Lactic acid bacteria with azelaic acid endurance capability and skin soothing and repairing capability

Abstract

The invention discloses lactic acid bacteria with azelaic acid endurance capacity and skin soothing and repairing capacity, and belongs to the technical field of microorganisms. The method comprises the following steps: adaptively domesticating a plurality of lactic acid bacteria in an azelaic acid stress environment, and screening to obtain the lactobacillus plantarum with high-concentration azelaic acid tolerance, namely the lactobacillus plantarum CCTCC NO: M 20232390. Metabolite obtained through fermentation of the lactobacillus plantarum has the effects of resisting inflammation, relieving, repairing and the like on the human cell level, and meanwhile, the lactobacillus plantarum has good safety.

Description

Technical Field

[0001] The present invention relates to a lactic acid bacterium with both azelaic acid tolerance and skin soothing and repairing abilities, belonging to the field of microbial technology. Background Art

[0002] Azelaic acid is a saturated straight-chain dicarboxylic acid, which has antibacterial, anti-inflammatory, whitening, regulating skin keratinization, oil control and other effects. It can be used as the main raw material of functional cosmetics and also as a medicine for treating skin diseases such as acne and chloasma. However, the dosage of azelaic acid in functional cosmetics or medicines generally needs to reach 10-20%, which is followed by irritation to the skin. How to reduce and even eliminate the irritation of functional cosmetics to the skin has always been the focus of concern for cosmetics developers.

[0003] As a type of probiotic bacteria, lactic acid bacteria have been favored by many cosmetics brands in recent years. There are mainly two categories of lactic acid bacteria that can be applied in cosmetics. One category is Lactobacillus, such as Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus reuteri, etc.; the other category is Bifidobacterium, which is named in the form of Saccharomyces boulardii in the list of used cosmetics raw materials. The main active ingredients of Lactobacillus fermentation products are lactic acid, as well as proteins, enzymes, vitamins, other organic substances, etc. According to the big data of Meili XiuXing, its main effects are: moisturizing, removing spots and whitening, removing acne, and anti-aging. At present, there is no report on the soothing effect of the Lactobacillus fermentation product on the irritation of azelaic acid products to the skin and the repair of the skin.

[0004] To soothe the irritation of products containing azelaic acid, it is necessary to adaptively domesticate lactic acid bacteria in an environment containing azelaic acid, so that lactic acid bacteria express related active substances that resist the irritation of azelaic acid. When lactic acid bacteria are in an azelaic acid stress environment, they may initiate a series of stress response mechanisms to adapt to this adverse condition. In this process, lactic acid bacteria may produce substances that resist the irritation of azelaic acid, thereby alleviating the irritating effect of azelaic acid on the skin. By screening and cultivating strains with azelaic acid tolerance and skin soothing and repairing abilities, their fermentation products can be applied to cosmetic formulations to reduce the irritation of azelaic acid to the skin, reduce its potential risks to the skin, and contribute to improving the overall safety of products. Therefore, starting from natural lactic acid bacteria, by adaptively domesticating lactic acid bacteria in an azelaic acid environment, obtaining a lactic acid bacterium with both azelaic acid tolerance and skin soothing and repairing abilities has very realistic scientific research and commercial significance. Summary of the Invention

[0005] In order to solve the above problems, the present invention adaptively domesticates lactic acid bacteria in an environment containing azelaic acid, and through domestication and screening, a Lactobacillus plantarum ZW with azelaic acid tolerance and skin soothing and repairing abilities is obtained.

[0006] The first object of the present invention is to provide a Lactobacillus plantarum ZW, taxonomically named Lactobacillus plantarum ZW, which is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M20232390, the deposit date being November 29, 2023, and the deposit address being Wuhan University, Wuhan, China.

[0007] The Lactobacillus plantarum CCTCC NO: M 20232390 has the following characteristics:

[0008] (1) Colony characteristics: round, milky white, opaque, with a diameter of about 1 - 2 mm, smooth edges, and a moist and shiny surface;

[0009] (2) Morphological characteristics: short rod-shaped, Gram-positive staining, no flagella, and no spore production;

[0010] (3) Growth characteristics: the optimal fermentation pH is 5.5, the temperature is 37 °C, the fermentation cycle is short, the logarithmic growth phase starts at 6 h, and the stationary phase is entered after 10 h;

[0011] (4) This strain can grow normally in MRS medium containing 5% azelaic acid and can still survive at azelaic acid concentrations of 10% and 20%;

[0012] (5) The fermentation product of this strain has a significant anti-inflammatory effect and can significantly inhibit the expression of inflammatory factors such as NO, TNF-α, and IL-6 in RAW264.7 cells;

[0013] (6) The fermentation product of this strain has a significant soothing effect and can significantly reduce the calcium ion influx rate of HaCaT cells caused by capsaicin stimulation;

[0014] (7) The fermentation product of this strain has a significant ability to promote the migration of skin cells, and the migration rate increases with the increase of the addition amount.

[0015] In one embodiment, the Lactobacillus plantarum ZW is obtained by adaptive domestication and screening in an azelaic acid environment.

[0016] In one embodiment, the domestication and screening are specifically as follows:

[0017] (1) First, activate Lactobacillus plantarum CCTCC NO: M 20232390;

[0018] (2) Collect the bacterial cells, disperse them, pick the bacterial liquid and streak it on a solid medium containing a certain concentration of azelaic acid, and then incubate it statically;

[0019] (3) Pick a single colony and transfer it to a liquid medium containing a certain concentration of azelaic acid for acclimation culture;

[0020] (4) Repeat steps (2)-(3) multiple times. Each time, increase the azelaic acid concentration in the solid medium and the liquid medium in a gradient manner until the azelaic acid concentration reaches 5-10% (w / w);

[0021] (5) Transfer the microorganism obtained in step (4) that can grow in a liquid medium containing 5-10% (w / w) azelaic acid to a liquid medium containing 5-10% (w / w) azelaic acid for further acclimation. And every 3-8 generations, gradually lower the pH of the liquid medium to below 4.0, and subculture repeatedly for more than 20 times, so that the growth ability of the microorganism in it is more than 80% of the growth ability of the microorganism in MRS medium.

[0022] In one embodiment, the azelaic acid concentration in the liquid medium in step (3) is 0.5% (w / w).

[0023] In one embodiment, the gradient increase in step (4) means that each time the azelaic acid concentration in the medium is increased by 0.5% (w / w) until the azelaic acid concentration reaches 5% (w / w).

[0024] In one embodiment, in step (5), the pH of the liquid medium is gradually lowered by 0.5 each time.

[0025] In one embodiment, in step (5), the growth ability is compared according to the OD 600 value after cultivation.

[0026] The second object provided by the present invention is to provide a microbial preparation containing Lactobacillus plantarum CCTCC NO: M 20232390 of the present invention or prepared from the microorganism of Lactobacillus plantarum CCTCC NO: M 20232390 described in the present invention.

[0027] In one embodiment, the microbial preparation uses Lactobacillus plantarum CCTCC NO: M 20232390 as the main microorganism.

[0028] In one embodiment, the preparation is a seed solution for preparing CCTCC NO: M 20232390 and then obtained by scale-up culture.

[0029] In one embodiment, the microbial preparation includes the culture, dry matter, inactivated matter and / or disrupted matter of Lactobacillus plantarum CCTCC NO: M 20232390; preferably, the preparation is a solid preparation or a liquid preparation.

[0030] In one embodiment, the culture is the supernatant of the culture broth obtained by culturing Lactobacillus plantarum CCTCC NO: M 20232390 (or the filtrate of the fermentation product).

[0031] In one embodiment, the dried product is a powder prepared by drying Lactobacillus plantarum CCTCC NO: M 20232390, and the drying treatment includes but is not limited to heat drying, spray drying, vacuum freeze drying, fluidized bed drying or vacuum drying.

[0032] In one embodiment, the inactivated product is inactivated cells obtained by inactivating Lactobacillus plantarum CCTCC NO: M 20232390, and the inactivation treatment includes but is not limited to heat treatment or lyophilization treatment.

[0033] In one embodiment, the disrupted product is the disrupted product obtained by disrupting the cultured Lactobacillus plantarum CCTCC NO: M 20232390.

[0034] The third object of the present invention is to provide the application of Lactobacillus plantarum CCTCC NO: M 20232390 or a microbial preparation containing the Lactobacillus plantarum CCTCC NO: M 20232390 in the preparation of a product; optionally, the product is a product containing azelaic acid component.

[0035] In one embodiment, the microbial preparation includes the culture, dried product, inactivated product and / or disrupted product of Lactobacillus plantarum CCTCC NO: M 20232390; preferably, the preparation is a solid preparation or a liquid preparation.

[0036] In one embodiment, the culture is the supernatant of the culture broth obtained by culturing Lactobacillus plantarum CCTCC NO: M 20232390 with a fermentation medium.

[0037] In one embodiment, the dried product is a powder prepared by drying Lactobacillus plantarum CCTCC NO: M 20232390, and the drying treatment includes but is not limited to heat drying, spray drying, vacuum freeze drying, fluidized bed drying or vacuum drying.

[0038] In one embodiment, the inactivated product is inactivated cells obtained by inactivating Lactobacillus plantarum CCTCC NO: M 20232390, and the inactivation treatment includes but is not limited to heat treatment or lyophilization treatment.

[0039] In one embodiment, the disrupted product is the disrupted product obtained by disrupting Lactobacillus plantarum CCTCC NO: M 20232390.

[0040] In one embodiment, the product includes, but is not limited to, drugs and cosmetics.

[0041] In one embodiment, the drug further contains pharmaceutically acceptable excipients, and the excipients include, but are not limited to, at least one of diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, cosolvents, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostatic agents or buffers.

[0042] In one embodiment, the drug further contains other drugs with soothing and repairing functions.

[0043] In one embodiment, the drug includes, but is not limited to, injections, oral liquids, tablets, capsules, dripping pills, and sprays.

[0044] In one embodiment, the administration methods include, but are not limited to, subcutaneous injection, intravenous injection, oral administration, topical application, respiratory inhalation administration, local administration, and sublingual administration.

[0045] In one embodiment, the dosage form of the cosmetics can be formulated in the form of solutions, topical ointments, emulsifiable foams, nutritive emollients, soft emollients, fillers, soft water, milky lotions, cosmetic bases, perfumes, soaps, liquid detergents, bath agents, sunscreens, sun oils, suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactants-containing detergents, oils, foundations, liquid foundations, wax foundations, patches and sprays.

[0046] In one embodiment, the cosmetics further contain at least one cosmetically acceptable carrier.

[0047] In one embodiment, the carrier can be oil, water, surfactant, humectant, lower alcohol, thickener, chelating agent, pigment, preservative, perfume.

[0048] The fourth object of the present invention is to provide a cosmetic, and the cosmetic contains a microbial preparation of Lactobacillus plantarum CCTCC NO: M20232390; optionally, the cosmetic is a cosmetic containing azelaic acid component.

[0049] In one embodiment, the microbial preparation contains the culture supernatant (or called fermentation product filtrate) of Lactobacillus plantarum CCTCC NO: M 20232390.

[0050] In one embodiment, the cosmetic is a skin care product.

[0051] In one embodiment, the cosmetic dosage form can be formulated in the form of a solution, topical ointment, cream foam, nourishing emollient, soft emollient, filler, soft water, milky lotion, cosmetic base, fragrance, soap, liquid detergent, bath agent, sunscreen, sunblock oil, suspension, emulsion, paste, gel, lotion, powder, soap, surfactant-containing cleaner, oil, foundation, liquid foundation, wax foundation, patch and spray.

[0052] In one embodiment, the cosmetic further comprises at least one cosmetically acceptable carrier.

[0053] In one embodiment, the carrier can be oil, water, surfactant, humectant, lower alcohol, thickening agent, chelating agent, pigment, preservative, fragrance.

[0054] The fifth object of the present invention is to provide a medicine, which contains Lactobacillus plantarum CCTCC NO: M20232390 or a microbial preparation containing Lactobacillus plantarum CCTCC NO: M 20232390.

[0055] In one embodiment, the medicine further contains a pharmaceutically acceptable excipient, and the excipient includes but is not limited to at least one of a diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH regulator, antioxidant, bacteriostatic agent or buffer.

[0056] In one embodiment, the medicine contains azelaic acid.

[0057] In one embodiment, the medicine further contains other drugs with soothing and repairing functions.

[0058] In one embodiment, the medicine includes but is not limited to injections, oral liquids, tablets, capsules, dripping pills, sprays.

[0059] In one embodiment, the administration methods include but are not limited to subcutaneous injection, intravenous injection, oral administration, topical application, respiratory inhalation administration, topical administration, sublingual administration.

[0060] The sixth object of the present invention is to provide a method for preparing a product composition, which includes: adding the culture, dried product, inactivated product and / or fragmented product of Lactobacillus plantarum CCTCC NO: M 20232390 or Lactobacillus plantarum CCTCC NO: M 20232390 to a product matrix to obtain the product composition.

[0061] Biological material preservation:

[0062] Lactobacillus plantarum ZW, classified and named as Lactobacillus plantarum ZW, was deposited at the China Center for Type Culture Collection on November 29, 2023, with the deposit number CCTCC NO: M 20232390 and the deposit address being Wuhan University, Wuhan, China.

[0063] Beneficial effects:

[0064] The Lactobacillus plantarum ZW provided by the present invention has the characteristic of high azelaic acid tolerance. This strain can grow normally in MRS medium containing 5% azelaic acid and can still survive at azelaic acid concentrations of 10% and 20%. The fermentation products of this strain have significant anti-inflammatory effects and can significantly inhibit the expression of inflammatory factors such as NO, TNF-α, and IL-6 in lipopolysaccharide-stimulated RAW264.7 cells. The fermentation products of this strain have significant soothing effects and can significantly reduce the calcium influx rate of HaCaT cells caused by capsaicin stimulation. The fermentation products of this strain have a significant ability to promote the migration of skin cells, and the migration rate increases with the increase in the addition amount. At the same time, the fermentation products of this strain have good safety. Therefore, Lactobacillus plantarum ZW has broad application prospects in soothing and repairing products, such as in cosmetic formulations, and is particularly suitable for functional products with a high content of azelaic acid. Description of the drawings

[0065] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0066] Among them:

[0067] Figure 1 It is a colony morphology diagram of Lactobacillus plantarum ZW.

[0068] Figure 2 It is an effect diagram of the ability of the fermentation products of Lactobacillus plantarum ZW to inhibit the expression of NO in cells.

[0069] Figure 3 It is an effect diagram of the ability of the fermentation products of Lactobacillus plantarum ZW to inhibit the expression of TNF-α in cells.

[0070] Figure 4 It is an effect diagram of the ability of the fermentation products of Lactobacillus plantarum ZW to inhibit the expression of IL-6 in cells.

[0071] Figure 5Effect diagram of the ability of the fermentation product of Lactobacillus plantarum ZW to inhibit intracellular calcium ion influx.

[0072] Figure 6 Effect diagram of the ability of the fermentation product of Lactobacillus plantarum ZW to promote cell migration.

[0073] Figure 7 Comparison diagram of the empty area of cell scratching.

[0074] Figure 8 Result diagram of the chicken embryo chorioallantoic membrane vascular test. Detailed implementation mode

[0075] To make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description of the specific implementation mode of the present invention is made in combination with the embodiments of the specification.

[0076] Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar promotions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0077] The percentages involved in the following embodiments are v / v units unless otherwise specified.

[0078] I. Media involved in the following embodiments:

[0079] 1. Adaptive acclimation medium:

[0080] Yeast extract powder 4g, peptone 10g, beef extract powder 5g, glucose 20g, dipotassium hydrogen phosphate 2g, ammonium citrate 2g, sodium acetate 5g, magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween-80 1g. Add the required concentration of azelaic acid according to different acclimation conditions, and finally make up the volume to 1000g with water. Adjust the pH according to different acclimation conditions and sterilize at 121°C for 20 min. For solid adaptive acclimation medium, add 20g of agar powder.

[0081] 2. Lactic acid or acetic acid tolerance comparison medium:

[0082] Yeast extract powder 4g, peptone 10g, beef extract powder 5g, glucose 20g, dipotassium hydrogen phosphate 2g, ammonium citrate 2g, sodium acetate 5g, magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween-80 1g, agar powder 20g. Finally make up the volume to 900g with water and sterilize at 121°C for 20 min. When the medium cools down to 60°C, add 100g of lactic acid or acetic acid (i.e., the final concentration is 10%) to the medium according to the comparison conditions.

[0083] 3. Fermentation medium (MRS medium):

[0084] Liquid medium: 4 g of yeast extract powder, 10 g of peptone, 5 g of beef extract powder, 20 g of glucose, 2 g of dipotassium hydrogen phosphate, 2 g of ammonium citrate, 5 g of sodium acetate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 1 g of Tween-80, add water to make up to 1000 g, adjust the pH to 6.2 ± 0.2, sterilize at 121 °C for 20 min. For solid medium, add 20 g of agar powder.

[0085] II. Materials involved in the following examples:

[0086] High-glucose DMEM medium was purchased from Cytiva, USA;

[0087] Nitric oxide detection kit, Mouse TNF-α ELISA Kit, and Mouse IL-6 ELISA Kit were all purchased from Beyotime Biotechnology Co., Ltd.;

[0088] Lactobacillus plantarum R1, Lactobacillus plantarum R2, Lactobacillus plantarum R3, Lactobacillus plantarum R4, Lactobacillus plantarum R5, Lactobacillus plantarum R6, Lactobacillus plantarum R7, Lactobacillus plantarum R8, Lactobacillus plantarum R9, and Lactobacillus plantarum R10 were all from the self-built strain bank of Jiangsu Ruiting Biotechnology Co., Ltd.;

[0089] Lactobacillus plantarum R11 was purchased from China Center for Industrial Culture Collection, with the preservation number of CICC 6240, belonging to the type strain.

[0090] Mouse mononuclear macrophage leukemia cells (RAW264.7) and human immortalized keratinocytes (HaCaT) were both purchased from Beina Biology.

[0091] Example 1: Adaptive domestication and screening of lactic acid bacteria in azelaic acid environment

[0092] (1) Select 11 kinds of lactic acid bacteria, including: Lactobacillus plantarum R1, Lactobacillus plantarum R2, Lactobacillus plantarum R3, Lactobacillus plantarum R4, Lactobacillus plantarum R5, Lactobacillus plantarum R6, Lactobacillus plantarum R7, Lactobacillus plantarum R8, Lactobacillus plantarum R9, Lactobacillus plantarum R10, and Lactobacillus plantarum R11.

[0093] (2) Oscillate and melt the cryopreservation tubes of the above 11 kinds of lactic acid bacteria in a 37 °C water bath, and then transfer them to MRS medium in a laminar flow hood. Shake flask culture for 48 h at 100 r / min and 37 °C.

[0094] (3) Let the lactic acid bacteria liquid cultured in the shake flask stand for 15 minutes, obtain the upper layer of bacterial suspension by the pouring method, centrifuge at 2000 r / min for 10 minutes, and collect the precipitated bacteria.

[0095] (4) The precipitated bacteria cells were redispersed with sterile normal saline. The bacterial liquid was picked up with an inoculation loop and streaked on a solid adaptive domestication medium containing 0.5% (w / w) azelaic acid, and then statically cultured at 37 °C for 48 h.

[0096] (5) From the single colonies isolated by streaking, the single colony with the largest colony area was selected, picked up with an inoculation loop and transferred to a liquid adaptive domestication medium containing 0.5% azelaic acid. It was cultured in a shaking flask at 100 r / min and 37 °C for 24 h.

[0097] (6) Repeat the operations in (3) to (5). Each time it was repeated, the content of azelaic acid in the adaptive domestication medium was increased by 0.5% (w / w) until the content of azelaic acid reached 5% (w / w). The survival of 11 lactic acid bacteria in the adaptive domestication medium containing different concentrations of azelaic acid is shown in Table 1.

[0098] (7) The microorganisms that could grow in the adaptive domestication medium containing 5% (w / w) azelaic acid obtained from Table 1 were transferred to a liquid adaptive domestication medium containing 5% (w / w) azelaic acid for continuous domestication. The pH was adjusted to 5.5, 5.0, 4.5, and 4.0 every 5 generations respectively, and subcultured 20 times repeatedly to make the growth ability of the microorganisms in it similar to the growth ability of the microorganisms in MRS medium. The growth ability is shown in Table 2.

[0099] (8) The strain Lactobacillus plantarum R10 with restored growth ability was collected for cultivation and identification. The domesticated Lactobacillus plantarum R6 and the domesticated Lactobacillus plantarum R10, as well as the undomesticated Lactobacillus plantarum R10 and the undomesticated commercially sourced Lactobacillus plantarum R11 were preserved.

[0100] The domesticated Lactobacillus plantarum R10, Lactobacillus plantarum R6, and the undomesticated commercially sourced Lactobacillus plantarum R11 were respectively inoculated into 200 mL of fermentation medium and cultured in a shaking flask at 100 r / min and 37 °C until OD 600 = 5; They were respectively inoculated into 1 L of fermentation medium at an inoculation amount of 5% (v / v) and cultured in a shaking flask at 100 r / min and 37 °C for 48 h; The bacteria cells were removed by centrifugation, and the fermentation broth was collected as the test sample for efficacy testing.

[0101] Table 1 Inhibitory effects of different concentrations of azelaic acid on lactic acid bacteria

[0102]

[0103] Note: In the table, "-" represents that the microorganism did not grow on the solid adaptive domestication medium, and "+" represents that the microorganism grew into single colonies on the solid adaptive domestication medium.

[0104] Table 2 Growth of Lactobacillus in 5% azelaic acid liquid adaptation domestication medium

[0105]

[0106]

[0107] As can be seen from the results in Table 2, before and after domestication, the growth of Lactobacillus plantarum R10 in MRS medium was basically the same. However, in the domestication medium, Lactobacillus plantarum R10, which was first screened with high-concentration azelaic acid and then domesticated for 20 generations in a low-pH environment, had a much higher ability to tolerate high-concentration azelaic acid and low-pH environment than the undomesticated Lactobacillus plantarum R10.

[0108] Example 2: Comparison of the tolerance of Lactobacillus to high-concentration azelaic acid environment

[0109] (1) Prepare a solid adaptation domestication medium and adjust the azelaic acid content to 10% and 20% respectively.

[0110] (2) Select the domesticated Lactobacillus plantarum R10 and Lactobacillus plantarum R6, and adjust the biomass to 10 8 CFU / mL respectively. Take 200 μL each and inoculate and spread evenly on the surface of the solid adaptation domestication medium, and culture at 37 °C for 48 h. Read the number of single colonies and calculate the actual colony number (CFU / mL). The results are shown in Table 3.

[0111] As can be seen from the results in Table 3, after adaptation domestication, Lactobacillus plantarum R10 can still grow in the system containing 10% and 20% azelaic acid, but Lactobacillus plantarum R6 can only grow in the system containing 10% azelaic acid and the colony number is low.

[0112] Table 3 Comparison of the tolerance of Lactobacillus to high-concentration azelaic acid

[0113]

[0114] Note: "-" in the table represents that the microorganism did not grow on the solid adaptation domestication medium.

[0115] Example 3: Comparison of the tolerance of Lactobacillus to lactic acid and acetic acid environment

[0116] (1) Prepare a lactic acid or acetic acid tolerance comparison medium, where the concentration of lactic acid or acetic acid is 10%.

[0117] (2) Select the domesticated Lactobacillus plantarum R10 and Lactobacillus plantarum R6, as well as the undomesticated commercial Lactobacillus plantarum R11, and adjust the biomass to 10 8CFU / mL. 200 μL was taken respectively for inoculation and evenly spread on the surface of the culture medium, and cultured at 37 °C for 48 h. The number of single colonies was read and the actual number of colonies (CFU / mL) was calculated. The results are shown in Table 4.

[0118] As can be seen from the results in Table 4, after adaptive domestication, Lactobacillus plantarum R10 could still grow in systems containing 10% lactic acid and acetic acid respectively, but Lactobacillus plantarum R6 could only grow in the system containing 10% lactic acid and the number of colonies was low. Commercially sourced Lactobacillus plantarum R11 could grow in the system containing 10% lactic acid but the number of colonies was very low, and it could not grow in the system containing 10% acetic acid. Thus, it can be seen that Lactobacillus plantarum R10 after adaptive domestication not only has the ability to tolerate azelaic acid and low pH environment, but also has a certain ability to tolerate other small molecule organic acids.

[0119] Table 4 Comparison of the abilities of lactic acid bacteria to tolerate high concentrations of lactic acid and acetic acid

[0120]

[0121] Example 4: Isolation, culture and identification of Lactobacillus plantarum ZW

[0122] The Lactobacillus plantarum R10 after adaptive domestication in Example 1 was transferred to solid MRS medium for culture for 48 h. White colonies similar to lactic acid bacteria were selected, and the morphological observation and physiological and biochemical characteristic identification of the strain were carried out.

[0123] The Lactobacillus plantarum R10 after adaptive domestication has the following cell characteristics:

[0124] (1) Colony morphology: round, milky white, opaque, about 1 - 2 mm in diameter, with smooth edges, wet and protruding surface with luster, and the colony morphology is as Figure 1 shown;

[0125] (2) Microscopic examination morphology: short rod-shaped, Gram-positive staining, no flagella, no spore production;

[0126] (3) Growth characteristics: The optimum fermentation pH is 5.5, the temperature is 37 °C, the fermentation period is short, the logarithmic growth phase starts at 6 h, and the stationary phase starts after 10 h.

[0127] The Lactobacillus plantarum R10 after adaptive domestication was identified by 16S rDNA gene sequence. The sequence was subjected to Blast sequence alignment analysis and phylogenetic tree homology analysis on the NCBI database. Finally, it was identified as Lactobacillus plantarum, and it was deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 20232390. It was named Lactobacillus plantarum ZW, the deposit date was November 29, 2023, and the deposit address was Wuhan University, Wuhan, China.

[0128] Example 5: Anti-inflammatory effect of the fermentation product of Lactobacillus plantarum ZW

[0129] Mouse monocyte macrophage leukemia cells (RAW264.7) were grown in complete medium (high-glucose DMEM medium containing 10% FBS and 1% double antibody). The cells were digested with trypsin and inoculated into 6-well plates at a density of 2×10 5 cells / well, 2 mL per well, and cultured in an incubator at 37 °C and 5% CO2. After 24 h, the medium was aspirated and the residual culture solution was washed away with PBS. The Blank group was added with 2 mL of high-glucose DMEM medium;

[0130] The Control group was added with 1 μg / mL lipopolysaccharide (LPS) prepared with 2 mL of high-glucose DMEM medium;

[0131] The sample groups were respectively added with 2 mL of the following solutions prepared with high-glucose DMEM medium and the fermentation broth obtained in Example 1: Fermentation broth of domesticated Lactobacillus plantarum R10 (i.e., Lactobacillus plantarum ZW, hereinafter referred to as ZW) with concentrations of 0.005% (v / v, the same below), 0.01%, 0.05%, and 0.1% (containing 1 μg / mL of LPS), fermentation broth of domesticated Lactobacillus plantarum R6 (hereinafter referred to as R6) with a concentration of 0.1% (containing 1 μg / mL of LPS), fermentation broth of undomesticated Lactobacillus plantarum R10 (hereinafter referred to as R10) with a concentration of 0.1% (containing 1 μg / mL of LPS), and fermentation broth of commercially sourced Lactobacillus plantarum R11 (hereinafter referred to as R11) with a concentration of 0.1% (containing 1 μg / mL of LPS), with 3 parallels in each group;

[0132] Incubate in the incubator for 24 h, then aspirate the cell supernatant, centrifuge, take the supernatant, and use a nitric oxide detection kit, Mouse TNF-α ELISA Kit, and Mouse IL-6 ELISA Kit to detect the cell inflammatory factors NO, TNF-α, and IL-6, and investigate the inhibitory ability of the lactic acid bacteria fermentation product on the expression of the inflammatory factors NO, TNF-α, and IL-6 in RAW264.7 cells. The test results are shown in Table 5.

[0133] The results showed that:

[0134] The fermentation products of ZW had the ability to significantly inhibit the expression of NO inflammatory factor in RAW264.7 cells at concentrations from 0.005% to 0.1%; the fermentation products of R10 had a certain inhibitory ability at a concentration of 0.1%, but the ability was inferior to that of ZW; the fermentation products of R6 did not show this inhibitory ability at a concentration of 0.1%; the fermentation products of R11 did not show this inhibitory ability at a concentration of 0.1%( Figure 2 ).

[0135] The fermentation products of ZW had the ability to significantly inhibit the expression of TNF-α inflammatory factor in RAW264.7 cells at concentrations from 0.005% to 0.1%; the fermentation products of R10 had a certain inhibitory ability at a concentration of 0.1%, but the ability was inferior to that after domestication; the fermentation products of R6 had this inhibitory ability at a concentration of 0.1%, and its inhibitory ability was weaker than that of ZW at a concentration of 0.01%; the fermentation products of R11 had a certain inhibitory ability at a concentration of 0.1%, but the ability was inferior to that of ZW and R10( Figure 3 ).

[0136] The fermentation products of ZW had the ability to significantly inhibit the expression of IL-6 inflammatory factor in RAW264.7 cells at concentrations from 0.01% to 0.1%; the fermentation products of R10 had a certain inhibitory ability at a concentration of 0.1%, but the ability was inferior to that after domestication; the fermentation products of R6 did not show this inhibitory ability at a concentration of 0.1%; the fermentation products of R11 had a certain inhibitory ability at a concentration of 0.1%, but the ability was inferior to that of ZW and R10( Figure 4 ).

[0137] Table 5 The ability of Lactobacillus plantarum fermentation products to inhibit the expression of inflammatory factors in cells

[0138]

[0139]

[0140] Therefore, the anti-inflammatory ability of Lactobacillus plantarum ZW after adaptive domestication with different concentrations of azelaic acid and low pH conditions was significantly improved. In comparison, the anti-inflammatory abilities of Lactobacillus plantarum R6 and commercially sourced Lactobacillus plantarum R11 after adaptive domestication were inferior to that of ZW.

[0141] Example 6: Soothing effect of the fermentation products of Lactobacillus plantarum ZW

[0142] Stimulating human immortalized keratinocytes (HaCaT) with capsaicin can activate the opening of calcium channels on the cell membrane, allowing extracellular calcium ions to flow into the cell and inducing a stress response in the skin. Different concentrations of lactic acid bacteria fermentation products were co-cultured with HaCaT cells, and the weakening of intracellular calcium ion fluorescence intensity relative to the Control group was detected to investigate the soothing ability of lactic acid bacteria fermentation products on the stimulation response of HaCaT cells. The specific steps are as follows:

[0143] Inoculate HaCaT cells at a density of 2×10 4 cells / well into a 6-well plate, 2 mL per well, and culture in an incubator at 37°C and 5% CO2 for 18 h. Aspirate the culture medium and wash away the residual culture solution with PBS. Add 2 mL of high-glucose DMEM medium to the Blank group, add 2 mL of 1 μmol / L capsaicin prepared with high-glucose DMEM medium to the Control group, and add the following solutions prepared with high-glucose DMEM medium and the fermentation broth obtained in Example 1 to the sample groups: ZW fermentation products with concentrations of 0.005%, 0.01%, 0.05%, and 0.1% (containing 1 μmol / L capsaicin), R6 fermentation products with a concentration of 0.1% (containing 1 μmol / L capsaicin), R10 fermentation products with a concentration of 0.1% (containing 1 μmol / L capsaicin), and R11 fermentation products with a concentration of 0.1% (containing 1 μmol / L capsaicin), and co-culture for 20 min. Subsequently, add the Fluo-4AM probe for staining, protect from light, and incubate at 37°C for 30 min. After staining, digest the cells with trypsin and transfer them to a black microplate, and perform fluorescence detection at an excitation wavelength of 490 nm and an emission wavelength of 525 nm.

[0144] The test results are shown in Table 6 and Figure 5 as follows.

[0145] The results show that:

[0146] The fermentation products of Lactobacillus plantarum ZW have a significant ability to inhibit the influx of calcium ions in HaCaT cells at concentrations from 0.005% to 0.1%; the R10 fermentation products also have a certain inhibitory ability at a concentration of 0.1%, but the ability is less than that of ZW; the R6 and R11 fermentation products both have a certain inhibitory ability at a concentration of 0.1%, but their inhibitory abilities are weaker than those of the ZW fermentation products at a concentration of 0.01%.

[0147] Table 6 The ability of Lactobacillus plantarum fermentation products to inhibit the influx of calcium ions in cells

[0148]

[0149]

[0150] Example 7: Repairing Effect of the Fermentation Product of Lactobacillus plantarum ZW

[0151] HaCaT cells were grown in complete medium (high-glucose DMEM medium containing 10% FBS and 1% double antibody). The cells were digested with trypsin and seeded in 6-well plates at a density of 2×10 5 cells / well, 2 mL per well, and cultured in an incubator at 37°C and 5% CO2. After 24 h, the confluent HaCaT cells were scratched. After scratching, 2 mL of high-glucose DMEM medium was added to the Control group, and the following solutions prepared with high-glucose DMEM medium and the fermentation broth obtained in Example 1 were added to the sample groups: ZW fermentation products at concentrations of 0.01%, 0.1%, and 0.5%, R6 fermentation product at a concentration of 0.5%, R10 fermentation product at a concentration of 0.5%, and R11 fermentation product at a concentration of 0.5%. There were 3 parallels in each group, and they were incubated in the incubator for 24 h. Subsequently, photos were taken under a microscope, and the ImageJ software was used to calculate the vacant area of the cell scratch. The reduction of the vacant area can prove the ability of the lactic acid bacteria fermentation product to promote cell migration, and further indicate its repair ability for damaged skin. The test results are as Figure 6 and Figure 7 shown.

[0152] The results showed that:

[0153] The ZW fermentation product had a significant ability to promote cell migration at concentrations of 0.01%, 0.1%, and 0.5%; the R10 fermentation product also had a significant ability to promote cell migration at a concentration of 0.5%, but its ability was less than that of ZW; the R6 fermentation product had a certain ability to promote cell migration at a concentration of 0.5%, and its promoting ability was weaker than that of the ZW fermentation product at a concentration of 0.01%; at the same time, commercially sourced R11 also had a certain ability to promote cell migration, but its effect was also less than that of the ZW fermentation product (Table 7).

[0154] Table 7 Ability of Lactobacillus plantarum Fermentation Products to Promote Cell Migration

[0155]

[0156] Example 8: Safety Evaluation of the Fermentation Product of Lactobacillus plantarum ZW

[0157] (1) Human Skin Patch Test

[0158] It was carried out according to the method specified in the "Technical Specifications for Cosmetics Safety" (2015 Edition).

[0159] Volunteer requirements: 30 normal subjects (aged 20 - 40), who do not have unhealed inflammatory skin diseases, have no scars, pigmentation, atrophy, nevus flammeus or other defects on the skin test sites, have not participated in other clinical trials, are not highly sensitive in constitution, and have not undergone patch tests in the past month.

[0160] The fermentation broth of the domesticated Lactobacillus plantarum R10 (i.e., Lactobacillus plantarum ZW) obtained in Example 1 was diluted with distilled water to obtain a sample solution with a concentration of 50%.

[0161] Pipette 0.020 - 0.025 mL of the sample solution onto the filter paper attached to the patch tester, place the filter paper in the patch tester, and at the same time use the same volume of distilled water as a blank control. Apply the patch tester with the test substance on the flexor side of the forearm with non-irritating tape, and gently press with the palm to make it evenly adhere to the skin, and maintain for 24 h.

[0162] After removing the patch tester, wait for 30 min. After the indentation disappears, observe the skin reaction. If the skin reaction is negative, observe the skin reaction results again at 24 h and 48 h respectively.

[0163] The experimental results are shown in Table 8:

[0164] Table 8 Results of human skin patch test

[0165]

[0166] Note: -: Negative reaction; ±: Suspected reaction (weak erythema); +: Weak positive reaction (erythema reaction: erythema, infiltration, edema).

[0167] All 30 subjects in the human patch test had a grade 0 reaction. According to the relevant regulations of the Cosmetics Safety and Technology Specification, the ZW fermentation product will not cause adverse reactions to human skin at a concentration of 50% and below. It can be seen that this fermentation product has high human safety.

[0168] (2) Chicken embryo chorioallantoic membrane vascular assay (HET-CAM assay)

[0169] The chicken embryo chorioallantoic membrane vascular assay evaluates irritation by detecting damage to the chorioallantoic membrane. The specific method is as follows:

[0170] Purchase 0-day-old chicken embryos, with each embryo weighing 50 - 60 g. During the incubation process, turn the embryos once a day. When incubated to 5 days old, check the development status of the chicken embryos. When incubated to 6 days old, peel off part of the eggshell to expose the white egg membrane; carefully remove the white egg membrane with forceps to ensure that the chorioallantoic membrane (CAM) of the chicken embryo is not damaged and the blood vessels on the membrane do not bleed. Respectively, directly drop 0.1 mL of the positive control, negative control, and test sample onto the surface of the CAM, observe the reaction of the CAM, and record the time when each stimulation effect (including three reaction degrees of bleeding, blood coagulation, and blood vessel lysis) appears within 0 min, 2.5 min, and 5 min, accurate to seconds, and record the degree of the reaction.

[0171] The positive control sample is: 1 mol / L NaOH (sodium hydroxide) solution or 1% SDS (sodium dodecyl sulfate) solution;

[0172] The negative control sample is: 0.9% sterilized normal saline (NaCl) solution;

[0173] The test sample is: a sample solution with a concentration of 50% obtained by diluting the fermentation broth of the domesticated Lactobacillus plantarum R10 (i.e., Lactobacillus plantarum ZW) obtained in Example 1 with distilled water.

[0174] The experimental results are as Figure 8 shown:

[0175] After adding 0.9% sterilized normal saline, there were no obvious changes in the capillaries of the chicken embryos;

[0176] After adding 1% SDS solution, the capillaries of the chicken embryos gradually disappeared and bled;

[0177] After adding 1 mol / L NaOH solution, the capillaries of the chicken embryos dissolved and significant bleeding occurred;

[0178] After adding 50% ZW fermentation product, there were no obvious changes in the capillaries of the chicken embryos.

[0179] Therefore, the ZW fermentation product has no obvious irritation and corrosion at a concentration of 50% and below, and has high safety.

[0180] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A Lactobacillus plantarum, characterized in that, The above-mentioned Lactobacillus plantarum was deposited at the China Center for Type Culture Collection on November 29, 2023, with the deposit number CCTCC NO: M 20232390.

2. The Lactobacillus plantarum according to claim 1, characterized in that, The above-mentioned Lactobacillus plantarum was obtained by adaptive domestication and screening in an azelaic acid environment; Optionally, the domestication and screening are specifically as follows: (1) First, activate Lactobacillus plantarum CCTCC NO: M 20232390; (2) Collect the bacterial cells, disperse them, pick the bacterial liquid and streak it on a solid medium containing a certain concentration of azelaic acid, and incubate it statically; (3) Pick a single colony and transfer it to a liquid medium containing a certain concentration of azelaic acid for domestication culture; (4) Repeat steps (2) to (3) multiple times, and each time the concentration of azelaic acid in the solid medium and the liquid medium is increased in a gradient manner until the azelaic acid concentration reaches 5-10% w / w; (5) Transfer the microorganism obtained in the previous step that can grow in the liquid medium containing 5-10% w / w azelaic acid to the liquid medium containing 5-10% w / w azelaic acid for continuous domestication, and every 3-8 generations, gradually reduce the pH of the liquid medium to below 4.0, and subculture repeatedly for more than 20 times, so that the growth ability of the microorganism in it is more than 80% of the growth ability of the microorganism in MRS medium.

3. A microbial preparation, characterized in that, It contains the Lactobacillus plantarum CCTCC NO: M20232390 described in claim 1 or is prepared from the microorganism of the Lactobacillus plantarum CCTCC NO: M 20232390 described in claim 1.

4. The preparation according to claim 3, characterized in that, The preparation uses the Lactobacillus plantarum CCTCC NO: M20232390 as the main microorganism.

5. The preparation according to claim 3 or 4, characterized in that, The preparation includes the culture, dry matter, inactivated matter, and / or broken matter of Lactobacillus plantarum CCTCC NO: M20232390; preferably, the culture is the supernatant of the culture solution or the fermentation product filtrate obtained by culturing Lactobacillus plantarum CCTCC NO: M 20232390; preferably, the dry matter is a powder prepared by drying Lactobacillus plantarum CCTCC NO: M 20232390; preferably, the inactivated matter is inactivated cells obtained by inactivating Lactobacillus plantarum CCTCC NO: M 20232390; preferably, the broken matter is the broken matter obtained by breaking the cultured Lactobacillus plantarum CCTCC NO: M 20232390.

6. The application of the Lactobacillus plantarum CCTCC NO: M 20232390 described in claim 1 or the microbial preparation containing the Lactobacillus plantarum CCTCC NO: M 20232390 described in claim 1 in the preparation of a product; optionally, the product contains azelaic acid; optionally, the product has a skin soothing and repairing function; optionally, the product includes but is not limited to drugs and cosmetics.

7. A cosmetic, characterized in that, The cosmetic contains the microbial preparation of Lactobacillus plantarum CCTCC NO: M20232390 as claimed in claim 1; optionally, the cosmetic is a cosmetic containing azelaic acid; optionally, the microbial preparation is the culture supernatant of Lactobacillus plantarum CCTCC NO: M20232390 as claimed in claim 1.

8. The cosmetic according to claim 7, characterized in that, The dosage form of the cosmetic can be formulated in the form of solutions, topical ointments, emulsifiable foams, nutritive emollients, soft emollients, fillers, soft water, milky lotions, cosmetic bases, perfumes, soaps, liquid detergents, bath agents, sunscreens, suntan oils, suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing detergents, oils, foundations, liquid foundations, wax foundations, patches and sprays; the cosmetic further contains at least one cosmetically acceptable carrier; optionally, the carrier is oil, water, surfactant, humectant, lower alcohol, thickener, chelating agent, pigment, preservative, perfume.

9. A drug, characterized in that, The drug contains Lactobacillus plantarum CCTCC NO: M20232390 as claimed in claim 1 or the microbial preparation containing Lactobacillus plantarum CCTCC NO: M20232390 as claimed in claim 1; optionally, the drug is a drug containing azelaic acid.

10. A method for preparing a product composition, characterized in that, The method includes: adding the Lactobacillus plantarum CCTCC NO: M20232390 as claimed in claim 1 or the culture, dried product, inactivated product and / or disrupted product of Lactobacillus plantarum CCTCC NO: M20232390 as claimed in claim 1 to a product matrix to obtain the product composition.

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