is August 21, 2023; the preservation number is CCTCC NO: M 20231510; the classification name is: Lacticaseibacillusparacasei LPH01.

Propionibacterium acnes ATCC 6919: purchased from American Type Culture Collection (ATCC) and used as an indicator strain.

2 Experimental methods

2.1 Preparation and concentration of lactic acid bacteria fermentation broth

The activated lactic acid bacteria were inoculated into 200 mL of LMRS liquid culture medium at a 2% (V/V) inoculation rate, cultured at 37°C for 24 hours, centrifuged at 8000 rpm for 10 minutes at 4°C, discarded the bacteria, and the fermentation supernatant was collected. After rotary evaporation at 80 rpm for 30 minutes at 60°C, the supernatant was placed in a -80°C ultra-low temperature refrigerator for 24 hours. Then, the supernatant was placed in a vacuum freeze dryer for 48 hours to obtain a lyophilized powder of the lactic acid bacteria fermentation supernatant.

2.2 Screening of lactic acid bacteria that inhibit Propionibacterium acnes

The Oxford cup double-layer plate method was used to screen lactic acid bacteria that inhibit Propionibacterium acnes. 8 mL of 2% sterilized agar was added to the sterile plate. After solidification, 5 sterile Oxford cups were placed in the agar layer. Prepare a semi-solid BHI medium (containing 1% agar by volume), sterilize it, wait for it to cool to about 45°C, add the logarithmic phase Propionibacterium acnes suspension to the BHI semi-solid medium (the final concentration of the indicator bacteria is 1×10 ⁷ CFU/mL), mix well, and draw 15 mL of the medium containing the indicator bacteria onto the agar layer. After the semi-solid medium solidifies, remove the Oxford cup with sterile tweezers, open the lid of the plate, and place it in a sterile operating table for 0.5h to evaporate the water. Use a pipette to draw 100 μL of lactic acid bacteria fermentation supernatant (dissolve the lyophilized powder with sterile water and dilute it to 400 mg/mL, then filter and sterilize it with a 0.22 μm water filter membrane) and add it to the Oxford cup hole. After sealing the plate, place it in a 4°C refrigerator for 4 hours to allow the lactic acid bacteria fermentation supernatant to fully diffuse. Finally, place it in a constant temperature incubator at 37°C and culture it anaerobically for 48 hours. The diameter of the inhibition zone was determined by the cross method (parallel experiments were performed 3 times, and the average of the 3 measurements was calculated). A blank control was set up: 100 μL MRS medium (MRS liquid medium freeze-dried powder was dissolved in sterile water and diluted to 400 mg/mL, and then filtered and sterilized with a 0.22 μm water filter membrane. The lactic acid bacteria with the best antibacterial effect were screened for further testing.

2.3 Morphological identification

The activated strain was streaked on the MRS plate and cultured at 37°C for 24 hours, and the morphology of the single colony was observed and photographed. At the same time, a single colony was picked up with an inoculation loop and evenly smeared on a glass slide. After Gram staining, the bacterial morphology was observed under a microscope and photographed.

2.4 Biochemical identification with API50 kit

The carbohydrate identification kit (API 50CH) was used to identify the biochemical characteristics of the selected strains. The strains were inoculated in MRS liquid medium and cultured at 37°C for 24 hours. 2 mL of bacterial suspension was centrifuged at 4°C and 4000 rpm for 5 minutes. The bacterial supernatant was discarded, washed twice with sterile saline, and resuspended in 2 mL of sterile saline. 0.5 mL of bacterial suspension was inoculated into API 50CH liquid medium. After mixing evenly, 90 μL was inoculated into the reagent strip, sealed with 50 μL of sterile liquid paraffin, and finally 80 μL of sterile water was added to each groove of the culture box. The reagent strip was placed in the culture box and cultured at 37°C for 48 hours before observing the color change. The final results will be entered into the API LAB plus system for strain identification.

2.5 16S rDNA amplification and DNA sequencing

The DNA of the purified strain was extracted using a bacterial genomic DNA extraction kit. A 25 μL PCR reaction system was used, namely: 1 μL bacterial genomic DNA, 1 μL forward primer 27F (SEQ ID NO.1: 5'-AGAGAGTTTGGCTCAG-3'), 1 μL 1492R (SEQ ID NO.2: 5'-GGTTACTTGTTACGATT-3'), 12.5 μL 2×Taq PCR MasterMix, 9.5 μL ddH ₂ O. The PCR reaction program was: 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, 30 cycles in total; 72°C end extension for 5 min. After the PCR reaction was completed, a 1.5% agarose gel was used to test the DNA fragments amplified by PCR, and the reaction was performed at 105 V for 45 min. The gel plate after electrophoresis was placed on a gel imaging system for observation and photography. The successfully amplified samples were sent to BGI for sequencing, and the sequencing results were analyzed for homology comparison using the BLAST program in NCBI.

2.6 Construction of phylogenetic tree

Strains with 16S rDNA gene sequence homology greater than 99% with the test strain in Gene Bank and standard strains in Genome were used as reference sequences, and the neighbor-joining method in MEGA 5.0 software was used to construct a phylogenetic tree.

3 Results and analysis

3.1 Antibacterial activity of strain LPH01 against Propionibacterium acnes

The fermentation liquid obtained by fermenting Lactobacillus paracasei LPH01 preserved in this laboratory was further prepared into freeze-dried powder, and then its antibacterial activity against Propionibacterium acnes was determined. As shown in Figure 1, strains U22 and h11 had no inhibitory effect on Propionibacterium acnes, strain S22 showed a strong antibacterial effect (inhibition zone diameter of 10.57±0.35mm), and strain LPH01 had the best antibacterial effect on Propionibacterium acnes, with an inhibition zone diameter of 24.65±0.27mm. This shows that Lactobacillus paracasei LPH01 is a potential strain with antagonism to Propionibacterium acnes.

3.2 Morphological identification results of strain LPH01

After purification, strain LPH01 formed single colonies in MRS medium. The colony morphology was almost uniform, with a smooth and moist surface, convex, neat edges, round, and milky white (Figure 2A). After Gram staining, purple cell morphology was observed under a microscope, and it was determined to be Gram-positive bacteria (G+), which were rod-shaped and uniform in size under a microscope (Figure 2B).

3.316S rDNA gene sequencing analysis results

After the 16S rDNA PCR amplification of strain LPH01 was successful, sequencing was completed by Huada Gene Technology Co., Ltd. The bidirectional sequence of the 16S rDNA gene amplification product of LPH01 is shown in SEQ ID NO.3. The results of 16SrDNA homology analysis showed that the strain numbered LPH01 had a 100% homology with the known Lacticaseibacillusparacasei (Accession: MT515983.1) in the Gene Bank database. The strain LPH01 and Lactobacillus paracasei were clustered together with 100% bootstrap support rate, indicating that the strain LPH01 was most closely related to Lactobacillus paracasei. Therefore, based on the colony morphology, physiological and biochemical characteristics and its genetic characteristics, the bacterium was identified as Lacticaseibacillusparacasei and named Lacticaseibacillusparacasei LPH01. The established phylogenetic tree is shown in Figure 3.

Example 2: Study on the physicochemical properties of the fermentation broth of strain LPH01

1 Experimental Materials

Lactobacillus paracasei LPH01 strain: CCTCC NO:M 20231510.

Propionibacterium acnes ATCC 6919: purchased from American Type Culture Collection (ATCC) and used as an indicator strain.

2 Experimental methods

2.1 Determination of minimum inhibitory concentration (MIC)

The MIC of Lactobacillus paracasei LPH01 was determined by the two-fold dilution method to explore the inhibitory effect of its fermentation supernatant on Propionibacterium acnes. First, the lyophilized powder of the fermentation supernatant of strain LPH01 was diluted twice with sterile water, and the concentrations after dilution were 400, 200, 100, 50, 25, 12.5, and 6.25 mg/mL. 100 μL of fermentation supernatant of each concentration was added to a 96-well culture plate, and then 100 μL of indicator bacteria suspension was added (final concentration was 1×10 ⁷ CFU/mL). At the same time, a negative control (fermentation supernatant was added without indicator bacteria solution) and a positive control (indicator bacteria solution was added without fermentation supernatant) were set. After the 96-well culture plate was placed at 37°C for 48 hours, the absorbance at 600 nm was measured, and the lowest concentration with an inhibition rate ≥90% or no bacterial growth was taken as the MIC. The experiment was measured in parallel 3 times.

2.2 Effect of heat treatment on the antibacterial effect of lactic acid bacteria metabolites

The lyophilized powder of the fermentation supernatant of Lactobacillus paracasei LPH01 was diluted with sterile water to 25 mg/mL, divided into several portions, and heated at 60, 80, 100, and 121°C for 30 min, cooled to room temperature, and sterilized by filtration with a 0.22 μm water filter membrane. 100 μL of Propionibacterium acnes suspension (final concentration of 1×10 ⁷ CFU/mL) and 100 μL of fermentation broth after different heat treatments (final concentration of 12.5 mg/mL) were added to the 96-well plate. After anaerobic culture at 37°C for 48 hours, the absorbance at 600 nm was measured. The fermentation broth that had not been heat treated was used as a control to determine the effect of heat treatment on the antibacterial effect of the metabolites of strain LPH01.

2.3 Effect of pH treatment on the antibacterial effect of lactic acid bacteria metabolites

The lyophilized powder of LPH01 fermentation broth of Lactobacillus paracasei was diluted with sterile water to 25 mg/mL, and then divided into several portions. The pH value of the fermentation broth was adjusted to 2, 3, 4, 5, 6, 7, 8, and 9 with 1 mol/L NaOH or 1 mol/L HCl, and then sterilized by filtration with a 0.22 μm water filter membrane. 100 μL of Propionibacterium acnes suspension (final concentration of 1×10 ⁷ CFU/mL) and 100 μL of fermentation broth treated with different pH values (final concentration of 12.5 mg/mL) were added to 96-well plates. After anaerobic culture at 37°C for 48 hours, the absorbance at 600 nm was measured. The fermentation broth without pH adjustment was used as a control to determine the effect of pH treatment on the antibacterial effect of the metabolites of strain LPH01.

2.4 Effect of enzyme treatment on the antibacterial effect of lactic acid bacteria metabolites

The lyophilized powder of LPH01 fermentation broth of Lactobacillus paracasei was diluted to 25 mg/mL with sterile water, and divided into several portions. The pH of the fermentation broth was adjusted to the optimal pH of different enzymes, and pepsin (pH 2.0), trypsin (pH 7.0), papain (pH 7.0), proteinase K (pH 7.0), alkaline protease (pH 9.0) and flavor protease (pH 7.0) were added respectively to make the final concentration of each protease 1.0 mg/mL. After incubation at 37°C for 2 hours, the fermentation broth was heated at 100°C for 5 minutes to inactivate the enzymes, and finally the pH was adjusted back to the initial pH of the fermentation broth (pH 4.0). Filter sterilization was performed with a 0.22 μm water filter membrane, and 100 μL of Propionibacterium acnes suspension (final concentration of 1×10 ⁷ CFU/mL) and 100 μL of fermentation broth treated with different enzymes (final concentration of 12.5 mg/mL) were added to 96-well plates. The 96-well culture plate was placed in 37°C anaerobic culture for 48 hours, and the absorbance at 600 nm was measured. The fermentation broth without enzyme treatment was used as a control to determine the effect of enzyme treatment on the antibacterial effect of the metabolites of strain LPH01.

3 Results and analysis

3.1 Minimum inhibitory concentration

As shown in Figure 4, when the concentration of the lyophilized powder of LPH01 fermentation broth was greater than 12.5 mg/mL, there was no bacterial growth, and the inhibition rate of Propionibacterium acnes exceeded 90%; when the concentration of the lyophilized powder of the fermentation broth was less than 12.5 mg/mL, the bacterial growth gradually increased, and the inhibitory activity against Propionibacterium acnes gradually decreased. The results show that the minimum inhibitory concentration of LPH01 fermentation broth against Propionibacterium acnes is 12.5 mg/mL.

3.2 Effect of heat treatment on the antibacterial effect of lactic acid bacteria metabolites

After the fermentation liquid of LPH01 of Lactobacillus paracasei was treated at different temperatures for 30 minutes, its inhibitory effect on Propionibacterium acnes was determined, and the fermentation liquid without heat treatment was used as the control. The results are shown in Figure 5. As the treatment temperature increases, the fermentation liquid still maintains a high inhibitory effect on Propionibacterium acnes. Even after high temperature treatment at 121°C, its inhibitory effect is not significantly different from that of the control group (p>0.05). The results show that the metabolites of LPH01 of Lactobacillus paracasei have certain high temperature resistance characteristics and strong thermal stability.

3.3 Effect of pH treatment on the antibacterial effect of lactic acid bacteria metabolites

The inhibitory effect of LPH01 fermentation broth of Lactobacillus paracasei on Propionibacterium acnes at different pH values is shown in Figure 6. LPH01 has good antibacterial activity in the pH range of 2 to 4. When the pH is 5, the antibacterial activity of LPH01 on Propionibacterium acnes decreases significantly, and the antibacterial activity gradually decreases with the increase of pH. When pH>6, it promotes the growth of Propionibacterium acnes. The results show that the metabolites of LPH01 are relatively stable in an acidic environment, and the antibacterial active substances are easily inactivated in an alkaline environment. It can be seen that the metabolites of LPH01 are sensitive to pH.

3.4 Effect of enzyme treatment on the antibacterial effect of lactic acid bacteria metabolites

Six representative proteases, including pepsin, trypsin, papain, proteinase K, alkaline protease and flavor protease, were selected to explore the sensitivity of Lactobacillus paracasei LPH01 fermentation broth to proteases. The results are shown in Figure 7. After the fermentation broth was treated with protease for 2 hours, its antibacterial activity against Propionibacterium acnes was not significantly different from that of the control group (p>0.05). The results show that the metabolites of Lactobacillus paracasei LPH01 are not easily degraded by proteases. Example 3. Mechanism of antibacterial action of the fermentation broth of strain LPH01 against Propionibacterium acnes

1 Experimental Materials

Lactobacillus paracasei LPH01 strain: CCTCC NO:M 20231510.

Propionibacterium acnes ATCC 6919: purchased from American Type Culture Collection (ATCC) and used as an indicator strain.

2 Experimental methods

2.1 Estimation of molecular weight of antibacterial substances in the fermentation broth of strain LPH01

The lyophilized powder of the fermentation supernatant of Lactobacillus paracasei LPH01 was diluted to 100 mg/mL with sterile water. 10 mL of sample solution was added to a 10KDa ultrafiltration centrifuge tube, and centrifuged at 4000 rpm for 40 min at 4 ° C to obtain fermentation broth components of >10KDa and <10KDa. Then the fermentation broth component <10KDa was repeatedly ultrafiltered with a 3KDa ultrafiltration tube to obtain fermentation broth components of 3-10KDa and <3KDa. The different components were collected in a 50mL centrifuge tube and placed at -80 ° C overnight. Then it was placed in a vacuum freeze dryer and dried for 48 hours, the lyophilized powder of the fermentation broth was collected and the mass of each component was determined. The lyophilized powder of the fermentation broth of each component was re-dissolved in sterile water to determine the inhibitory effect of different components of the fermentation broth of strain LPH01 on Propionibacterium acnes. The fermentation broth without fractionation was used as a control, and the experiment was measured in parallel 3 times. In addition, the MIC values of different components of the fermentation broth of Lactobacillus paracasei LPH01 were determined by the two-fold dilution method.

2.2 Effect of antibacterial substances in the fermentation broth of strain LPH01 on the ability of Propionibacterium acnes to degrade triglycerides

Collect P. acnes in the logarithmic growth phase, wash three times with PBS, and adjust the cell density to 2×10 ⁷ CFU/mL, and mix with equal volumes of LPH01 and subfractions with molecular weight >10KDa (final concentrations of 1/4MIC, 1/2MIC, and 1MIC). Add triglyceride standard to the bacterial suspension at 2% and incubate anaerobically at 37°C for 48h. After incubation, centrifuge the culture solution at 3000rpm for 10min at 4°C, and collect the supernatant. Take 10μL of supernatant, add 1mL of working solution, mix well, incubate at 37°C for 10min, and measure the triglyceride content at 500nm.

2.3 Quantitative analysis of the inhibitory substances produced by lactic acid bacteria on the extracellular lipase gene of indicator bacteria

In order to investigate the effects of the fermentation broth of strain LPH01 and subfractions with molecular weights greater than 10 KDa on P. acnes, P. acnes in the logarithmic growth phase were collected, washed three times with PBS, and the cell density was adjusted to 2×10 ⁷ CFU/mL. They were mixed with equal volumes of LPH01 and subfractions with molecular weights greater than 10 KDa (final concentrations of 1/4 MIC, 1/2 MIC, and 1 MIC), respectively, and incubated anaerobically at 37°C for 48 h. After the incubation, the culture broth was centrifuged at 8000 rpm for 10 min at 4°C to collect the bacteria. Total RNA was extracted using the Trizol reagent method and the concentration and purity were tested. Total RNA was reversed into cDNA according to the instructions of the cDNA reverse transcription kit, and then sequence amplification was performed using a 20 μL PCR system. The PCR reaction conditions were pre-denaturation at 95°C for 1 min; denaturation at 95°C for 20 s, annealing at 60°C for 20 s, and extension at 72°C for 30 s, for 45 cycles. The experiment used the 2- ^△△Ct method to calculate the relative content of the target genes in Table 1, and 16S RNA was used as the internal reference gene.

Table 1 Primer sequences

3 Results and analysis

3.1 Estimation of molecular weight of antibacterial substances in the fermentation broth of strain LPH01

The results are shown in Figure 8. The fermentation broth after ultrafiltration and fractionation still has antibacterial activity. Among them, the yield of the fermentation broth with a molecular weight of >10KDa is the highest (52.87%), followed by the fermentation broth with a molecular weight of 3-10KDa and <3KDa, with yields of 26.97% and 9.90%, respectively. The antibacterial effect of the fermentation broth subcomponents with different molecular weights was determined by re-dissolving them into the original system. The results showed that when the concentration of the fermentation broth of strain LPH01 was 25mg/mL, there was no significant difference in the antibacterial effect of the fermentation broth with a molecular weight of >10KDa and that of the fermentation broth without fractionation, and the antibacterial effect was significant, with a minimum inhibitory concentration of 12.5mg/mL. It can be seen that the molecular weight of the antibacterial substance in the fermentation broth of strain LPH01 is greater than 10KDa.

3.2 Effect of antibacterial substances in the fermentation broth of strain LPH01 on the ability of Propionibacterium acnes to degrade triglycerides

Sebum is mainly composed of triglycerides, glycerides, squalene and other cholesterols, among which triglycerides have the highest content, accounting for about 50%. Triglycerides can be decomposed by bacteria such as Propionibacterium acnes, and a large amount of free fatty acids are generated, which further stimulate the inflammatory response of hair follicles and surrounding areas, thereby causing acne. Therefore, Propionibacterium acnes was used as an indicator bacteria to explore the effect of antibacterial substances in the fermentation liquid of strain LPH01 on the ability of Propionibacterium acnes to degrade triglycerides. The experimental results are shown in Figure 9. Compared with the control group, 1/2-2MIC of strain LPH01 fermentation liquid and subfractions of >10KDa can significantly inhibit the degradation of triglycerides by Propionibacterium acnes (p<0.05). After treating Propionibacterium acnes with 1/4MIC of strain LPH01 fermentation liquid and subfractions of >10KDa, the degradation rate of triglycerides was not statistically significant compared with the control group (p>0.05). The results showed that the fermentation broth of strain LPH01 and the subfractions >10KDa could inhibit the growth of Propionibacterium acnes, further inhibit the conversion of triglycerides, reduce the production of free fatty acids, and alleviate inflammatory damage.

3.3 Quantitative analysis of the antibacterial substances in the fermentation broth of strain LPH01 on the extracellular lipase gene

Propionibacterium acnes lipase is considered to be one of the virulence factors involved in the pathogenesis of acne. It can change the lipid composition of human skin, induce the colonization of Propionibacterium acnes and the occurrence of acne. Lipase can decompose triglycerides in sebum into glycerol and free fatty acids. Fatty acids are highly inflammatory, chemotactic and irritating to sebaceous follicle cells. Therefore, inhibiting the expression of lipase-related genes is crucial for improving acne. The expression levels of Propionibacterium acnes lipase-related genes (GehA, PPA2105, PPA1761 and PPA1796) under the action of LPH01 fermentation broth and subfractions >10KDa were detected by qRT-PCR. The results are shown in Figure 10. At a concentration of 1/4MIC-1MIC, the fermentation broth of strain LPH01 and subfractions >10KDa can significantly inhibit the expression of GehA, PPA2105, PPA1761 and PPA1796 genes, and the difference is statistically significant compared with the control group (p<0.05). This finding suggests that LPH01 fermentation broth and >10KDa subfractions can effectively inhibit the expression of P. acnes lipase-related genes, thereby reducing P. acnes colonization and inflammation.

In summary, the present invention illustrates the antibacterial activity and mechanism of Lactobacillus paracasei LPH01 against Propionibacterium acnes, and conducts a preliminary exploration of antibacterial substances. The results show that Lactobacillus paracasei LPH01 has obvious antibacterial activity, and the antibacterial active ingredients of its fermentation broth are heat-resistant, acid-resistant, and insensitive to proteases. After ultrafiltration and classification of the fermentation broth of strain LPH01, the antibacterial effect of the molecular weight >10KDa is the best. In addition, the fermentation broth of strain LPH01 shows the ability to resist triglyceride degradation and inhibit the expression of lipase-related virulence factor genes. Therefore, in the food and pharmaceutical industries, strain LPH01 may be a natural antibacterial agent and has a certain effect on improving common acne.

Application Examples

Application Example 1: Preparation of facial mask essence using Lactobacillus paracasei LPH01 fermentation liquid

Weigh 0.05g disodium EDTA, 4g glycerol, 4g butylene glycol, 0.2g sodium hyaluronate, 0.5g niacinamide, 0.5g p-hydroxyacetophenone, 5g freeze-dried powder of Lactobacillus paracasei LPH01 fermentation broth and appropriate amount of water, add and stir in a certain proportion, and add 0.3g carbomer while stirring in a water bath until transparent to prepare a facial mask essence (100g). Biocellulose and Tencel fiber are selected as the carrier of the anti-acne mask.

Application Example 2: Preparation of anti-acne cream using Lactobacillus paracasei LPH01 fermentation liquid

Weigh 5.0 g of vaseline, 4.0 g of octadecyl alcohol, 1.5 g of monoglyceride, 3.0 g of paraffin oil and 0.5 g of sodium lauryl sulfate, add to an appropriate amount of distilled water, heat to above 80°C, stir to dissolve, cool, add 2.5 g of glycerol, 5 g of freeze-dried powder of Lactobacillus paracasei LPH01 fermentation liquid and an appropriate amount of fragrance, stir evenly to obtain an O/W type acne cream preparation (100 g).

Application Example 3: Preparation of anti-acne essence using Lactobacillus paracasei LPH01 fermentation liquid

Weigh 0.3 g of p-hydroxyacetophenone, 0.2 g of phenoxyethanol, 0.5 g of hexylene glycol, 2% butylene glycol, 0.02 g of disodium EDTA, 0.1 g of carbomer, 0.1 g of allantoin, 0.2 g of aminomethyl propanol, 5 g of freeze-dried powder of Lactobacillus paracasei LPH01 fermentation broth and appropriate amount of water to prepare an anti-acne essence (100 g).

Application Example 4: Preparation of acne patch using Lactobacillus paracasei LPH01 fermentation liquid

15 g of hydrogenated viscous resin, 20 g of thermoplastic elastomer, 5 g of freeze-dried powder of Lactobacillus paracasei LPH01 fermentation broth, 5 g of β-nicotinamide mononucleotide, 5 g of distilled peppermint essential oil, 5 g of melaleuca essential oil, 5 g of far-infrared mineral powder and 10 g of sodium carboxymethyl cellulose were weighed to prepare an anti-acne hydrocolloid, and finally the obtained product was cut, embossed, die-cut and packaged to finally obtain a prepared acne patch.

Application Example 5: Preparation of anti-acne cleanser using Lactobacillus paracasei LPH01 fermentation liquid

Weigh 5 g of sodium fatty alcohol polyoxyethylene ether sulfate, 3 g of coconut oil amidopropyl betaine, 15 g of alkyl glycoside, 1 g of ethylene glycol distearate, 0.2 g of allantoin, 0.2 g of disodium ethylenediaminetetraacetic acid, 0.4 g of freeze-dried aloe vera gel, 2 g of dimethyldiallylammonium chloride, 5 g of freeze-dried powder of Lactobacillus paracasei LPH01 fermentation broth, a small amount of preservatives, an appropriate amount of flavor and water to prepare an anti-acne cleanser (100 g).

The above description is only a specific embodiment of the present invention, but the features of the present invention are not limited thereto. Any changes or modifications that can be easily thought of by anyone familiar with the technology within the field of the present invention should be included in the scope of the patent application of the present invention.

Claims (10)

1. A strain of Lacticaseibacillus paracasei LPH01, deposited in China Center for Type Culture Collection with the deposit number of CCTCC NO:M 20231510. 2. A bacterial agent, the active ingredient of which comprises the Lactobacillus paracasei described in claim 1 ( Lacticaseibacillus paracasei )LPH01 or its fermentation broth. 3. The bacterial agent according to claim 2, characterized in that the bacterial agent is a freeze-dried powder. 4. The bacterial agent according to claim 2, characterized in that the bacterial agent is obtained by ultrafiltration and classification of the fermentation liquid obtained by fermenting the Lactobacillus paracasei according to claim 1. 5. The bacterial agent according to claim 2 or 3, characterized in that the fermentation broth is prepared by inoculating the activated bacteria into a liquid culture medium, culturing at 30-37°C to the logarithmic phase, and collecting the fermentation supernatant by centrifugation. 6. The bacterial agent according to claim 5, characterized in that the culture medium is MRS liquid culture medium. 7. Use of the Lacticaseibacillus paracasei LPH01 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a product for inhibiting Propionibacterium acnes. 8. Use of the Lacticaseibacillus paracasei LPH01 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a product for preventing and/or treating acne. 9. The use according to claim 8, characterized in that the product is a medicine or a skin care product. 10. A product for inhibiting Propionibacterium acnes or for preventing and/or treating acne, characterized in that the active ingredient of the product comprises the Lacticaseibacillus paracasei LPH01 according to claim 1 or the bacterial agent according to claim 2; the product is a medicine or a skin care product.