WO2023035634A1 - 一株高产胞外多糖的鼠李糖乳杆菌及其在缓解皮肤损伤中的应用 - Google Patents

一株高产胞外多糖的鼠李糖乳杆菌及其在缓解皮肤损伤中的应用 Download PDF

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WO2023035634A1
WO2023035634A1 PCT/CN2022/090284 CN2022090284W WO2023035634A1 WO 2023035634 A1 WO2023035634 A1 WO 2023035634A1 CN 2022090284 W CN2022090284 W CN 2022090284W WO 2023035634 A1 WO2023035634 A1 WO 2023035634A1
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exopolysaccharide
lactobacillus rhamnosus
vhprobi
uvb
strain
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English (en)
French (fr)
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彭书东
段治
吴松洁
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青岛蔚蓝生物股份有限公司
青岛蔚蓝生物集团有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/004—Aftersun preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of screening and application of probiotics, and in particular relates to a strain of Lactobacillus rhamnosus with high exopolysaccharide production and its application in alleviating skin damage.
  • Human skin mainly consists of two layers of tissue, the epidermis and the dermis.
  • the solar ultraviolet irradiated to human skin contains 90-99% UVA and 1-10% UVB. Under the same dose, UVB damages the skin 1000 times higher than UVA.
  • UVB mainly acts on the keratinocytes (HaCaT) of the epidermis, causing damage to intracellular proteins, DNA and membrane lipids, causing metabolic disorders and photodamage to the skin, and in severe cases, skin cancer.
  • Using traditional sunscreens against UV rays may cause side effects such as skin inflammation and allergies. Therefore, the use of natural products produced by plants, animals and microorganisms for anti-ultraviolet effect has attracted attention.
  • Lactic acid bacteria are ubiquitous probiotics and are considered as "generally safe bacteria", which have been widely used in food, medicine, cosmetics and other fields. Lactic acid bacteria can produce natural metabolites such as organic acids, antimicrobial peptides, and exopolysaccharides. Lactic acid bacteria exopolysaccharide (Exopolysaccharide, EPS) refers to the macromolecules produced by lactic acid bacteria during their growth and metabolism and secreted outside the cell wall, mainly capsular polysaccharides attached to the cell wall and mucus polysaccharides entering the fermentation broth. Studies in recent years have found that lactic acid bacteria and their exopolysaccharides have good application effects in anti-tumor, anti-inflammation, immune regulation and antibacterial. The research on the function of lactic acid bacteria and their exopolysaccharides in anti-ultraviolet damage is also a hotspot in this field.
  • the object of the present invention is to provide a novel strain of Lactobacillus rhamnosus (Lactobacillus rhamnosus) and application thereof.
  • the Lactobacillus rhamnosus has a high yield of exopolysaccharide, has good antioxidant activity, can effectively inhibit the skin from secreting pro-inflammatory cytokines, and alleviates the damage of ultraviolet rays to the skin by UVB, and the effect is remarkable.
  • Lactobacillus rhamnosus VHProbi O17 (Lactobacillus rhamnosus VHProbi O17), which has been preserved in the Chinese typical culture preservation of Wuhan University, Wuhan, China on May 24, 2021 Center, its deposit number is CCTCC NO: M2021589.
  • One aspect of the present invention provides the application of Lactobacillus rhamnosus VHProbi O17 in the preparation of products for preventing and alleviating skin damage.
  • the product is a cosmetic.
  • One aspect of the present invention provides the application of Lactobacillus rhamnosus VHProbi O17 in the production of exopolysaccharide.
  • the invention also provides an exopolysaccharide, which is produced and obtained by using Lactobacillus rhamnosus VHProbi O17 as a fermentation strain.
  • the exopolysaccharide is composed of 6 monosaccharides of D-galactosamine hydrochloride, arabinose, glucosamine hydrochloride, galactose, glucose and mannose, and the molar ratio is 0.05:0.03:0.09:0.05:0.19: 1.98.
  • the exopolysaccharide contains ⁇ -mannopyranose.
  • the molecular weight of the exopolysaccharide is 82.4kDa.
  • the present invention also provides the application of the exopolysaccharide in the preparation of products for preventing and alleviating skin damage.
  • the present invention also provides a cosmetic, which contains Lactobacillus rhamnosus VHProbi O17 and/or exopolysaccharide produced by fermentation of Lactobacillus rhamnosus VHProbi O17.
  • the Lactobacillus rhamnosus VHProbi O17 provided by the invention has a strong antioxidant activity, and the scavenging rates of DPPH and hydroxyl radicals reach 51.73% and 26.04% respectively.
  • Lactobacillus rhamnosus VHProbi O17 has no toxic effect on skin cells, and also has a certain growth-promoting effect, which can effectively alleviate the damage of cells caused by UVB radiation.
  • the decline in viability was significantly reduced.
  • the amount of bacteria was 8 ⁇ 10 8 CFU/mL
  • the activity of HaCaT cells after UVB irradiation increased by 76.6% compared with that without bacteria pretreatment.
  • the strain can also significantly inhibit UVB-induced cell secretion to produce pro-inflammatory response cytokines IL-6 and IL-8, and the secretion of IL-6 and IL-8 has been reduced by 34.4% and 54.2% respectively, which has achieved unexpected technical Effect.
  • Lactobacillus rhamnosus VHProbi O17 can produce a new exopolysaccharide OP-2, which is composed of six monosaccharides: D-galactosamine hydrochloride, arabinose, glucosamine hydrochloride, galactose, glucose and mannose. Can effectively relieve UVB damage to the skin.
  • Lactobacillus rhamnosus VHProbi O17 provided by the invention and the exopolysaccharide produced therefrom can be widely used in cosmetics and have broad prospects.
  • Lactobacillus rhamnosus named Lactobacillus rhamnosus VHProbi O17 (Lactobacillus rhamnosus VHProbi O17), has been preserved in the Chinese Type Culture Collection Center of Wuhan University, Wuhan, China on May 24, 2021, and its preservation number is CCTCC NO : M2021589.
  • Fig. 1 is the bacterium colony figure and the Gram stain figure of bacterial strain O17; Wherein A is the colony figure, B is the Gram stain figure;
  • Fig. 2 is the protein fingerprint of bacterial strain O17;
  • Fig. 3 is the Riboprinter fingerprint of bacterial strain O17;
  • Fig. 4 is the RAPD fingerprint of bacterial strain O17
  • Fig. 5 is the rep-PCR fingerprint of bacterial strain O17
  • Figure 6 is a comparison chart of the effect of Lactobacillus rhamnosus VHProbi O17 on alleviating UVB damage to skin cells;
  • Fig. 7 is the growth curve of Lactobacillus rhamnosus VHProbi O17 and the production curve of exopolysaccharide;
  • Figure 8 is a DEAE- ⁇ Sphere elution chromatogram of exopolysaccharide produced by Lactobacillus rhamnosus VHProbi O17;
  • Fig. 9 is the ion chromatogram analysis figure of exopolysaccharide OP-2;
  • Figure 10 is an infrared spectrum analysis diagram of exopolysaccharide OP-2;
  • Fig. 11 is the gel column analysis diagram and standard curve diagram of exopolysaccharide OP-2; wherein A is the gel column analysis diagram, and B is the standard curve diagram;
  • Figure 12 is a comparison chart of the effect of exopolysaccharide OP-2 on alleviating UVB damage to skin cells
  • Fig. 13 is a comparison chart of the inhibition of UVB-induced skin cytokine production by exopolysaccharide OP-2.
  • the screening method of the present invention is not limited to the description in the examples. Known methods that can achieve the purpose of screening are all available.
  • the screening descriptions in the examples are only descriptions of the present invention, and are not limitations on the protection scope of the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.
  • MRS agar medium pure water 1000mL, peptone 10g, beef extract 10g, yeast extract 5.0g, sodium acetate 5g, glucose 5g, potassium dihydrogen phosphate 2g, Tween 80 1.0mL, diamine citrate 2.0g , calcium carbonate 20g, magnesium sulfate heptahydrate 0.58g, manganese sulfate heptahydrate 0.25g, agar 15g, adjust pH to 6.2-6.5, autoclave at 121°C for 15min.
  • Embodiment 2 strain identification
  • the strain O17 was inoculated on the MRS agar medium, and after culturing at 37°C for 48 hours, the O17 single colony was smooth, off-white, short rod-shaped under the microscope, and both ends were even. See Figure 1 for the O17 single colony and the photo under the optical microscope.
  • inoculum amount of 0.1% fresh bacterial solution was inoculated in MRS liquid medium, and after culturing at 37°C and 150 rpm for 48 hours, the bacterial cells were collected, washed 4 times with sterile water, and dried on the surface. Then take a small amount of fresh bacteria and evenly spread it on the target plate in the form of a thin film, add 1 ⁇ L of lysate to cover the sample, after drying, add 1 ⁇ L of matrix solution to cover the sample, after drying, put the sample target into the mass spectrometer for identification .
  • the co-crystallized film formed by the sample and the matrix is irradiated with laser light to ionize the protein in the sample, and the ions are accelerated to fly through the flight tube under the action of an electric field of 10-20KV, and proteins of different molecular weights are detected according to the flight time of the detector.
  • the software Autof Acquirer V2.0.59 was used to complete the data analysis.
  • the strain O17 had obvious ion peaks at m/z 2946.082, 3476.573, 3791.097, 4692.724, 5892.635, 6953.051, 7581.626, 9382.474, etc.
  • the results are shown in Figure 2. After data comparison analysis, it was found that the protein expression similarity between strain O17 and Lactobacillus rhamnosus was the highest.
  • Table 2 16s rDNA PCR amplification system table
  • the 16s rDNA sequence SEQ ID NO:1 of the O17 strain was obtained by sequencing, and the sequence was compared in the NCBI database. The results showed that the similarity between SEQ ID NO:1 and Lactobacillus rhamnosus was the highest. Therefore, the O17 strain was preliminarily determined to be Lactobacillus rhamnosus.
  • a 1.5% agarose gel plate was prepared, and DL2000 DNA Marker was used as the result control, and the voltage was stabilized at 100V for 80 minutes, and finally the electrophoresis was detected by a gel imaging system.
  • the RAPD fingerprint of strain O17 is shown in Figure 4.
  • the voltage is 100V, and the electrophoresis time is 80min to detect the amplification result.
  • the rep-PCR fingerprint of strain O17 is shown in FIG. 5 .
  • the O17 strain provided by the present invention is a new strain of Lactobacillus rhamnosus, and it is named Rhamnosus Lactobacillus rhamnosus VHProbi O17.
  • Use this culture medium as the inoculum inoculate it into 50mL of MRS liquid medium according to the inoculation amount of 2%, and let it stand Cultivate for 24 hours to obtain the culture medium of the strain.
  • Aspirate 1 mL of bacterial liquid to collect the bacterial cells wash the bacterial cells with 1 mL of PBS buffer solution twice, and then add 2 mL of PBS solution to resuspend the bacterial cells for use.
  • Clearance % (A sample - A control ) / (A blank - A control ) x 100%.
  • a control is the absorbance of the mixed solution of ferrous sulfate, hydrogen peroxide and sodium salicylate
  • a blank is the absorbance of the mixed solution of ferrous sulfate and sodium salicylate.
  • Lactobacillus rhamnosus VHProbi O17 provided by the present invention can effectively scavenge DPPH and hydroxyl radicals, and the scavenging rates reach 51.73% and 26.04% respectively. This shows that Lactobacillus rhamnosus VHProbi O17 has a strong antioxidant activity and has achieved unexpected technical effects.
  • Embodiment 4 Lactobacillus rhamnosus VHProbi O17 anti-UVB radiation effect
  • HaCaT keratinocytes were anaerobically cultured in high-glucose DMEM medium containing 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin. Culture conditions: add 5% (v/v) carbon dioxide in a carbon dioxide incubator at 37° C., and change the medium every 3 days.
  • Lactobacillus rhamnosus VHProbi O17 in 100mL MRS medium ferment aerobically at 37°C for 40h; centrifuge at 6000 ⁇ g for 10min, discard the supernatant; wash the bacteria twice with PBS buffer solution; add pure water to prepare the concentrations of 1.6 ⁇ 10 7 , 8 ⁇ 10 7 , 4 ⁇ 10 8 , 2 ⁇ 10 9 and 1 ⁇ 10 10 CFU/mL bacterial liquid; heat the bacterial liquid at 100°C for 20 minutes to inactivate the bacterial cells; Bacteria solutions with different concentrations were lyophilized and dissolved in equal volumes of DEME medium.
  • the HaCaT cells were cultured in a 24-well plate for 24 hours, and the culture medium was removed; the above-mentioned inactivated Lactobacillus rhamnosus VHProbi O17 (bacteria amount: 0, 1 ⁇ 10 8 , 2 ⁇ 10 8 , 4 ⁇ 10 8 , 8 ⁇ 10 8 and 16 ⁇ 10 8 CFU/mL) DMEM medium, incubate for 2 hours; then replace each well with 500 ⁇ L fresh DMEM medium, and incubate for 24 hours.
  • the above-mentioned inactivated Lactobacillus rhamnosus VHProbi O17 bacteria amount: 0, 1 ⁇ 10 8 , 2 ⁇ 10 8 , 4 ⁇ 10 8 , 8 ⁇ 10 8 and 16 ⁇ 10 8 CFU/mL
  • MTT thiazolium blue
  • UVB induces HaCat cells to secrete pro-inflammatory cytokines
  • HaCaT cells were cultured in a 24-well plate for 24 hours, and the medium was removed; the above-mentioned VHProbi O17 containing inactivated Lactobacillus rhamnosus was added (the bacterial counts were 0, 1 ⁇ 10 8 , 2 ⁇ 10 8 , 4 ⁇ 10 8 , 8 ⁇ 10 8 CFU/mL) in DMEM medium, incubate for 2 h; aspirate the medium, add 150 ⁇ L of PBS buffer to each well; use ultraviolet lamp irradiation, the ultraviolet intensity is 250 ⁇ w/cm 2 , and the irradiation dose is 15 mJ/ cm. After culturing in DMEM medium for 24 h, cell viability was detected by MTT assay. HaCaT cells that were not pretreated with UV irradiation and Lactobacillus rhamnosus VHProbi O17 were used as controls.
  • pretreatment with inactivated Lactobacillus rhamnosus VHProbi O17 could significantly inhibit UVB-induced secretion of pro-inflammatory cytokines IL-6 and IL-8 from HaCaT cells.
  • UVB irradiation the contents of IL-6 and IL-8 in the culture supernatant of HaCaT cells increased sharply, with the highest reaching 478.41pg/mL and 522.583pg /mL;
  • the production of IL-6 and IL-8 was greatly reduced, by 34.4% and 54.2%, respectively (Fig. 6C, 6D).
  • the Lactobacillus rhamnosus VHProbi O17 provided by the present invention has no toxicity to cells, and can effectively alleviate the damage of cells caused by UVB radiation, and has achieved unexpected technical effects.
  • the activated Lactobacillus rhamnosus VHProbi O17 was inoculated in 100 mL of MRS medium, cultured at 37 °C for 56 h, 5 mL of the bacterial liquid was taken every 8 h, and centrifuged at 6000 ⁇ g for 10 min to obtain the supernatant. The supernatant was dialyzed with 1000Da dialysis bags for 48 hours, and the pure water was replaced every 12 hours. After dialysis, the content of exopolysaccharide was detected by phenol-sulfuric acid method. The results are shown in Figure 7. Lactobacillus rhamnosus VHProbi O17 reached a stable phase at 32 hours, and the production of exopolysaccharide reached the maximum at 40 hours, which was 0.603 mg/mL.
  • Lactobacillus rhamnosus VHProbi O17 was inoculated in 1L of MRS medium, aerobically fermented at 37°C for 40h, centrifuged at 6000 ⁇ g for 10min, and the supernatant was obtained. Concentrate the supernatant 5 times under reduced pressure, then add 2 times the volume of cold absolute ethanol, and let it stand at 4°C overnight; centrifuge at 12000 ⁇ g for 15 minutes to obtain a crude polysaccharide precipitate; repeat the alcohol precipitation step once. Add pure water to dissolve the crude polysaccharide precipitate, add 4% (w/v) trichloroacetic acid, let stand at 4°C for 2 hours, and then centrifuge at 8000 ⁇ g for 15 minutes to remove protein.
  • the dialyzed solution was connected to a chromatographic column (1.2 ⁇ 11.4cm) filled with DEAE- ⁇ Sphere filler, and the polysaccharide was eluted step by step with pure water and different concentrations of NaCl (0.05, 0.1, 0.2, 0.4, 0.6M),
  • the flow rate was 1mL/min, and then 5mL of eluent was collected in each tube, and the absorbance of each tube at 490nm was detected by the phenol-sulfuric acid method; the collection tubes containing polysaccharides were combined, and the samples were dialyzed and then lyophilized and concentrated for further research.
  • Mobile phase A (0.1M NaOH), mobile phase B (0.1M NaOH, 0.2M NaAc), flow rate 0.5mL/min; column temperature 30°C; elution gradient: 0min A/B (95:5, v/v ), 30min A/B (80: 20v/v), 30.1min A/B (60: 40v/v), 45min A/B (60: 40v/v), 45.1min A/B (95: 5v/v ), 60min A/B (95:5v/v). Finally, the chromatographic data analysis was performed using the software Chromeleon 7.2, and the results are shown in Figure 9.
  • the exopolysaccharide OP-2 was formulated into a 5mg/mL solution, centrifuged at 12000 ⁇ g for 10min, and the supernatant was filtered through a 0.22 ⁇ m microporous membrane, and then the sample was transferred to a 1.8mL injection vial.
  • a 20uL sample was injected into a high performance liquid chromatograph for analysis, equipped with a BRT105-104-102 tandem gel column and differential detector. According to the standard product (molecular weight: 1152, 5000, 11600, 23800, 48600, 80900, 148000, 273000, 409800, 667800Da) curve, the calculation formula is obtained to calculate the molecular weight of each sample, as shown in Figure 11.
  • the exopolysaccharide OP-2 produced by Lactobacillus rhamnosus VHProbi O17 provided by the invention is composed of D-galactosamine hydrochloride, arabinose, glucosamine hydrochloride, galactose, glucose and mannose 6 Monosaccharide composition, the molar ratio is 0.05:0.03:0.09:0.05:0.19:1.98. Infrared spectrum scanning results showed that exopolysaccharide OP-2 had ⁇ -mannopyranose. In addition, through gel column analysis, it was found that the molecular weight of exopolysaccharide OP-2 was 82.4kDa.
  • HaCatT cells were cultured in a 24-well plate for 24 hours, the medium was removed, and DMEM medium containing 0, 0.5, 1, 2, 4, 8, 16 mg/mL of purified exopolysaccharide OP-2 was added, and incubated for 2 hours ; Then each well was replaced with 500 ⁇ L of fresh DMEM medium and cultured for 24 hours. After cell culture, the cell morphology was observed by an inverted microscope, and the toxicity of exopolysaccharide OP-2 to HaCaT cells was evaluated by MTT method.
  • HaCat cells cultured in a 24-well plate for 24 hours were first incubated in DMEM medium containing 0, 1, 2, 4, 8 and 16 mg/mL exopolysaccharide OP-2 for 2 hours; Add 150 ⁇ L of PBS buffer solution; use ultraviolet light to irradiate, the ultraviolet intensity is 250 ⁇ w/cm 2 , and the irradiation doses are 7.5, 15 and 30 mJ/cm 2 . After irradiation, HaCat cells were cultured in DMEM medium for 24 hours, and the cell viability was detected by MTT method. HaCat cells that were not pretreated with UV irradiation and exopolysaccharide OP-2 were used as control.
  • IL-1 ⁇ As an example, after UVB induction, the concentration of IL-1 ⁇ in the supernatant of HaCat cells increased from 1.41pg/mL to 74.97pg/mL; After the HaCat cells were induced by UVB, the IL-1 ⁇ content in the supernatant was reduced by 20%-91%, and the lowest was 6.73pg/mL ( FIG. 13A ). Similarly, IL-6 and IL-8 also had similar results, and the contents were reduced by 67% and 23% respectively (Fig. 13C, 13D), which achieved unexpected technical effects.
  • the Lactobacillus rhamnosus VHProbi O17 provided by the present invention has strong antioxidant activity, and the scavenging rates for DPPH and hydroxyl radicals reach 51.73% and 26.04%, respectively.
  • the strain has no toxic effect on skin cells, and also has a certain growth-promoting effect.
  • Lactobacillus rhamnosus VHProbi O17 can effectively reduce the damage of UVB radiation to cells, significantly inhibit UVB-induced cell secretion and production of pro-inflammatory cytokines IL-6 and IL-8, and the secretion of IL-6 and IL-8 decreased respectively 34.4% and 54.2%.
  • Lactobacillus rhamnosus VHProbi O17 can produce a new exopolysaccharide OP-2, which is composed of six monosaccharides: D-galactosamine hydrochloride, arabinose, glucosamine hydrochloride, galactose, glucose and mannose.
  • the exopolysaccharide can significantly inhibit the secretion of IL-1 ⁇ , IL-6 and IL-83 cytokines from skin cells induced by UVB, and alleviate the damage of UVB to the skin.
  • the Lactobacillus rhamnosus VHProbi O17 provided by the invention and the exopolysaccharide produced by it can be widely used in cosmetics and have broad prospects.

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Abstract

本发明提供了一株新型鼠李糖乳杆菌(Lactobacillus rhamnosus)及其应用。所述的鼠李糖乳杆菌能高产胞外多糖，具有良好的抗氧化活性，并且能有效抑制皮肤分泌促炎症反应细胞因子，缓解紫外线UVB对皮肤的损伤，其保藏号为CCTCC NO：M2021589。所述鼠李糖乳杆菌对机体无毒害作用，有望用于制备防晒化妆品，应用前景广阔。

Description

一株高产胞外多糖的鼠李糖乳杆菌及其在缓解皮肤损伤中的应用
本申请要求于2021年09月07日提交中国专利局、申请号为202111043542.3、发明名称为“一株高产胞外多糖的鼠李糖乳杆菌及其在缓解皮肤损伤中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于益生菌筛选与应用技术领域，具体涉及一株高产胞外多糖的鼠李糖乳杆菌及其在缓解皮肤损伤中的应用。
背景技术
人体皮肤主要包含表皮层和真皮层2层组织。辐照到人体皮肤的太阳紫外线含有90-99％UVA和1-10％的UVB。在相同剂量下，UVB对皮肤的损伤程度比UVA高1000倍。并且UVB主要作用于表皮层的角质细胞(HaCaT)，引发细胞内蛋白、DNA和膜脂的损伤，造成代谢紊乱引起皮肤的光损伤，严重者造成皮肤癌。使用传统的防晒霜抗紫外可能会引起皮肤炎症和过敏等副作用。因此利用植物、动物和微生物产生的天然产物进行抗紫外作用受到了关注。目前已有研究发现天然类黄酮水飞蓟素通过上调ER alpha和ERbeta蛋白途径，保护细胞免受UVB损伤；海藻糖通过抑制MMP的表达来抵抗UVB诱导的皮肤老化；来自成团泛菌的胞外多糖能够保护HaCaT细胞免受UVB的损伤。
乳酸菌是普遍存在的益生菌，被认为是“一般安全菌”，其已广泛用于食品、药品和化妆品等领域。乳酸菌能够产生有机酸、抗菌肽、胞外多糖等天然代谢产物。乳酸菌胞外多糖(Exopolysaccharide，EPS)指的是乳酸菌在其生长以及代谢的过程中产生并且分泌到细胞壁外部的大分子，主要是依附细胞壁的荚膜多糖和进入发酵液中的黏液多糖。近些年的研究发现，乳酸菌及其胞外多糖在抗肿瘤，抗炎，调节免疫和抗菌等方面具有很好的应用效果。乳酸菌及其胞外多糖在抗紫外损伤方面的功能研究也是本领域的一个热点。
发明内容
本发明的目的是提供一株新型鼠李糖乳杆菌(Lactobacillus  rhamnosus)及其应用。所述的鼠李糖乳杆菌高产胞外多糖，具有良好的抗氧化活性，并且能够有效抑制皮肤分泌促炎症反应细胞因子，缓解紫外线UVB对皮肤的损伤，效果显著。
本发明一方面提供了一种鼠李糖乳杆菌，命名为鼠李糖乳杆菌VHProbi O17(Lactobacillus rhamnosus VHProbi O17)，已于2021年5月24日保藏于中国武汉武汉大学的中国典型培养物保藏中心，其保藏号为CCTCC NO：M2021589。
本发明一方面提供了鼠李糖乳杆菌VHProbi O17在在制备用于预防和缓解皮肤损伤的制品中的应用。
所述的制品为化妆品。
本发明一方面提供了鼠李糖乳杆菌VHProbi O17在生产胞外多糖中的应用。
本发明还提供了一种胞外多糖，是以鼠李糖乳杆菌VHProbi O17为发酵菌株生产获得的。
所述的胞外多糖由D-氨基半乳糖盐酸盐、阿拉伯糖、盐酸氨基葡萄糖、半乳糖、葡萄糖和甘露糖6种单糖组成，其摩尔比为0.05：0.03：0.09：0.05：0.19：1.98。
所述的胞外多糖含有β-吡喃甘露糖。
所述的胞外多糖的分子量为82.4kDa。
本发明还提供了所述胞外多糖在制备用于预防和缓解皮肤损伤的制品中的应用。
本发明还提供了一种化妆品，包含有鼠李糖乳杆菌VHProbi O17和/或鼠李糖乳杆菌VHProbi O17发酵生产的胞外多糖。
本发明提供的鼠李糖乳杆菌VHProbi O17具有很强的抗氧化活性，对DPPH和羟自由基的清除率分别达到51.73％和26.04％。
鼠李糖乳杆菌VHProbi O17对皮肤细胞无毒性影响，还具有一定的促生长作用，能有效缓解UVB辐照对于细胞的损伤。经灭活的鼠李糖乳杆菌VHProbi O17预处理后的HaCaT细胞，再被UVB辐照后，其活力下降幅度明显减小。其中，当菌量为8×10 8CFU/mL时，HaCaT细胞被UVB辐照后 的活性比不加菌预处理时提高了76.6％。该菌株还能显著抑制UVB诱导细胞分泌产生促炎症反应细胞因子IL-6和IL-8，IL-6和IL-8的分泌量分别降低了34.4％和54.2％，取得了意料不到的技术效果。
鼠李糖乳杆菌VHProbi O17能产生一种新的胞外多糖OP-2，由D-氨基半乳糖盐酸盐、阿拉伯糖、盐酸氨基葡萄糖、半乳糖、葡萄糖和甘露糖6种单糖组成，可以有效缓解UVB对皮肤的损伤。当辐照剂量为15mJ/cm 2时，不加胞外多糖预处理的HaCaT细胞经UVB辐照后的活性仅为27.2％，而当胞外多糖OP-2的添加量达16mg/mL时，预处理后的HaCaT细胞经UVB辐照后的活性高达65.7％，效果非常显著。胞外多糖OP-2还能有效抑制UVB诱导皮肤细胞分泌IL-1α、IL-6和IL-83种细胞因子。胞外多糖OP-2预处理后的HaCat细胞，再经UVB诱导，其上清液中IL-1α含量降低了20％-91％，最低降至6.73pg/mL，IL-6和IL-8含量也分别降低了67％和23％，取得了意料不到的技术效果。
本发明提供的鼠李糖乳杆菌VHProbi O17及其所产的胞外多糖可广泛应用于化妆品中，前景广阔。
生物保藏说明
鼠李糖乳杆菌，命名为鼠李糖乳杆菌VHProbi O17(Lactobacillus rhamnosus VHProbi O17)，已于2021年5月24日保藏于中国武汉武汉大学的中国典型培养物保藏中心，其保藏号为CCTCC NO：M2021589。
附图说明
图1为菌株O17的菌落图及革兰氏染色图；其中A为菌落图，B为革兰氏染色图；
图2为菌株O17的蛋白指纹图；
图3为菌株O17的Riboprinter指纹图谱；
图4为菌株O17的RAPD指纹图；
图5为菌株O17的rep-PCR指纹图；
图6为鼠李糖乳杆菌VHProbi O17缓解UVB对皮肤细胞损伤效果的对比图；
图7为鼠李糖乳杆菌VHProbi O17的生长曲线和胞外多糖的产生曲 线；
图8为鼠李糖乳杆菌VHProbi O17产胞外多糖的DEAE-μSphere洗脱色谱图；
图9为胞外多糖OP-2的离子色谱分析图；
图10为胞外多糖OP-2的红外光谱分析图；
图11为胞外多糖OP-2的凝胶柱分析图和标准曲线图；其中A为凝胶柱分析图，B为标准曲线图；
图12为胞外多糖OP-2缓解UVB对皮肤细胞损伤效果的对比图；
图13为胞外多糖OP-2抑制UVB诱导皮肤细胞因子产生的对比图。
具体实施方式
本发明所述筛选方法并不局限于实施例所述，已知的能够达到筛选目的的方法均可以，实施例的筛选说明只是对本发明的说明，并不是对本发明保护范围的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
下面结合具体实施例，对本发明做进一步阐述。
实施例1 高产胞外多糖的菌株筛选
配制MRS琼脂培养基：纯水1000mL，蛋白胨10g，牛肉浸取物10g，酵母提取物5.0g，乙酸钠5g，葡萄糖5g，磷酸二氢钾2g，吐温80 1.0mL，柠檬酸二胺2.0g，碳酸钙20g，七水硫酸镁0.58g，七水硫酸锰0.25g，琼脂15g，调pH 6.2-6.5，121℃高压灭菌15min。
将菌种库中保存的200余株乳酸菌分别活化后，接种于MRS平板，37℃培养48h，观察菌株“粘稠”表型并检测菌落被接种环接触时形成丝的长度。最终筛选出一株菌落粘稠度高、拉丝最长的乳酸菌，命名为O17。
实施例2 菌株鉴定
2.1菌落形态鉴定
将菌株O17接种于MRS琼脂培养基上，37℃培养48h后，可见O17单菌落光滑，呈灰白色，显微镜下为短棒状，两端平齐，O17单菌落及光学显微镜下照片见图1。
2.2 MALDI-TOF-MS检测菌株的全蛋白表达
按照0.1％的接种量在MRS液体培养基中接种新鲜菌液，37℃，150rpm培养48h后，收集菌体，无菌水洗涤4次，晾干表面水分。然后取少量新鲜菌体以薄膜的形式均匀涂布于靶板上，加1μL裂解液覆盖样品，晾干后，再加1μL基质溶液覆盖样品，晾干后，将样品靶放入质谱仪进行鉴定。用激光辐照样品与基质形成的共结晶薄膜，使样品中蛋白质电离，离子在10～20KV电场作用下加速飞过飞行管道，根据到达检测器的飞行时间不同检测不同分子量蛋白质。利用软件Autof Acquirer V2.0.59完成数据分析，菌株O17在m/z 2946.082，3476.573，3791.097，4692.724，5892.635，6953.051，7581.626，9382.474等处有明显的离子峰，结果如图2所示。数据比对分析后发现菌株O17与鼠李糖乳杆菌的蛋白表达相似性最高。
2.3分子生物学鉴定
2.4.1 16s rDNA序列分析
1、基因组DNA提取
参照天根细菌基因组DNA提取试剂盒(目录号：DP302)操作。
2、16s rDNA基因扩增
1)引物序列：
27F：AGAGTTTGATCCTGGCTCA；
1492R：GGTTACCTTGTTACGACTT。
2)反应体系(50μL)
表2：16s rDNA PCR扩增体系表
Figure PCTCN2022090284-appb-000001
3)电泳验证PCR产物核酸电泳结果为1500bp左右时符合要求。
4)PCR产物测序
通过测序获得O17菌株的16s rDNA序列SEQ ID NO:1，并将该序列在NCBI数据库中进行比对。结果显示SEQ ID NO:1与鼠李糖乳杆菌(Lactobacillus rhamnosus)的相似性最高，因此，初步确定O17菌株为鼠李糖乳杆菌(Lactobacillus rhamnosus)。
SEQ ID NO:1：
Figure PCTCN2022090284-appb-000002
2.4.2 Riboprinter指纹图谱
用一根取菌棒从琼脂培养基平板上沾取已纯化好的单菌落，将其放入有缓冲液的样品管中，用手持搅拌器搅拌使其在缓冲液中悬浮，然后将样 品架放入加热器中灭活后放入Riboprinter系统中，样品经过DNA制备、转膜、成像检测及数据处理后，得到菌株O17的Riboprinter指纹图谱(图3)。
2.4.3 RAPD和rep-PCR指纹图谱鉴定
1、RAPD指纹图谱鉴定
1)引物序列：M13(5’-GAGGGTGGCGGTTCT-3’)；
2)RAPD反应体系
表3 RAPD反应体系
Figure PCTCN2022090284-appb-000003
3)电泳
制备1.5％的琼脂糖凝胶板，DL2000 DNA Marker作为结果对照，稳压100V电泳80min，最后利用凝胶成像系统检测电泳图。菌株O17的RAPD指纹图谱如图4所示。
2、rep-PCR指纹图谱
1)rep-PCR引物
Figure PCTCN2022090284-appb-000004
2)rep-PCR的反应体系
表4 rep-PCR的反应体系
Figure PCTCN2022090284-appb-000005
Figure PCTCN2022090284-appb-000006
3)电泳
DL2000 DNA Marker作为结果对照。电压100V，电泳时间80min检测扩增结果。菌株O17的的rep-PCR指纹图谱如图5所示。
综上，结合O17菌株的菌落形态特征，蛋白指纹图谱，以及分子生物学鉴定结果，可以得出结论，本发明提供的O17菌株为一株新的鼠李糖乳杆菌，并将其命名为鼠李糖乳杆菌VHProbi O17(Lactobacillus rhamnosus VHProbi O17)。
实施例3 鼠李糖乳杆菌VHProbi O17的抗氧化活性
1、菌株清除DPPH自由基能力测定
1)PBS菌悬液制备
将生长状态优良的单菌落接种于3mL的MRS液体培养基中，37℃条件下培养24h，以此培养液为接种液，按照2％的接种量接种于50mL的MRS液体培养基中，静置培养24h，获得菌株的培养液。吸取1mL菌液收集菌体后用1mLPBS缓冲液洗涤菌体2遍后再加入2mLPBS溶液重悬菌体备用。
2)菌株清除DPPH自由基能力的测定
取1mL待测菌株的PBS菌悬液，加入1mL 0.4mM的现配的DPPH自由基溶液，混合均匀后然后置于室温温度下遮光反应30min，然后测定样品在波长517nm处的吸光度A样品，测3次平行。对照组样品以等体积PBS溶液和DPPH·乙醇混合液，并以等体积PBS菌悬液和乙醇混合液空白调零。清除率按下列公式计算：清除率％＝[1-(A 样品-A 空白)/A 对 照]×100％。
2、菌株清除羟自由基能力的测定
将100ul 5mM的水杨酸钠-乙醇溶液，100ul 5mM的硫酸亚铁，500μL去离子水和200ul乳酸菌PSP溶液菌悬液混匀后加入100μL过氧化氢溶液(3mM)，37℃水浴15min后，4000rpm离心10min收集上清在510nm 波长处测量样品吸光度。羟自由基清除率按照下列公式进行计算。
清除率％＝(A 样品-A 控制)/(A 空白-A 控制)×100％。
其中：A 控制为硫酸亚铁、过氧化氢和水杨酸钠混合溶液的吸光度，A 空白为硫酸亚铁和水杨酸钠混合溶液的吸光度。
表5 抗氧化活性
Figure PCTCN2022090284-appb-000007
从表5的数据可以看出，本发明提供的鼠李糖乳杆菌VHProbi O17能有效清除DPPH和羟自由基，清除率分别达到51.73％和26.04％。从而说明，鼠李糖乳杆菌VHProbi O17具有很强的抗氧化活性，取得了意料不到的技术效果。
实施例4 鼠李糖乳杆菌VHProbi O17抗UVB辐射的效果
1、HaCaT细胞培养
将HaCaT角质细胞在含10％(v/v)FBS和1％(v/v)青霉素-链霉素的高糖DMEM培养基中进行厌氧培养。培养条件：在二氧化碳培养箱中加有5％(v/v)的二氧化碳，37℃，培养基每3天换一次。
2、鼠李糖乳杆菌VHProbi O17对细胞的毒性分析
将鼠李糖乳杆菌VHProbi O17接种于100mLMRS培养基中，37℃有氧发酵40h；6000×g离心10min，弃上清；将菌体用PBS缓冲液清洗2次；加纯水配制浓度分别为1.6×10 7、8×10 7、4×10 8、2×10 9和1×10 10CFU/mL的菌液；将菌液分别在100℃条件下加热20min，使菌体灭活；将不同浓度的菌液冻干后备用，并分别用等体积的DEME培养基溶解。
将HaCaT细胞在24孔板中培养24h，移除培养基；分别加入上述含灭活鼠李糖乳杆菌VHProbi O17(菌量为0，1×10 8，2×10 8，4×10 8，8×10 8和16×10 8CFU/mL)的DMEM培养基，孵育2h；然后每孔更换为500μL新鲜DMEM培养基，培养24h。通过MTT法评估鼠李糖乳杆菌VHProbi O17对细胞的毒性，具体为：在24孔板中每孔加入50μL、3mg/mL的MTT(噻唑蓝)溶液，避光孵育3h；从孔中吸出溶液，并且每孔中加入 500μL的二甲亚砜溶解并使用酶标仪在450nm处检测吸光度。
3、UVB诱导HaCat细胞分泌促炎症细胞因子
将HaCaT细胞在24孔板中培养24h，移除培养基；分别加入上述含灭活鼠李糖乳杆菌VHProbi O17(菌量分别为0，1×10 8，2×10 8，4×10 8，8×10 8CFU/mL)的DMEM培养基，孵育2h；吸出培养基，每孔中加入150μL PBS缓冲液；使用紫外灯辐照，紫外强度为250μw/cm 2，辐照剂量为15mJ/cm。用DMEM培养基培养24h后，通过MTT法检测细胞活性。以未被紫外辐照和鼠李糖乳杆菌VHProbi O17预处理的HaCaT细胞作对照。
4、细胞因子分析
取上述经紫外辐照后的细胞培养上清液，利用ELISA试剂盒分别检测促炎症反应细胞因子白细胞介素-6(IL-6)和白细胞介素-8(IL-8)的浓度，参考操作指示进行。结果如图6所示。
由图6的结果可知，在菌量≤8×10 8CFU/mL时，灭活的鼠李糖乳杆菌VHProbi O17对HaCaT细胞生长无抑制作用；在2×10 8～8×10 8CFU/mL范围内，随着菌量的增加，灭活的鼠李糖乳杆菌VHProbi O17对细胞生长有一定的促进作用(图6A)。
UVB辐照后，HaCaT细胞的活性均得到不同程度的下降。但经灭活的鼠李糖乳杆菌VHProbi O17预处理后的HaCaT细胞，再被UVB辐照后，其活力下降幅度明显减小。其中，当菌量为8×10 8CFU/mL时，HaCaT细胞经UVB辐照后的活性比不加菌预处理时提高了76.6％(图6B)。
此外，用灭活的鼠李糖乳杆菌VHProbi O17预处理可以显著抑制UVB诱导HaCaT细胞分泌促炎症反应细胞因子IL-6和IL-8。经UVB辐照后，HaCaT细胞培养上清液中IL-6和IL-8含量急剧增加，最高达到478.41pg/mL和522.583pg/mL；但用菌量为8×10 8CFU/mL的灭活鼠李糖乳杆菌VHProbi O17预处理后，IL-6和IL-8产生量大幅减少，分别降低了34.4％和54.2％(图6C，6D)。
综上所述，本发明提供的鼠李糖乳杆菌VHProbi O17对细胞没有毒性，且能有效缓解UVB辐照对于细胞的损伤，取得了意料不到的技术效果。
实施例5 鼠李糖乳杆菌VHProbi O17产胞外多糖的制备
1、胞外多糖的制备
将活化后的鼠李糖乳杆菌VHProbi O17接种于100mL MRS培养基中，37℃培养56h，每8h取5mL菌液，6000×g离心10min，得到上清液。将上清液分别用1000Da透析袋透析48h，每12h更换一次纯水。透析完成后用苯酚-硫酸法检测胞外多糖的含量。结果如图7所示，鼠李糖乳杆菌VHProbi O17在32h时达到稳定期，在40h时胞外多糖产量达到最大值，为0.603mg/mL。
2、胞外多糖的分离提取
将鼠李糖乳杆菌VHProbi O17接种于1L的MRS培养基中，37℃有氧发酵40h，6000×g离心10min，得到上清液。将上清液减压浓缩5倍，再加入2倍体积的冷的无水乙醇，4℃静置过夜；12000×g离心15min，得到粗多糖沉淀；重复醇沉步骤1次。将粗多糖沉淀加纯水溶解后加入4％(w/v)的三氯乙酸，4℃静置2h后，8000×g离心15min，除去蛋白。除蛋白后，用6mol/L的NaOH调溶液的pH为4，避免多糖在低pH环境下降解，再加入2倍体积的冷的无水乙醇并静置过夜，12000×g离心15min得到粗多糖。将得到的粗多糖溶解后用1000Da透析袋透析48h，每12h更换一次纯水，透析结束后用1mol/L的NaOH调溶液的pH为6.5。
透析后的溶液接入填有DEAE-μSphere填料的层析柱中(1.2×11.4cm)，多糖用纯水和不同浓度的NaCl分步洗脱(0.05，0.1，0.2，0.4，0.6M)，流速为1mL/min，之后按每管5mL收集洗脱液，并用苯酚-硫酸法检测每管在490nm的吸光度；合并含多糖的收集管，样品透析后冻干浓缩用于下一步研究。
结果如图8所示：0.1M NaCl洗脱下来的胞外多糖活性最佳，量最多，记为OP-2。
实施例6 鼠李糖乳杆菌VHProbi O17产胞外多糖的结构表征
1)单糖组成
在密闭管中，将约5mg的胞外多糖OP-2样品在105℃下用2M三氟乙酸水解6h并用氮气干燥样品。之后加入甲醇洗涤并吹干，重复洗涤3 次。将残余物重新溶解在去离子水中，并通过0.22μm微孔滤膜过滤后，使用ICS5000离子色谱检测分析。色谱参数：采用Dionex TMCarboPac TM PA10(250×4.0mm，10um)液相色谱柱；进样量为5uL。流动相A(0.1M NaOH)，流动相B(0.1M NaOH，0.2M NaAc)，流速0.5mL/min；柱温为30℃；洗脱梯度：0min A/B(95:5，v/v)，30min A/B(80：20v/v)，30.1min A/B(60：40v/v)，45min A/B(60：40v/v)，45.1min A/B(95：5v/v)，60min A/B(95：5v/v)。最后利用软件Chromeleon 7.2进行色谱数据分析，结果如图9所示。
2)红外光谱分析
将干燥的2mg胞外多糖OP-2与200mg KBr粉末混合，研磨均匀并压片。FT-IR spectrometry被用于样品的红外扫描，波长范围4000-400cm -1。记录红外光谱图，如图10所示。
3)分子量
将胞外多糖OP-2配制成5mg/mL溶液，12000×g离心10min，上清液用0.22μm的微孔滤膜过滤，然后将样品转置于1.8mL进样小瓶中。20uL的样品被注射入高效液相色谱仪中进行分析，色谱仪带有一根BRT105-104-102串联凝胶柱和示差检测器。根据标准品(分子量：1152，5000，11600，23800，48600，80900，148000，273000，409800，667800Da)曲线，得出计算公式进而计算出每个样品的分子量大小，如图11所示。
从色谱分析结果可知，本发明提供的鼠李糖乳杆菌VHProbi O17产生的胞外多糖OP-2由D-氨基半乳糖盐酸盐、阿拉伯糖、盐酸氨基葡萄糖、半乳糖、葡萄糖和甘露糖6种单糖组成，摩尔比为0.05：0.03：0.09：0.05：0.19：1.98。红外光谱扫描结果显示，胞外多糖OP-2存在β-吡喃甘露糖。另外，通过凝胶柱分析发现，胞外多糖OP-2的分子量为82.4kDa。
上述结果显示，鼠李糖乳杆菌VHProbi O17产生的胞外多糖OP-2与已知的胞外多糖相比，结构组成均不相同，因此该多糖是一种新的乳酸菌胞外多糖。
实施例7 胞外多糖OP-2对HaCaT细胞的影响
1、HaCaT细胞培养
参考实施例4。
2、胞外多糖OP-2对细胞的毒性
将HaCatT细胞在24孔板中培养24h，移除培养基，再分别加入含0，0.5，1，2，4，8，16mg/mL纯化的胞外多糖OP-2的DMEM培养基，孵育2h；然后每孔更换为500μL的新鲜DMEM培养基培养24h。细胞培养结束后，通过倒置显微镜观察细胞形态，采用MTT法评估胞外多糖OP-2对HaCaT细胞的毒性。
从图12A的结果可知，经胞外多糖OP-2处理后的HaCat细胞活性没有明显下降，从而说明胞外多糖OP-2对HaCat细胞没有毒性影响。
3、UVB诱导HaCat细胞
将在24孔板中培养24h的HaCat细胞先在分别含0、1、2、4、8和16mg/mL胞外多糖OP-2的DMEM培养基中孵育2h；然后吸出培养基，每孔中加入150μL PBS缓冲液；使用紫外灯辐照，紫外强度为250μw/cm 2，辐照剂量分别为7.5，15和30mJ/cm 2。辐照后，将HaCat细胞在DMEM培养基中继续培养24h后，采用MTT法检测细胞活性。以未被紫外辐照和胞外多糖OP-2预处理的HaCat细胞作对照。
从图12B，12C，12D的结果可知，UVB辐照可以显著降低HaCaT细胞的活性，但经胞外多糖OP-2预处理后，UVB对细胞的损伤被缓解。当辐照剂量为15mJ/cm 2时，不加胞外多糖预处理的HaCaT细胞经UVB辐照后的活性仅为27.2％，而当胞外多糖OP-2的添加量达16mg/mL时，预处理后的HaCaT细胞经UVB辐照后的活性高达65.7％，取得了意料不到的技术效果。
4、细胞因子分析
取上述辐照剂量为15mJ/cm 2的HaCat细胞上清液，利用ELISA试剂盒分别检测其中四种促炎症细胞因子IL-1α，IL-1β，IL-6，IL-8的浓度，结果如图13所示。
从图13的结果可知，经过UVB诱导后，HaCaT细胞分泌的促炎症反应因子IL-1α、IL-6和IL-8急剧增多，而炎症因子IL-1β的分泌量没有显著变化。而经胞外多糖OP-2预处理后的HaCaT细胞，再经UVB诱导，其分泌 IL-1α、IL-6和IL-8均受到一定的抑制。以IL-1α为例，UVB诱导后，HaCat细胞上清中IL-1α的浓度从1.41pg/mL增加到了74.97pg/mL；而通过胞外多糖OP-2(1-16mg/mL)预处理后的HaCat细胞，再经UVB诱导，其上清液中IL-1α含量降低了20％-91％，最低降至6.73pg/mL(图13A)。同样地，IL-6和IL-8也具有类似的结果，含量分别降低了67％和23％(图13C、13D)，取得了意料不到的技术效果。
综上所述，本发明提供的鼠李糖乳杆菌VHProbi O17具有很强的抗氧化活性，对DPPH和羟自由基的清除率分别达到51.73％和26.04％。该菌株对皮肤细胞无毒性影响，还具有一定的促生长作用。鼠李糖乳杆菌VHProbi O17能有效减轻UVB辐照对于细胞的损伤，显著抑制UVB诱导细胞分泌产生促炎症反应细胞因子IL-6和IL-8，IL-6和IL-8的分泌量分别降低了34.4％和54.2％。鼠李糖乳杆菌VHProbi O17能产生一种新的胞外多糖OP-2，由D-氨基半乳糖盐酸盐、阿拉伯糖、盐酸氨基葡萄糖、半乳糖、葡萄糖和甘露糖6种单糖组成。该胞外多糖可以显著抑制UVB诱导皮肤细胞分泌IL-1α、IL-6和IL-83种细胞因子，缓解UVB对皮肤的损伤。本发明提供的鼠李糖乳杆菌VHProbi O17及其所产生的胞外多糖可广泛应用于化妆品中，前景广阔。
以上对本发明所提供的一株高产胞外多糖的鼠李糖乳杆菌及其在缓解皮肤损伤中的应用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (10)

  1. 一种鼠李糖乳杆菌，其特征在于，所述鼠李糖乳杆菌的保藏号为CCTCC NO：M2021589。
  2. 权利要求1所述的鼠李糖乳杆菌在制备用于预防或缓解皮肤损伤的制品中的应用。
  3. 如权利要求2所述的应用，其特征在于，所述的制品为化妆品。
  4. 一种胞外多糖，其特征在于，所述胞外多糖是以权利要求1所述的鼠李糖乳杆菌为发酵菌株生产获得的。
  5. 如权利要求4所述的胞外多糖，其特征在于，所述胞外多糖由D-氨基半乳糖盐酸盐、阿拉伯糖、盐酸氨基葡萄糖、半乳糖、葡萄糖和甘露糖6种单糖组成。
  6. 如权利要求5所述的胞外多糖，其特征在于，所述胞外多糖中D-氨基半乳糖盐酸盐、阿拉伯糖、盐酸氨基葡萄糖、半乳糖、葡萄糖和甘露糖的摩尔比为0.05：0.03：0.09：0.05：0.19：1.98。
  7. 如权利要求5或6所述的胞外多糖，其特征在于，所述胞外多糖含有β-吡喃甘露糖。
  8. 如权利要求5或6所述的胞外多糖，其特征在于，所述胞外多糖的分子量为82.4kDa。
  9. 权利要求4-8任一所述的胞外多糖在制备用于预防或缓解皮肤损伤的制品中的应用。
  10. 一种化妆品，其特征在于，所述化妆品包含有权利要求1所述的鼠李糖乳杆菌和/或权利要求4-8任一所述的胞外多糖。
PCT/CN2022/090284 2021-09-07 2022-04-29 一株高产胞外多糖的鼠李糖乳杆菌及其在缓解皮肤损伤中的应用 WO2023035634A1 (zh)

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