CN114657106A - Lactobacillus plantarum and application thereof in preventing and treating acne - Google Patents
Lactobacillus plantarum and application thereof in preventing and treating acne Download PDFInfo
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- CN114657106A CN114657106A CN202210423122.6A CN202210423122A CN114657106A CN 114657106 A CN114657106 A CN 114657106A CN 202210423122 A CN202210423122 A CN 202210423122A CN 114657106 A CN114657106 A CN 114657106A
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Abstract
The invention provides a new lactobacillus plantarum, the preservation number of which is CCTCC NO: m2021594. The provided lactobacillus plantarum is separated from pickle fermentation liquor, and can relieve skin inflammation, improve skin health condition, and prevent and relieve acne symptoms. The lactobacillus plantarum VHProbi E15 strain provided by the invention has strong tolerance to artificial intestinal juice; the strain is sensitive to common antibiotics such as erythromycin, ampicillin and the like; can ferment glucose to produce acid without producing gas. The coaggregation test and the zone of inhibition test prove that the lactobacillus plantarum VHProbi E15 can generate obvious inhibition effect on the propionibacterium acnes. The acne-removing essence cream prepared by taking lactobacillus plantarum VHProbi E15 lysate as the only effective component is proved to be capable of effectively reducing the severity of moderate acne through human body tests. The lactobacillus plantarum VHProbi E15 provided by the invention has no toxic action on organisms, can be added into skin care products for treating acne, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of probiotic screening and application, and particularly relates to lactobacillus plantarum with an acne treatment effect and application thereof.
Background
Skin is an epithelial surface of the human body that interacts with microorganisms, and this surface is a dynamic interface, rather than a water impermeable barrier, and the skin's microbiome can extend to the dermis and dermal fat. The human skin is a unique environment and has a microorganism group different from other primates, and the skin microorganism group has the characteristics of high diversity, specific part and stability; particularly, the method has the characteristics that certain species are dominant, and the overall diversity degree is low. Microorganisms of healthy skin belong to 19 different phyla and 200 genera, while microorganisms of different parts of the body skin are different in composition, such as propionibacterium acnes, which is a common bacterium in skin areas with developed sebaceous glands, and staphylococcus and streptococcus, which are dominant in skin of different environments, such as dry skin areas; the common nature of skin microorganisms between different sites reflects the commonality between skin physiology.
After studying the pathogenesis of acne, researchers generally consider that the pathogenesis mainly comprises: endocrine dysfunction, increased androgen secretion causes increased sebaceous secretion from sebaceous glands; abnormal keratinization of the pilosebaceous canal leads to narrowing of the caliber, and obstruction of excretion of sebum and normal exfoliated cells; a topical pathogen infection. The current acne treatment drugs include retinoic acid, antibiotics, corticosteroid hormones, anti-androgen drugs, anti-seborrhea drugs, and physical therapy includes visible light and laser therapy. However, most of the existing therapies have the problems of high treatment cost, long treatment period and large toxic and side effects of medicines. Therefore, it is imperative to find a new acne relief regimen.
Probiotics are living microorganisms that, when administered in sufficient quantities, can confer health benefits on the host. The probiotics serving as a new generation of skin disease antibacterial biological agent has the potential of replacing antibiotic therapy, and can make up for the defects of drug resistance increase, high recurrence rate and the like caused by antibiotic and hormone drug therapy. The Fabbrocini et al reported that the level of IL-10, an anti-inflammatory cytokine, in serum, was significantly increased after one month of oral administration of the probiotic mixture to patients with acne vulgaris, indicating that oral administration of probiotic can act as an adjuvant therapy for acne vulgaris by modulating the inflammatory response. Rahmayani et al found that the expression of genes related to insulin signaling was restored and the appearance of acne was improved in patients taking a liquid containing Lactobacillus rhamnosus SP1 compared to patients taking a liquid without the strain. Although the world health organization specifies that viable microorganisms can be considered probiotics, there is evidence that heat-inactivated probiotics may also exert an immunomodulatory effect. Therefore, the use of probiotic preparations as active ingredients for the prevention and treatment of acne has become a research focus in recent years. The postbiotic prepared from the probiotics can be used as a potential treatment strategy for skin diseases, and has important social benefit and economic value in developing probiotic strains with the effect of preventing and treating acne.
Disclosure of Invention
The invention aims to provide a novel lactobacillus plantarum (Lactplantibibacillus plantarum), which is separated from pickle fermentation liquor and can relieve skin inflammation, improve skin health condition and prevent and relieve acne symptoms.
The lactobacillus plantarum provided by the invention is lactobacillus plantarum VHProbi E15 strain (Lactplantibacillus plantarius VHProbi E15), which has been preserved in China center for type culture collection at 24 months and 5 months in 2021, and the preservation number is CCTCC NO: m2021594.
The 16s rDNA sequence of the Lactobacillus plantarum VHProbi E15 strain is SEQ ID NO. 1.
The Riboprinter fingerprint of the Lactobacillus plantarum VHProbi E15 strain provided by the invention is shown in figure 3; the MALDI-TOF ribosomal protein molecular weight spectrum is shown in FIG. 4; the RAPD fingerprint is shown in figure 5; the rep-PCR fingerprint is shown in FIG. 6.
The lactobacillus plantarum provided by the invention can be used for preparing functional foods, health products, medicines or skin care products.
The lactobacillus plantarum provided by the invention can be used for preparing products with antioxidant function.
The lactobacillus plantarum provided by the invention can be used for preparing products for preventing or treating acne.
The invention also provides a product for preventing or treating acne, which comprises live bacteria of Lactobacillus plantarum VHProbi E15 strain and/or fermentation products thereof.
The invention also provides a product for preventing or treating acne, which comprises a lysate of lactobacillus plantarum VHProbi E15 strain.
The lactobacillus plantarum VHProbi E15 strain provided by the invention has strong tolerance to artificial intestinal juice; the strain is sensitive to common antibiotics such as erythromycin, ampicillin and the like; the strain can grow at 15 ℃, does not grow at 45 ℃, has the maximum tolerant salt concentration of 7 percent, and can ferment glucose to produce acid and produce no gas; the degradation rate of cholesterol is 16.24%; skin cell adhesion was 2.65. The coaggregation test and the zone of inhibition test prove that the lactobacillus plantarum VHProbi E15 can generate obvious inhibition effect on the propionibacterium acnes.
Human body tests prove that the acne-removing essence cream prepared by taking the lactobacillus plantarum VHProbi E15 lysate as the only effective component can effectively reduce the severity of moderate acne, obviously reduce the acne area of a patient after being used for 4 weeks, and lighten the skin color of a subject. The percutaneous water loss rate is obviously reduced, and the skin grease is obviously reduced. In addition, the pore area/AOI area (mm) of the subject2) The water content of the skin cuticle and the pH value of the skin are improved compared with the basic value. The acne-removing essence cream can effectively exert the effects of controlling oil and removing acne and repairing skin barriers after being used for 4 weeks.
The lactobacillus plantarum VHProbi E15 provided by the invention has no toxic action on organisms, can be added into skin care products for treating acne, and has wide application prospect.
Drawings
FIG. 1 is a photograph of a single colony of the E15 strain;
FIG. 2 is a graph showing the result of identifying API 50CH of E15 strain;
FIG. 3 is a Riboprinter fingerprint of E15 strain;
FIG. 4 is a MALDI-TOF ribosomal protein fingerprint spectrum of E15 strain;
FIG. 5 is a RAPD fingerprint of E15 strain;
FIG. 6 is a rep-PCR fingerprint of E15 strain;
FIG. 7 is a graph showing the results of a coaggregation test of E15 strain with Propionibacterium acnes;
FIG. 8 is a graph showing the results of the Oxford cup test for inhibition of Propionibacterium acnes by E15 strain;
fig. 9 is an illustration of improvement in facial acne in a subject.
Detailed Description
The lactobacillus plantarum VHProbi E15 provided by the invention meets the requirements of regulations, and is identified by heterogeneous taxonomy, so that the lactobacillus plantarum VHProbi E15 is a newly discovered strain. The lactobacillus plantarum VHProbi E15 provided by the invention can effectively prevent and relieve mild and moderate acne, and has an effect of relieving acne by singly using the strain without compounding with prebiotics and/or other probiotics; has important application value.
The lactobacillus plantarum VHProbi E15 is preserved in China center for type culture Collection of Wuhan university at 24/5/2021 by the applicant, and the preservation number is CCTCC NO: m2021594.
The screening method of the present invention is not limited to the examples, and any known method capable of achieving the screening purpose is possible, and the screening description of the examples is only illustrative of the present invention and is not limiting the scope of the present invention. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and scope of the invention.
The present invention will be described in detail with reference to specific examples.
Example 1 isolation screening of Lactobacillus plantarum VHProbi E15
1. Preliminary screening
Preparing MRS agar culture medium, adjusting pH to 6.2-6.5, and autoclaving at 121 deg.C for 15 min.
Taking 1mL of pickle fermentation liquor, diluting the pickle fermentation liquor by using sterile normal saline, putting the pickle fermentation liquor into a sterile sample bag, beating the pickle fermentation liquor by using a homogenizer and uniformly mixing the pickle fermentation liquor; and (3) taking 100 mu L of the uniformly mixed solution, diluting in a gradient manner, coating the uniformly mixed solution on an MRS agar culture medium, performing anaerobic culture at 37 ℃ for 48h, and performing microscopic examination on a single colony grown on a plate. According to the microscopic examination result, the applicant screens 18 potential lactobacilli in total, and the potential lactobacilli are named as E1, E2, … …, E13, E14, E15, E16, E17 and E18.
2. Double sieve
Preparing 1L MRS liquid culture medium, autoclaving at 121 deg.C for 15min, cooling, adding 3.2g pig mucosa pepsin, shaking for dissolving, and placing in 37 deg.C water bath shaker for 1 hr to obtain acid-resistant culture medium.
Respectively inoculating the 18 screened lactobacillus strains E1, E2, … …, E13, E14, E15, E16, E17 and E18 into the acid-resistant culture medium according to 6 percent of inoculation amount, carrying out anaerobic standing culture at 37 ℃ for 48h, and taking the fermentation liquor for counting the bacterial amount.
The result shows that the E15 strain has the maximum viable bacteria amount after being sieved again by an acid-resistant culture medium, and the logarithmic value is as high as 7.28Log CFU/mL. Thus indicating that the E15 strain has the highest acid resistance.
Example 2 Strain identification
1. Colony morphology identification
The E15 strain is inoculated on MRS agar medium, after anaerobic culture at 37 ℃ for 24h, the single colony of E15 is seen to be milk white, the diameter of the colony is about 2-3mm, the surface is moist, smooth and circular, and the picture of the colony is shown in figure 1.
2. Physiological and biochemical characteristic identification
The inoculation solution in this example was prepared as follows: under the aseptic condition, taking a proper amount of fresh E15 bacterial liquid, centrifuging for 5min at 5000rpm/min, washing for 2 times by using PBS buffer solution, then re-suspending the bacteria by using the same volume of PBS buffer solution, and diluting by 50 times to obtain inoculation liquid.
2.1 temperature resistance test
Taking a proper amount of fresh bacterial liquid (24h, 37 ℃), centrifuging for 5min at 5000rpm, washing with the PSP solution once, resuspending with the same volume of the PSP solution, and diluting by 50 times to obtain inoculation liquid.
Inoculating the inoculum solution into 10mL of MRS liquid culture medium according to the inoculation amount of 10%, using 5mL of MRS liquid culture medium without inoculation as a control, respectively culturing in a 15 ℃ constant-temperature incubator for 7 days, culturing in a 45 ℃ constant-temperature incubator for 2 days, and observing whether the bacterial solution becomes turbid.
The results showed that the E15 strain grew normally at 15 ℃ and did not grow at 45 ℃.
2.2 salinity tolerance test
Under sterile conditions, 190 μ L of BSM liquid medium with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% was added to 96-well plates, 3 replicates per salt concentration, followed by 10 μ L of inoculum solution, and wells without inoculum were used as controls. 50 μ L of autoclaved paraffin oil was added to each well to prevent evaporation of water during the culture. Culturing at 37 deg.C, and observing whether the culture medium turns turbid.
The results showed that the E15 strain had a maximum salt tolerance of 7%.
2.3 carbon source metabolism test
Carbon source metabolism experiments are carried out on the E15 strain by using the API 50CH reagent strip, and the details of the experimental method and the result interpretation refer to the specification of the API 50CH kit. The identification result of the E15 strain is as follows: the% ID is 99 and the T value is 1, the API result is lactobacillus plantarum, the identification comment is excellent identification, and the results of the metabolic test are shown in fig. 2.
2.4 acid and gas evolution test for glucose
The media formulations used in this example were as follows:
peptone 0.5 g; yeast extract 0.3 g; tween 800.1 mL; 0.5mL of salt solution A; 0.5mL of saline solution B; 0.5g of sodium acetate; 2.5g of glucose; 0.05mL of 2% bromocresol green (w/v); 100mL of distilled water; the pH value is 6.8-7.0.
The prepared culture medium is subpackaged into large test tubes containing inverted small test tubes, 3 mL/tube, and autoclaved for 15min at 121 ℃.
Salt solution composition A: KH (Perkin Elmer)2PO4 10g、K2HPO41.0g, dissolved in distilled water and fixed to 100 mL.
Salt solution B composition: MgSO (MgSO)4·7H2O 11.5g、MnSO4·2H2O 2.4g、FeSO4·7H20.68g of O, dissolving in distilled water, and fixing the volume to 100 mL.
Under aseptic conditions, inoculating the inoculum with 10% of inoculum size to culture medium, using non-inoculated culture medium as control, sealing the top with 2mL of sterile liquid paraffin, culturing at 37 deg.C for 24h, and observing whether the culture medium color changes.
The results show that: after culturing for 24h at 37 ℃, the culture medium turns from green to yellow, and no gas exists in the small inverted tube, which indicates that the E15 bacterial strain ferments glucose to produce acid and does not produce gas.
3. Molecular biological identification
3.116 s rDNA Gene sequence analysis
3.1.1 extraction of genomic DNA
Reference was made to the Tiangen bacterium genomic DNA extraction kit (catalog No.: DP 302).
3.1.2, 16s rDNA Gene amplification
The primer sequence is as follows:
27F：AGAGTTTGATCCTGGCTCA；
1492R：GGTTACCTTGTTACGACTT。
the 16s rDNA sequence SEQ ID NO 1 of the E15 strain is obtained by sequencing and is compared in an NCBI database, and the E15 strain is preliminarily determined to be the lactobacillus plantarum.
3.2 Riboprinter fingerprint
And (3) dipping the purified single colony from an agar culture medium plate by using a bacteria taking rod, putting the single colony into a sample tube with a buffer solution, stirring the single colony in the buffer solution by using a handheld stirrer to enable the single colony to be suspended in the buffer solution, putting a sample rack into a heater to inactivate, putting the sample rack into a Riboprinter system, and obtaining a bacteria identification result after DNA preparation, membrane conversion, imaging detection and data processing of the sample. The identification result shows that the E15 strain is Lactobacillus plantarum, and the Riboprinter fingerprint result is shown in FIG. 3.
3.3 MALDI-TOF-MS detection of ribosomal protein expression of strains
Inoculating fresh bacterial liquid into an MRS liquid culture medium according to the inoculation amount of 0.1%, culturing at 37 ℃ and 150rpm for 48h, collecting the thalli, washing with sterile water for 4 times, and airing the surface water. Then taking a small amount of fresh thalli to be evenly coated on a target plate in a film form, adding 1 mu L of lysate to cover the sample, airing, then adding 1 mu L of matrix solution to cover the sample, airing, and then putting the sample target into a mass spectrometer for identification. Irradiating a cocrystallized film formed by the sample and the matrix with laser to ionize protein in the sample, accelerating ions to fly through a flight pipeline under the action of a 10-20 KV electric field, and detecting the molecular weight of the protein according to different flight times of the ions reaching a detector. The protein fingerprint is obtained by using an Autof Analyzer v1.0 analysis software of Autofms 1000, and the ion peaks of the main ribosomal proteins of the E15 strain are as follows: m/z5201.363, 7887.113, 5734.785, 7303.595, 5185.929, 5421.786, 7878.099, 3943.720. The results of the identification are shown in FIG. 4.
3.4 RAPD and rep-PCR fingerprinting
3.4.1 RAPD fingerprint identification
1) The primer sequence is as follows: GAGGGTGGCGGTTCT is added.
2) RAPD reaction system
Table 1: RAPD reaction system table
3) Electrophoresis
Preparing a 1.5% agarose gel plate, using DL2000 DNA Marker as a result contrast, performing electrophoresis at a constant voltage of 100V for 80min, and finally detecting an electrophoretogram by using a gel imaging system. The RAPD fingerprint of E15 strain is shown in FIG. 5.
3.4.2 rep-PCR fingerprint
1) The primer sequence is as follows: CTACGGCAAGGCGACGCTGACG are provided.
2) rep-PCR reaction system
Table 2: rep-PCR reaction system table
3) Electrophoresis
DL2000 DNA Marker was used as a result control. The voltage is 100V, and the electrophoresis time is 80min to detect the amplification result. The rep-PCR fingerprint of the E15 strain is shown in FIG. 6.
3.5 Whole genome sequencing
Inoculating fresh E15 bacterial liquid into 500mL MRS broth according to the volume ratio of 1%, culturing at 37 deg.C for 20h, centrifuging at 8000rpm for 10min, and collecting thallus. The thallus is sent to a sequencing center to obtain the whole genome sequence of the thallus. The whole genome sequence was uploaded to the NCBI Gene database at GenBank accession numbers CP094961-CP 094962.
The results of colony morphology and physiological and biochemical characteristics of the E15 strain were uploaded to http:// www.tgw1916.net/bacterial _ log _ desktop. htmL, and compared with the results published in De Clerck E, et al. According to the identification result of molecular biology, the E15 strain is determined to be a new lactobacillus plantarum strain, and the new lactobacillus plantarum strain is named as lactobacillus plantarum VHProbi E15 (Lactplantibibacillus plantarum VHProbi E15).
Example 3 tolerance test of Lactobacillus plantarum VHProbi E15 to artificial gastric and intestinal juices
1. Preparation of artificial gastric juice
5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl were weighed, respectively, 1000mL of distilled water was added, pH3.0 was adjusted with dilute hydrochloric acid, and then sterilization was carried out at 115 ℃ for 20 min. Then 3.2g of pig mucosa pepsin is added before use, shaken up and dissolved, and placed in a water bath shaker at 37 ℃ for a water bath for 1h to simulate the temperature of a human body.
2. Preparation of artificial intestinal juice
Separately weighing peptone 5g, yeast extract 2.5g, glucose 1g, KH2PO46.8g and 3.0g of ox-gall salt, 77mL of 0.2mol/L NaOH solution is added, the volume is adjusted to 1000mL, the pH value is adjusted to 6.8 +/-0.1 by dilute hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized for 20min at 115 ℃. Then 1g pancreatin is added before use, shaken up and dissolved, and put into a water bath shaker at 37 ℃ for water bath for 1h to simulate the temperature of a human body.
3. Test method
2mL of fresh bacterial liquid is taken, centrifuged at 5000rpm/min for 5min to collect thalli, the thalli are washed for 3 times by using normal saline, and then 2mL of normal saline is used for resuspension to serve as inoculation liquid. Taking 1mL of inoculation liquid, adding the inoculation liquid into 24mL of artificial intestinal juice, placing the artificial intestinal juice in a water bath shaker (200rpm/min) at 37 ℃ for 3h, sampling 1mL, and detecting the amount of live bacteria.
The viable bacteria counting method is used for measuring the bacterial quantity according to the national standard GB 4789.35-2016-food microorganism test lactobacillus test, and the viable bacteria quantity (Log CFU/mL) of the bacterial strain after being digested by artificial intestinal juice is shown in a table 3.
Table 3: live bacteria scale after artificial gastrointestinal fluid digestion
As can be seen from Table 3, the viable count of the Lactobacillus plantarum VHProbi E15 screened by the method is only slightly reduced after being digested by artificial gastric juice, and is only reduced by about 1.2Log after being continuously digested by the artificial intestinal juice, which indicates that the strain has stronger tolerance to the artificial intestinal gastric juice.
Example 4 antibiotic resistance test of Lactobacillus plantarum VHProbi E15
1. Preparing antibiotics: ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline, and vancomycin were all prepared into 2048 μ g/mL stock solutions, and stored at-20 deg.C for further use. When in use, the stock solution is serially diluted into a use solution by 2 times of BMS liquid culture medium, and the gradient dilution concentration is 1-1024 mu g/mL for 11 gradients.
2. Preparing an inoculation solution: preparing inoculation liquid: taking a proper amount of fresh bacterial liquid (24-48 h, 40 ℃ culture), centrifuging for 5min at 5000rpm, washing once with sterile normal saline, then re-suspending the bacteria with the same volume of normal saline, and diluting by 50 times to obtain inoculation liquid.
3. Minimal inhibitory concentration MIC of antibiotic against Lactobacillus plantarum VHProbi E15 was determined by broth dilution.
adding BMS liquid culture medium without antibiotics into the 1 st column of a 96 pore plate, using the BMS liquid culture medium as a negative control, sequentially adding 190 mu L of BMS liquid culture medium containing antibiotics with different concentrations into the 2 nd to 12 th columns, then respectively inoculating 10 mu L of the inoculation liquid, making 3 parallel pores, and using 1 pore without adding bacteria liquid as a blank.
b. Cover by adding 50 μ L of paraffin oil to prevent evaporation of water.
c. And taking out the 96-well plate after shaking culture at 40 ℃ for 48h, measuring the OD600 value, and counting the MIC value of the antibiotic to the strain by using the result of 48h, wherein the specific result is shown in Table 4.
Table 4: antibiotic MIC value (mu g/mL) of Lactobacillus plantarum VHProbi E15
The results in table 4 show that the lactobacillus plantarum VHProbi E15 provided by the invention is sensitive to common antibiotics such as erythromycin and ampicillin, and has good biological safety.
Example 5 Lactobacillus plantarum VHProbi E15 in vitro Cholesterol degradation experiment
1. Preparation of cholesterol micelle solution: accurately weighing 1g of cholesterol, dissolving in absolute ethyl alcohol, diluting to 100mL, and filtering and sterilizing with a 0.22-micron microporous filter membrane under aseptic conditions.
2. 10.0g of peptone and 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 801.0 mL of Tween, 5.0g of sodium acetate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate, 1g of bile salt and 1000mL of distilled water are weighed, the pH value is adjusted to 7.3, sterilization is carried out at 115 ℃ for 30min, and then cholesterol solution is added to ensure that the final concentration of cholesterol is 0.1%. Inoculating fresh bacterial liquid of lactobacillus plantarum VHProbi E15 according to the inoculation amount of 0.1%, performing static culture at 37 ℃ for 48h, taking 0.2mL of bacterial liquid, adding 1.8mL of absolute ethyl alcohol, mixing uniformly, standing for 10min, centrifuging for 5min at 3000 rpm, and taking supernatant for measuring the cholesterol content. Method for measuring cholesterol GB/T5009.128-2003< determination of cholesterol in food >.
The results show that: the degradation rate of the lactobacillus plantarum VHProbi E15 on cholesterol provided by the invention reaches 16.24% (this is data without bile salts).
Example 6 Caco-2 cell adhesion assay of Lactobacillus plantarum VHProbi E15
Caco-2 cells at 2X 106Inoculating the inoculation amount of cells/hole in a six-hole plate, and culturing for 24h in a carbon dioxide incubator for cell adhesion experiments;
the stationary phase strains were resuspended to 5X 10 with MRS medium7CFU/mL；
Adding 1mL of the strain into a six-hole plate with cells attached to the wall, and culturing for 2h in a carbon dioxide incubator;
washing with PBS for 3 times to remove non-adhered bacteria;
adding 500ul of pancreatin for digestion for 3 minutes, adding 1.5mL of cell culture solution to stop digestion, repeatedly blowing, collecting the obtained solution into a sterile EP tube, performing gradient dilution on the collected solution by 10 times, 100 times, 1000 times and 10000 times, and coating plates for counting. The cells of the blank group were counted simultaneously. The adhesive ability of the test strain was calculated according to the following formula:
capacity for attachment (CFU/cells) — total number of bacteria attached per culture well/total number of cells per culture well.
The results show that: the cell adhesion of the lactobacillus plantarum VHProbi E15 provided by the invention is 2.65, and the standard deviation is 0.44.
Example 7 inhibition assay of Propionibacterium acnes by Lactobacillus plantarum VHProbi E15
1. Agglutination test
Taking the lactobacillus plantarum VHProbi E15 subjected to cryopreservation, performing aseptic operation, streaking to an MRS plate, and culturing at 37 ℃ for 48-72 h. The propionibacterium acnes ATCC6919 and the propionibacterium acnes ATCC11827 are inoculated to BHI broth culture medium and are subjected to anaerobic culture at 37 ℃ for 3-4 days.
Respectively taking a proper amount of the activated fresh bacterial liquid, centrifuging at 6000rpm for 4min, concentrating by a proper multiple, washing with tris salt buffer solution for 2 times, and then resuspending with the same buffer solution.
Taking 300 mu L of the plant lactobacillus suspension, adding the plant lactobacillus suspension into a 24-hole enzyme label plate, adding 300 mu L of the propionibacterium acnes mixed suspension to be used as an experimental group, taking another equal amount of plant lactobacillus suspension and buffer solution to be mixed to be used as a control group, and setting each control and sample to be 2 parallels. The 24-well plate was incubated in a microplate shaker at 400rpm for at least 24h at room temperature with shaking. During the period, the machine is stopped for 30min every 30min of oscillation, the initial orifice plate state and the orifice plate state during the stop at each time are recorded by photographing, and whether the agglutination phenomenon occurs or not is observed.
The results are shown in FIG. 7: the lactobacillus plantarum VHProbi E15 and Propionibacterium acnes were co-incubated for 30h10min before cross-agglutination occurred. Therefore, the lactobacillus plantarum VHProbi E15 can effectively antagonize propionibacterium acnes and has an obvious inhibition effect on the growth of propionibacterium acnes.
2. Zone of inhibition test
Taking the lactobacillus plantarum VHProbi E15 subjected to cryopreservation, performing aseptic operation, streaking to an MRS plate, and culturing at 37 ℃ for 48-72 h. After the plate grows out of single bacterium, the plate is aseptically picked into MRS broth culture medium and is statically cultured for 24 hours at 37 ℃. And (3) centrifuging 1mL of bacterial liquid at 5000rpm for 5min to obtain bacterial sludge, rinsing the bacterial sludge for 2 times by using sterile water, and re-dissolving by using 2mL of sterile 1% peptone solution for later use.
The Propionibacterium acnes ATCC11827 and Propionibacterium acnes ATCC6919 were inoculated into a reinforced Clostridium medium and cultured anaerobically at 37 ℃ for 48 hours.
Spreading culture medium, preparing 1.5% agar solution, sterilizing, pouring into flat plate, and spreading. After the agar is solidified, a proper number of aseptic oxford cups are evenly placed. Laying an upper layer of culture medium, wherein the upper layer of semi-solid culture medium: the reinforced clostridial medium was supplemented with 0.7% agar and 1% tween-80, followed by slight heating to dissolve tween-80. Sterilizing at 121 deg.C for 15min, and cooling to no scald for use (about 45 deg.C).
And (3) mixing 2 strains of propionibacterium acnes cultured for 48 hours in equal amount, fully and uniformly mixing, adding 0.4% (v/v) of the mixture into the upper-layer semisolid culture medium, uniformly mixing, and pouring 14mL of the mixture onto the lower-layer culture medium. After solidification, the oxford cup was removed and the added sample was marked on the bottom of the plate. 100uL of the thallus + peptone solution was added to the well, and 1% peptone solution was added as a control. And (5) observing the bacteriostatic result. After culturing for 72h, the plate is taken out, whether the zone of inhibition exists or not is observed, the picture is taken, and the size of the zone of inhibition is measured by using a ruler.
The results are shown in FIG. 8: the plates all have obvious inhibition zones, and the average diameter of the inhibition zones is 29.00 +/-1.00 mm. Therefore, the lactobacillus plantarum VHProbi E15 can generate obvious inhibition effect on the Propionibacterium acnes.
Example 8 anti-acne efficacy test of Lactobacillus plantarum VHProbi E15 lysate
1.1 preparation of Lactobacillus plantarum VHProbi E15 lysate
Lactobacillus plantarum VHProbi E15 was cultured using MRS broth to log phase;
fermentation medium: 2% of brown sugar, 3% of collagen peptide, 0.3% of yeast powder and 0.25% of diammonium hydrogen phosphate;
adding the activated strain into a fermentation culture medium according to the inoculation amount of 1%, and standing and fermenting for 24 hours at 37 ℃; after fermentation, collecting thalli in ice bath, breaking the walls for 30min by ultrasonic waves, and centrifuging to obtain supernate; heating the supernatant at 75 deg.C for 15min, and performing heat inactivation to obtain lysate.
1.2 preparation of the lactobacillus acne-removing essence cream:
with reference to the formulation described in table 5, the lactic acid bacteria acne-removing cream was formulated with deionized water. Wherein, the lactobacillus plantarum VHProbi E15 lysate with the content of 8 percent is the only effective component in the lactobacillus pox clearing essence cream.
TABLE 5 lactic acid bacteria acne-removing essence formula table
2. Population test protocol
20 healthy subjects with 18-25 years of age and mild or moderate acne on the face were selected. The subjects used the lactic acid bacteria acne-removing essence cream after cleaning the face every morning and evening for 4 weeks. And photographs of the collar beam were taken before and after the product was continuously used for 1 week, 2 weeks, 3 weeks, and 4 weeks, respectively, and the moisture content of the stratum corneum, the rate of percutaneous moisture loss, the oil content of the skin, and the pH of the skin were measured before and after the product was continuously used for 2 weeks, and 4 weeks, respectively. The environmental temperature of the test is 20.4-21.5 ℃, and the relative humidity is 47.7-51.9%.
3. Sample completion
3.1 inclusion criteria:
(1) healthy people, the age of which is 18-45 years old;
(2) both the left and right faces are associated with mild or moderate acne;
(3) the device can be well matched with testers, and the regularity of life can be kept during the research period;
(4) all contents of the informed consent can be read and understood, and the informed consent can be voluntarily signed;
(5) during the test period, the use of any cosmetics, drugs and health products which have an influence on the results was not agreed. 3.2 exclusion criteria, patients with any of the following conditions must be excluded from the study:
(1) the facial skin diseases may affect the judgment of the test result;
(2) those with high allergic constitution;
(3) a female who is pregnant, lactating, or intended to be pregnant during the test;
(4) severe center of gravity, liver and kidney function damage and severe immunologic hypofunction;
(5) those with mental disorders, severe endocrine disorders, and oral contraceptives;
(6) the clinical testers or other testers of the medicine are involved in 30 days, or the testers with the medicine which has influence on the test result in about 1 week;
(7) within 14 days, there were cosmetic products that could have an effect on the test results, both orally and externally;
(8) the test person can not be matched;
(9) the investigator considered inappropriate for attending the investigator;
(10) other corresponding exclusion criteria.
Table 6 subject information table
4. Test procedure
1) Cleaning the face of a subject, sucking water with a paper towel, and standing in an efficacy evaluation room with a temperature of 21 +/-1 ℃ and a relative humidity of 50 +/-10% for 20 min;
2) the technical personnel operates the CK-MPA and Visia-CR instruments to measure the basic value index of the tested part of the tested person.
5. Statistical method
The statistical analysis software was SPSS. The measured values at different time points were compared with the base values and the repeated measures anova test level α was 0.05.
The improvement rate is the change rate before use, and the calculation formula is as follows:
Δ 2 ═ W2-W0 using product for 2 weeks;
Δ 3 ═ W3-W0 using product 3 weeks;
the improvement rate of the product used for 1 week is 100% delta 1/W0;
the improvement rate of 2 weeks using the product is 100% Δ 2/W0;
the improvement rate of using the product for 3 weeks is 100% delta 3/W0;
the improvement rate of the product used for 4 weeks was Δ 4/W0 × 100%.
Wherein, W0-skin parameter base value before product application in test area;
w1 — test area use product 1 skin parameter value;
w2 — test area use product 2 week skin parameter value;
w3 — subject area used product 3 week skin parameter value;
w4-test area using product 4-week skin parameter value.
6. The result of the detection
6.1 acne area detection result
TABLE 7 acne area test results table
TABLE 8 statistical analysis results of area ratio of Visia-CR acne
Significance labeling method: "n.s." means no statistical difference, P.gtoreq.0.05; p <0.05 indicates significant differences ("+" indicates 0.01 ≦ P < 0.05; "+" indicates 0.001 ≦ P < 0.01; "+" indicates P < 0.001).
The smaller the acne area fraction, the less pronounced the redness. The acne area percentage of the subjects was significantly reduced compared to the basal value after 4 weeks of product use, with 59.1% of the subjects having a reduced area percentage of Visia-CR acne, with 61.5% of the subjects having a 50% reduction in Visia-CR acne area. The change in specific area of whelk in 4 weeks is shown in figure 9.
6.2 skin color test results
Table 9 skin color test results table
TABLE 10 statistical analysis of skin color results Table
Significance labeling method: "n.s." means no statistical difference, P.gtoreq.0.05; p <0.05 indicates significant differences ("+" indicates 0.01 ≦ P < 0.05; "+" indicates 0.001 ≦ P < 0.01; "+" indicates P < 0.001).
The smaller the skin color number, the less pronounced the redness. There was a lightening of skin color in 22.7% of subjects after 4 weeks of product use, but no significant difference.
6.3Pore area/AOI area (mm)2) Test results
TABLE 11 pore area/AOI area (mm2) test results Table
TABLE 12 pore area/AOI area (mm2) statistics Table
Significance labeling method: "n.s." means no statistical difference, P.gtoreq.0.05; p <0.05 indicates significant differences ("+" indicates 0.01 ≦ P < 0.05; "+" indicates 0.001 ≦ P < 0.01; "+" indicates P < 0.001).
Pore area/AOI area (mm)2) The smaller the number of pores, the less. Pore area/AOI area (mm) of subject2) Compared to the basal value, there was 31.8% of the subject pore area/AOI area (mm) after 4 weeks of use of the product2) Reduced, but not significantly different.
6.4 test results of moisture content of stratum corneum
TABLE 13 moisture content test results of stratum corneum
TABLE 14 statistical results of skin stratum corneum moisture content Table
Significance labeling method: "n.s." means no statistical difference, P.gtoreq.0.05; p <0.05 indicates significant differences ("+" 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 water is present in the skin. There was 59.1% of subjects who had increased the water content of the stratum corneum compared to the basal value after 4 weeks of product use, but without significant difference.
6.5 transdermal Water loss test
TABLE 15 test table for percutaneous water loss test
TABLE 16 statistical results of the percutaneous Water loss test
Significance labeling method: "n.s." means no statistical difference, P.gtoreq.0.05; p <0.05 indicates significant differences ("+" indicates 0.01 ≦ P < 0.05; "+" indicates 0.001 ≦ P < 0.01; "+" indicates P < 0.001).
The lower the value of skin transdermal water loss, the better the skin barrier function. The transdermal water loss of the subjects was significantly reduced compared to the basal value after 4 weeks of use of the product (P < 0.05). Wherein 68.2% of subjects have reduced transdermal water loss rate, and 66.7% of subjects have reduced transdermal water loss rate by more than 20%.
6.6 oil content test results
TABLE 17 table of oil and fat content test results
TABLE 18 statistical results of the oil content test
Significance labeling method: "n.s." means no statistical difference, P.gtoreq.0.05; p <0.05 indicates significant differences ("+" indicates 0.01 ≦ P < 0.05; "+" indicates 0.001 ≦ P < 0.01; "+" indicates P < 0.001).
The smaller the oil content value, the less the skin oil content. The subjects had a significant decrease in skin oil content after four weeks of product use compared to baseline (P <0.05), with a 72.7% decrease in skin oil content in the subjects.
6.7 skin pH balance
TABLE 19 pH value test results table
TABLE 20 PH value test statistical results table
Significance labeling method: "n.s." means no statistical difference, P.gtoreq.0.05; p <0.05 indicates significant differences ("+" indicates 0.01 ≦ P < 0.05; "+" indicates 0.001 ≦ P < 0.01; "+" indicates P < 0.001).
Normal skin is weakly acidic and is between 4.0 and 5.5. The subjects showed a 22.7% decrease in skin ph after four weeks of product use compared to baseline, but did not have a significant difference.
After the subjects suffering from the mild and moderate acne use the lactobacillus acne-removing essence cream for 4 weeks, the acne area is remarkably reduced (P)<0.01), wherein there is a 59.1% reduction in Visia-CR whelk area in the subject, wherein 61.5% of the subjects have a more than 50% reduction in whelk area. The transdermal water loss rate of the subjects was significantly reduced (P)<0.05), 68.2% of subjects had a reduction in the rate of transdermal water loss, and 66.7% of subjects had a reduction in the rate of transdermal water loss of more than 20%. The skin oil content of the subjects was significantly reduced after four weeks of product use compared to the basal value (P)<0.05) in which a reduction in the oil content of the skin of 72.7% of the subjects occurred. In addition, skin color, pore area/AOI area (mm) of the subject2) The water content of the stratum corneum and the pH value of the skin are improved compared with the basic value. Therefore, the lactobacillus plantarum VHProbi E15 lysate has obvious effect on improving the skin of mild and moderate acne patients, and can achieve the effects of controlling oil and removing acnes.
Sequence listing
<110> Islands Ulva Biometrics group, Inc. of Islands Ulva Biometrics group
<120> lactobacillus plantarum and application thereof in preventing and treating acne
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1432
<212> DNA
<213> Artificial Sequence (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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| US20170224750A1 (en) * | 2014-08-13 | 2017-08-10 | Nestec S.A. | Lactobacillus plantarum cncm i-4026 preparations and skin health |
| CN107177522A (en) * | 2016-03-09 | 2017-09-19 | 北京大伟嘉生物技术股份有限公司 | One plant height activity forage plant lactobacillus and its cultural method and application |
| WO2019098752A1 (en) * | 2017-11-16 | 2019-05-23 | 씨제이제일제당 (주) | Antibacterial cosmetic composition comprising culture of lactobacillus plantarum |
| US20190344100A1 (en) * | 2016-06-21 | 2019-11-14 | Yun NV | Dermatological preparations for maintaining and/or restoring healthy skin microbiota |
| CN113186139A (en) * | 2021-06-21 | 2021-07-30 | 西北民族大学 | Lactobacillus plantarum LR002 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20170224750A1 (en) * | 2014-08-13 | 2017-08-10 | Nestec S.A. | Lactobacillus plantarum cncm i-4026 preparations and skin health |
| CN107177522A (en) * | 2016-03-09 | 2017-09-19 | 北京大伟嘉生物技术股份有限公司 | One plant height activity forage plant lactobacillus and its cultural method and application |
| US20190344100A1 (en) * | 2016-06-21 | 2019-11-14 | Yun NV | Dermatological preparations for maintaining and/or restoring healthy skin microbiota |
| WO2019098752A1 (en) * | 2017-11-16 | 2019-05-23 | 씨제이제일제당 (주) | Antibacterial cosmetic composition comprising culture of lactobacillus plantarum |
| CN113186139A (en) * | 2021-06-21 | 2021-07-30 | 西北民族大学 | Lactobacillus plantarum LR002 and application thereof |
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| CN116083315A (en) * | 2023-01-17 | 2023-05-09 | 江南大学 | Lactobacillus plantarum CCFM1296 with regulated and controlled host HA content and progeny thereof |
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