WO2023035633A1 - 一株对皮肤损伤具有保护作用的鼠李糖乳杆菌及其应用 - Google Patents

一株对皮肤损伤具有保护作用的鼠李糖乳杆菌及其应用 Download PDF

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WO2023035633A1
WO2023035633A1 PCT/CN2022/090283 CN2022090283W WO2023035633A1 WO 2023035633 A1 WO2023035633 A1 WO 2023035633A1 CN 2022090283 W CN2022090283 W CN 2022090283W WO 2023035633 A1 WO2023035633 A1 WO 2023035633A1
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lactobacillus rhamnosus
skin
vhprobi
cells
strain
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French (fr)
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段治
吴松洁
崔洪昌
张景燕
郭超群
李凯玲
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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
    • A61K8/96—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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning 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
    • A61Q19/004—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
    • 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 relates to the technical field of functional microorganism screening and application, in particular to a Lactobacillus rhamnosus, in particular to a strain of Lactobacillus rhamnosus capable of protecting skin damage and its application.
  • Probiotics are a type of active microorganisms that are beneficial to the host by colonizing in the human body and changing the composition of the flora in a certain part of the host.
  • Probiotics were first discovered by Russian microbiologists in the early 20th century. It has been developed for more than a century, and its concept and efficacy have been recognized by scientists and consumers. Especially in terms of regulating the balance of intestinal flora, anti-pathogen effects, affecting the human immune system, and enhancing immunity, etc., it has been deeply rooted in the hearts of the people.
  • probiotic preparations are also increasingly used in healthy skin care, prevention and treatment of skin diseases, and skin anti-oxidative aging. The advantage is that this method is effective for users, and it is more natural without Side effects represent an emerging area of skin health.
  • the skin is the largest organ of the human body. It is in direct contact with the external environment. It is an important structural boundary and sensory interface of the human body. It is also an immunogenic organ. It is the first line of defense against external allergens and a biosensor. and fungal attack. Recent studies aimed at understanding skin barrier function have shown that the physical, immunological, and cell biological properties of the skin are closely related to the skin microbiota. Several clinical studies have shown that probiotics have positive effects on the skin, directly or indirectly. Evidence shows that probiotic therapy has great potential in the prevention and treatment of skin diseases such as skin eczema, atopic dermatitis, acne, allergic inflammation or skin sensitivity, UV-induced skin damage, and wound protection.
  • skin diseases such as skin eczema, atopic dermatitis, acne, allergic inflammation or skin sensitivity, UV-induced skin damage, and wound protection.
  • the object of the present invention is to provide a novel strain of Lactobacillus rhamnosus (Lactobacillus rhamnosus) and application thereof.
  • the Lactobacillus rhamnosus is isolated from the feces of healthy infants, and it has been verified that it has a protective effect on skin damage caused by various factors.
  • the present invention provides a strain of Lactobacillus rhamnosus, named as Lactobacillus rhamnosus VHProbi E06 (Lactobacillus rhamnosus VHProbi E06), which has been preserved in the Chinese Typical Culture Collection of Wuhan University, Wuhan, China on May 24, 2021 Center, its deposit number is CCTCC NO: M2021588.
  • One aspect of the present invention provides the application of Lactobacillus rhamnosus VHProbi E06 in the preparation of products for preventing or alleviating skin damage.
  • the product is cosmetic or medicine.
  • the present invention also provides a cosmetic with sunscreen or anti-aging effects, comprising Lactobacillus rhamnosus VHProbi E06 and/or a fermentation product of Lactobacillus rhamnosus VHProbi E06.
  • the present invention also provides a medicine for alleviating skin damage, comprising Lactobacillus rhamnosus VHProbi E06 and/or a fermentation product of Lactobacillus rhamnosus VHProbi E06.
  • Lactobacillus rhamnosus VHProbi E06 provided by the invention has strong acid resistance; does not produce hemolysin, does not dissolve blood cells, is sensitive to common antibiotics such as erythromycin and tetracycline, and has good biological safety; can tolerate higher salt degree, the maximum tolerated salt concentration is higher than 8%.
  • the Lactobacillus rhamnosus has strong antioxidant capacity and can effectively scavenge DPPH free radicals with a scavenging rate of 38.22%.
  • the lactobacillus rhamnosus can effectively promote the proliferation of skin keratinocytes (HaCat cells). Compared with the control group, the viability of HaCat cells in the two treatment groups added with Lactobacillus rhamnosus VHProbi E06 heat-inactivated cells was significantly improved.
  • the Lactobacillus rhamnosus can effectively relieve skin cell damage caused by Staphylococcus aureus infection.
  • the mortality of HaCaT cells in the control group infected by Staphylococcus aureus was as high as 56.6%, while the mortality of HaCaT cells in the treatment group of probiotics treated with heat-killed Lactobacillus rhamnosus VHProbi E06 dropped to 41.3%. significantly
  • the Lactobacillus rhamnosus can effectively alleviate skin cell damage caused by hydrogen peroxide oxidation. Compared with the control group, the viability of HaCat cells in the hydrogen peroxide injury group decreased significantly, only 49.7%; while the cell viability of the probiotic treatment group pretreated with inactivated Lactobacillus rhamnosus VHProbi E06 reached 59.1%, which increased obvious.
  • the Lactobacillus rhamnosus has significant anti-inflammatory and immunoregulatory effects on skin damage caused by UVB radiation, and can effectively relieve skin cell damage caused by UVB radiation.
  • the secretion levels of three pro-inflammatory cytokines, IL-1 ⁇ , IL-1 ⁇ and IL-8, in skin cells were significantly increased;
  • the secretion levels of IL-1 ⁇ , IL-1 ⁇ and IL-8 in the skin cells of the probiotic treatment group after pretreatment with inactivated Lactobacillus plumsus VHProbi E06 decreased significantly; and the vitality of the skin cells in the probiotic treatment group could reach 70.4 %, much higher than the UVB damage group without pretreatment, the effect is very significant.
  • the Lactobacillus rhamnosus can relieve and protect the damaged skin barrier of the 3D reconstructed human skin model stimulated by TritonX-100.
  • the transmembrane resistance value of the skin model with 0.1% TritonX-100 solution on the surface decreased significantly by 60.2%, while the skin of the probiotics treatment group pretreated with Lactobacillus rhamnosus VHProbi E06 inactivated cells
  • the decline of the transmembrane resistance value of the model was eased, only 50.6% lower than that of the control group.
  • the Lactobacillus rhamnosus VHProbi E06 provided by the invention has a certain protective effect on skin damage caused by various factors, can be widely used in cosmetics or medicines, and has broad application prospects.
  • Lactobacillus rhamnosus VHProbi E06 (Lactobacillus rhamnosus VHProbi E06), has been preserved in the Chinese Center for Type Culture Collection of Wuhan University in Wuhan, China on May 24, 2021, and its preservation number is CCTCC NO: M2021588.
  • Fig. 1 is the colony morphology figure of VL-2 bacterial strain
  • Figure 2 is the Riboprinter fingerprint of the VL-2 strain
  • Figure 3 is the RAPD fingerprint of the VL-2 strain
  • Fig. 4 is the rep-PCR fingerprint of VL-2 bacterial strain
  • Figure 5 is a comparison chart of HaCat cell viability in each group
  • Figure 6 is a comparison chart of HaCat cell death rate infected by Staphylococcus aureus
  • Figure 7 is a comparison chart of HaCat cell viability damaged by hydrogen peroxide oxidation
  • Figure 8 is a comparison chart of HaCat cell viability damaged by UVB
  • Figure 9 is a comparison of cytokines in the 3D skin model of UVB damage.
  • Figure 10 is a comparison chart of cell viability in a 3D skin model damaged by UVB;
  • Figure 11 is a comparison chart of the transmembrane resistance value of the 3D skin model damaged by TritonX-100.
  • 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 Man Rogosa Sharpe
  • agar medium purified water 1000mL, peptone 10g, beef extract 10g, yeast extract 5.0g, sodium acetate 5g, glucose 5g, potassium dihydrogen phosphate 2g, Tween 80 1.0mL, lemon Acid diamine 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.
  • the 9 strains of Lactobacillus VL-1, VL-2,..., VL-9 obtained by screening were inoculated in the above-mentioned acid-resistant medium according to the inoculum amount of 6%, and cultured at 37°C for 72 hours, and the fermentation liquid was taken for bacterial fermentation. volume count.
  • Embodiment 2 strain identification
  • VL-2 strain was inoculated on MRS agar medium and cultured at 37°C for 48h.
  • the colony morphology of VL-2 is shown in Figure 1.
  • the colony is milky white, the diameter of the colony is about 2 mm, the surface is wet, and it is short rod-shaped under the microscope. The two ends are round and usually appear alone in a short chain.
  • VL-2 bacterial liquid Under sterile conditions, take an appropriate amount of fresh VL-2 bacterial liquid, centrifuge at 5000rpm/min for 5min, wash twice with PBS buffer, resuspend the bacterial cells with the same volume of PBS buffer and dilute 50 times as a bacterial suspension.
  • the basal medium formula used in this experiment is as follows:
  • the 16s rDNA sequence SEQ ID NO:1 of the VL-2 strain was obtained by sequencing, and the sequence was compared in the NCBI database, and the VL-2 strain was preliminarily determined to be Lactobacillus rhamnosus. The sequence looks like this:
  • 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 the VL-2 strain is shown in Figure 3.
  • the DL2000 DNA Marker was used as the result control.
  • the voltage is 100V, and the electrophoresis time is 80min to detect the amplification result.
  • the rep-PCR fingerprint of the VL-2 strain is shown in FIG. 4 .
  • VL-2 strain is a new strain of Lactobacillus rhamnosus, which was named Lactobacillus rhamnosus VHProbi E06 (Lactobacillus rhamnosus VHProbi E06), which was released in 2021. It was deposited in the Chinese Type Culture Collection Center of Wuhan University in Wuhan, China on May 24, and its collection number is CCTCC NO: M2021588.
  • inoculum Inoculate the cryopreserved Lactobacillus rhamnosus VHProbi E06 strain on MRS agar medium by streaking, culture at 37°C for 24-48 hours, and subculture once in MRS liquid medium. Lactobacillus rhamnosus VHProbi E06 was inoculated into fresh MRS liquid medium at 37°C for 24-48 hours with an inoculation amount of 5%, and fresh bacterial liquid was obtained as the inoculum.
  • Streak culture Streak inoculate the test strain on the prepared blood cell plate, culture in a 37°C incubator, and observe whether the test bacteria has hemolysis for 24 to 48 hours.
  • inoculum solution take an appropriate amount of fresh bacterial solution (24h, 37°C culture), centrifuge at 5000rpm for 5min, wash once with sterile normal saline, resuspend the bacteria with the same volume of normal saline and dilute 50 times as the inoculum .
  • Lactobacillus rhamnosus VHProbi E06 provided by the present invention is sensitive to common antibiotics such as erythromycin and tetracycline, and has good biological safety.
  • Use this culture medium as the inoculum inoculate it into 50mL MRS liquid medium according to the inoculation amount of 2%, and culture it statically 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.
  • Lactobacillus rhamnosus VHProbi E06 provided by the invention can effectively scavenge DPPH free radicals, and the scavenging rate reaches 38.22%, which is significantly higher than Lactobacillus paracasei (L.paracasei) IMC-4 bacterial strain.
  • Lactobacillus rhamnosus VHProbi E06 was cultured in MRS liquid medium to the stationary phase, washed 3 times with sterile PBS, resuspended to 5 ⁇ 10 7 CFU/mL with sterile PBS, and heated in a water bath at 70°C for 20 minutes. Inactivated spare.
  • Human immortalized keratinocytes HaCat are cultured to the required amount in high-sugar DMEM (10% FBS) medium, trypsinized and counted, and the cell suspension is added to a 24-well plate, and the number of cells in each cell culture well is 2 ⁇ 10 5 , the amount of culture solution added to each well was 0.6ml. After being cultured in a carbon dioxide incubator (5% CO2, 37° C.) for 24 hours, subsequent experiments were carried out.
  • Cell viability (%) (absorbance of detection sample-absorbance of blank)/(absorbance of control group-absorbance of blank) ⁇ 100.
  • the original culture medium of HaCaT cells was replaced with non-resistance serum-free DMEM medium.
  • the experiment set up a control group and a probiotic treatment group, in which: 1 ⁇ L of Staphylococcus aureus bacterial solution was inoculated in each well of cells in the control group; , multiplicity of infection) value of 10 was added to Lactobacillus rhamnosus VHProbi E06 heat-inactivated thallus.
  • control group normal culture of cells
  • hydrogen peroxide injury group adding hydrogen peroxide solution with a final concentration of 0.5 mM
  • probiotic treatment group first inactivate Lactobacillus rhamnosus VHProbi E06 according to the ratio of MOI value 10
  • the bacteria were added to the HaCat cells, placed in a 37° C., 5% carbon dioxide incubator and incubated for 3 hours, and then a hydrogen peroxide solution with a final concentration of 0.5 mM was added.
  • the above groups of cells were placed in a 37°C, 5% carbon dioxide incubator to continue culturing for 1 hour.
  • the original culture medium was discarded for each group of cells, rinsed twice with PBS, and 0.6 mL of fresh culture medium was added to each well. Place in a 37°C, 5% carbon dioxide incubator to continue culturing for 16 hours. MTT solution with a final concentration of 0.3 mg/ml was added to each cell culture well to be tested, and placed in a 37° C., 5% carbon dioxide incubator for 3 hours. Carefully discard the supernatant, add 500ul of DMSO to each cell culture well of the 24-well plate, incubate at 37°C for 30min to fully dissolve the purple crystals, and measure the absorbance value at 490nm with a microplate reader. Three parallel samples were set up in each group for detection. The cell viability of each group was calculated separately.
  • Cell viability% (absorbance of detection sample-absorbance of blank)/(absorbance of control group-absorbance of blank) ⁇ 100.
  • the experiment set up the control group, the UVB damage group, and the probiotics treatment group in which:
  • Control group cells were cultured normally, without probiotics, and without UVB irradiation;
  • UVB damage group irradiate under UVB ultraviolet lamp, the light dose is 60mJ/cm2;
  • Probiotics treatment group First, the inactivated cells of Lactobacillus rhamnosus VHProbi E06 were added to HaCat cells at a ratio of MOI value of 100, and placed in a 37°C, 5% carbon dioxide incubator for 3 hours; then UVB ultraviolet Illumination under the lamp, the light dose is 60mJ/cm2;
  • the cells were placed in a 37°C, 5% carbon dioxide incubator to continue culturing for 16h.
  • MTT solution was added to each cell culture well to be tested, with a final concentration of 0.3mg/ml, and placed in a 37°C, 5% carbon dioxide incubator for 3h incubation. Carefully discard the supernatant, add 500ul of DMSO to each cell culture well of a 24-well plate, incubate at 37°C for 30min to fully dissolve the purple crystals, and detect the absorbance value at 490nm with a microplate reader. Three parallel samples were set up for detection in each group. The cell viability of each group was calculated separately.
  • Cell viability% (absorbance of detection sample-absorbance of blank)/(absorbance of control group-absorbance of blank) ⁇ 100.
  • the 3D reconstructed human skin model EpiSkin TM was purchased from Shanghai Sinopino Biotechnology Co., Ltd. It is an in vitro reconstructed human epidermal model obtained by culturing normal human keratinocytes on a collagen matrix through air-liquid culture. The model is histologically able to mimic the layered structure of normal human epidermis, including the stratum corneum with barrier function.
  • EpiSkin TM 3D skin model is divided into control group, UVB damage group, and probiotics treatment group, of which:
  • Control group evenly smear sterile PBS on the surface of the skin model without UVB irradiation
  • UVB damage group evenly smear sterile PBS on the surface of the skin model, incubate in a 5% carbon dioxide incubator at 37°C for 3 hours, then place it under a UVB ultraviolet lamp for light stimulation, and the light dose is 200mJ/cm 2 ;
  • Probiotics treatment group apply heat-inactivated cells of Lactobacillus rhamnosus VHProbi E06 evenly on the surface of the skin model, incubate in a 5% carbon dioxide incubator at 37°C for 3 hours, and then place it under a UVB ultraviolet lamp for light stimulation , the light dose is 200mJ/cm 2 ;
  • the skin model was placed in a 37°C, 5% carbon dioxide incubator to continue culturing for 72 hours.
  • pro-inflammatory cytokines and cell viability were detected respectively, and three parallel samples were set for detection in each group.
  • the 3D skin tissue was incubated for 3 h in the detection medium containing a final concentration of 0.3 mg/ml MTT, the skin tissue was cut and separated from the scaffold with a puncher, placed in a 1.5 ml EP tube, 1 ml DMSO was added, and The skin tissue was soaked for 2 hours under the condition of avoiding light, and vortexed to fully dissolve the purple crystals, and the absorbance value was detected at 490nm with a microplate reader. The viability of skin cells in each group was calculated, as shown in Figure 10.
  • Cell viability (%) (absorbance of detection sample-absorbance of blank)/(absorbance of control group-absorbance of blank) ⁇ 100.
  • TritonX-100 polyethylene glycol octyl phenyl ether
  • TritonX-100 is a non-ionic surfactant that can destroy lipid bilayers, remove the plasma membrane and inner membrane system of cells, and destroy the weak bonds between molecules. Some protein antigens. Treatment of skin samples with 0.1% TritonX-100 will cause damage to skin cells, and the transmembrane resistance of the skin after damage will decrease significantly.
  • EpiSkin TM 3D skin models were divided into control group, TritonX-100 injury group, and probiotics treatment group, among which:
  • Control group evenly smear sterile PBS on the surface of the skin model
  • TritonX-100 injury group evenly smear 0.1% TritonX-100 solution on the surface of the skin model
  • Probiotics treatment group first apply Lactobacillus rhamnosus VHProbi E06 heat-inactivated cells evenly on the surface of the skin model, incubate in a 5% carbon dioxide incubator at 37°C for 3 hours, and then apply 0.1% TritonX-100 solution ;
  • the detection method of the transmembrane resistance value is as follows: adjust the function switch of the EVOM2 transmembrane resistance instrument to OHMs, connect the electrode, and put the electrode into Hanks balanced salt solution (HBSS) preheated to 37°C for 20 minutes to balance. Remove the medium in the skin model culture plate, add preheated HBSS, add 0.5ml to each well of the upper layer, add 1.5ml to each well of the lower layer, equilibrate at 37°C for 20min, remove the HBSS, add 37°C preheated HBSS again, and measure Transmembrane resistance value. Repeat the above steps with a blank vector not seeded with skin cells to obtain a blank value. Calculate the transmembrane resistance value of each group of skin models.
  • HBSS Hanks balanced salt solution
  • the Lactobacillus rhamnosus VHProbi E06 provided by the present invention has strong acid resistance, does not produce hemolysin, cannot lyse blood cells, is sensitive to common antibiotics such as erythromycin and tetracycline, and has good biological safety.
  • the strain has strong antioxidant capacity and can effectively scavenge DPPH free radicals.
  • the strain can obviously promote the proliferation of HaCat cells; and can effectively reduce various skin damages caused by Staphylococcus aureus infection, hydrogen peroxide oxidation, ultraviolet UVB irradiation and TritonX-100 stimulation, and the effect is very significant.
  • the Lactobacillus rhamnosus VHProbi E06 and its fermentation product provided by the invention can be widely used in cosmetics or medicines, and have broad application prospects.
  • a strain of Lactobacillus rhamnosus provided by the present invention has a protective effect on skin damage and its application have been described in detail above.
  • This article uses specific examples to illustrate the principle and implementation of the present invention. The description of the above embodiments is only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Abstract

本发明涉及一株新型鼠李糖乳杆菌(Lactobacillus rhamnosus)VHProbi E06及其应用。所述鼠李糖乳杆菌的保藏号为CCTCC NO：M2021588，该菌株在皮肤角质形成细胞系HaCat及3D皮肤模型水平上，能够有效缓解由细菌感染、紫外线、氧化损伤及化学损伤等多种因素造成的皮肤伤害，可应用于化妆品或药品领域。

Description

一株对皮肤损伤具有保护作用的鼠李糖乳杆菌及其应用
本申请要求于2021年09月07日提交中国专利局、申请号为202111045021.1、发明名称为“一株对皮肤损伤具有保护作用的鼠李糖乳杆菌及其应用”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
本发明涉及功能微生物筛选与应用技术领域，具体涉及一种鼠李糖乳杆菌，特别是一株对皮肤损伤具有保护作用的鼠李糖乳杆菌及其应用。
背景技术
益生菌是通过定殖在人体内，改变宿主某一部位菌群组成的一类对宿主有益的活性微生物。益生菌最早由俄国微生物学家在20世纪初发现，发展至今已有一个多世纪，其概念以及功效已经被科学家和消费者所认同。尤其在调节肠道菌群平衡，抗病原体效应，影响人体免疫系统、增强免疫力等方面的功效已经深入人心。除此之外，近年来益生菌制剂也越来越多地用于健康皮肤护理、预防和治疗皮肤疾病以及皮肤抗氧化衰老等方面，其优点是这种方法对使用者有效，并且更天然没有副作用，代表了皮肤健康的一个新兴领域。
皮肤是人体最大的器官，直接与外环境接触，是人体重要的结构边界和感知界面，也是免疫原性器官，是抵御外界过敏原的第一道防线和生物传感器，最经常受到物理、化学、细菌及真菌的侵害。最近为了解皮肤屏障功能而进行的研究表明，皮肤的物理、免疫和细胞生物学特性与皮肤微生物菌群密切相关。多项临床研究表明，益生菌直接或间接地对皮肤有积极作用。有证据表明，益生菌疗法在预防和治疗皮肤湿疹、特应性皮炎、痤疮、过敏性炎症或皮肤过敏、紫外线引起的皮肤损伤、伤口保护等皮肤疾病方面具有巨大的潜力。
同时，实验和临床数据也证实了这样一种假设，即某些益生菌菌株或特定的细菌裂解物或提取物在肠道以外发挥作用，或直接局部应用于皮肤，并为皮肤层面带来益处。这种益生菌或提取物形式可以有助于加强皮肤屏障功能和调节皮肤免疫系统，促进皮肤的稳定状态。因此筛选优质的 皮肤益生菌菌株，尤其是可以直接应用于皮肤并且对皮肤具有保护功效的菌株，仍然是本领域的研究重点。
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。所述鼠李糖乳杆菌分离自健康婴儿粪便，经验证对多种因素造成的皮肤损伤均具有保护作用。
本发明一方面提供了一株鼠李糖乳杆菌，命名为鼠李糖乳杆菌VHProbi E06(Lactobacillus rhamnosus VHProbi E06)，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：M2021588。
本发明一方面提供了鼠李糖乳杆菌VHProbi E06在制备用于预防或缓解皮肤损伤的制品中的应用。
所述的制品为化妆品或药品。
本发明还提供了一种具有防晒或抗衰老功效的化妆品，包含鼠李糖乳杆菌VHProbi E06和/或鼠李糖乳杆菌VHProbi E06的发酵产物。
本发明还提供了一种用于缓解皮肤损伤的药品，包含鼠李糖乳杆菌VHProbi E06和/或鼠李糖乳杆菌VHProbi E06的发酵产物。
本发明提供的鼠李糖乳杆菌VHProbi E06耐酸性强；不产生溶血素，不溶解血细胞，对红霉素、四环素等常见的抗生素敏感，具有良好的生物安全性；能够耐受较高的盐度，最大耐受盐浓度高于8％。
所述鼠李糖乳杆菌具有较强的抗氧化能力，能有效清除DPPH自由基，清除率达到38.22％。
所述鼠李糖乳杆菌能有效促进皮肤角质形成细胞(HaCat细胞)增殖。与对照组相比，添加鼠李糖乳杆菌VHProbi E06热灭活菌体的两个处理组HaCat细胞的活力得到显著提高。
所述鼠李糖乳杆菌能有效缓解金黄色葡萄球菌感染造成的皮肤细胞损伤。被金黄色葡萄球菌侵染的对照组HaCaT细胞死亡率高达56.6％，而同时加入鼠李糖乳杆菌VHProbi E06热灭活菌体的益生菌处理组HaCaT细胞的死亡率下降至41.3％，效果非常显著
所述鼠李糖乳杆菌能有效缓解过氧化氢氧化造成的皮肤细胞损伤。与对照组相比，过氧化氢损伤组HaCat细胞的活力大幅下降，仅为49.7％；而经过鼠李糖乳杆菌VHProbi E06灭活菌体预处理的益生菌处理组细胞活力达到59.1％，提高明显。
所述鼠李糖乳杆菌对紫外线UVB照射造成的皮肤损伤具有显著的抗炎和免疫调节作用，能有效缓解UVB照射造成的皮肤细胞损伤。与对照组相比，经过UVB光照刺激后，皮肤细胞对IL-1α、IL-1β和IL-8三种促炎症反应细胞因子的分泌水平均有显著升高；而光照刺激前，先经鼠李糖乳杆菌VHProbi E06灭活菌体预处理后的益生菌处理组皮肤细胞对IL-1α、IL-1β及IL-8的分泌水平明显回落；且益生菌处理组皮肤细胞的活力能达到70.4％，远高于不经预处理的UVB损伤组，效果非常显著。
所述鼠李糖乳杆菌对经TritonX-100刺激造成的3D重建人体皮肤模型的皮肤屏障受损具有缓解保护作用。与对照组相比，表面涂抹0.1％TritonX-100溶液的皮肤模型的跨膜电阻值大幅下降了60.2％，而经鼠李糖乳杆菌VHProbi E06灭活菌体预处理后的益生菌处理组皮肤模型的跨膜电阻值的下降程度有所缓解，仅比对照组降低了50.6％。
本发明提供的鼠李糖乳杆菌VHProbi E06对多种因素造成的皮肤损伤均具有一定的保护作用，可广泛应用于化妆品或药品中，应用前景广阔。
生物保藏说明
鼠李糖乳杆菌VHProbi E06(Lactobacillus rhamnosus VHProbi E06)，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：M2021588。
附图说明
图1为VL-2菌株菌落形态图；
图2为VL-2菌株Riboprinter指纹图谱；
图3为VL-2菌株RAPD指纹图谱；
图4为VL-2菌株rep-PCR指纹图谱；
图5为各组HaCat细胞活力对比图；
图6为金黄色葡萄球菌感染的HaCat细胞死亡率对比图；
图7为过氧化氢氧化损伤的HaCat细胞活力对比图；
图8为UVB损伤的HaCat细胞活力对比图；
图9为UVB损伤的3D皮肤模型细胞因子对比图；
图10为UVB损伤的3D皮肤模型细胞活力对比图；
图11为TritonX-100损伤的3D皮肤模型跨膜电阻值对比图。
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 菌株的分离筛选
1.1乳酸杆菌初筛
配制MRS(Man Rogosa Sharpe)琼脂培养基：纯化水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。
依据2019版《人类遗传资源库伦理规范》，与样本提供者签订项目承诺书和知情同意书后，按照生物样本库标准操作规范，取1g来自1岁半的半年内未食用过益生菌制剂的健康婴儿的新鲜粪便，经无菌生理盐水稀释后放入无菌样品袋中，用匀浆仪拍打混匀；取100μL混匀液梯度稀释，涂布于MRS琼脂培养基后于37℃培养48h，待平板长出单菌落进行镜检。根据镜检结果，申请人共筛选出9株潜在乳酸杆菌，分别命名为VL-1，VL-2，……，VL-9。
1.2乳酸杆菌复筛
配置1L MRS液体培养基，115℃高压灭菌30min，待培养基冷却后，加入3.2g猪粘膜胃蛋白酶，摇匀溶解，置37℃水浴摇床中温水浴1h，制成耐酸性培养基。
将筛选得到的9株乳酸杆菌VL-1，VL-2，……，VL-9，按6％接种 量分别接种于上述耐酸性培养基中，37℃静置培养72h，取发酵液进行菌量计数。
结果显示，所述9株乳酸杆菌发酵液中活菌量的对数值分别为8.21、8.36、7.71、7.52、6.97、7.43、7.21、5.96、6.65Log CFU/mL，VL-2菌株经耐酸性培养基复筛后活菌量最多，菌量对数值高达8.36Log CFU/mL。从而说明，本发明筛选到的VL-2号菌株耐酸能力最高。
实施例2 菌株鉴定
2.1菌落形态鉴定
将VL-2菌株接种于MRS琼脂培养基上，37℃培养48h。VL-2的菌落形态如图1所示，菌落呈乳白色，菌落直径在2mm左右，表面湿润，显微镜下呈短杆状，二端呈圆形，通常单独出现，呈短链状。
2.2生理生化特性鉴定
在无菌条件下，取适量新鲜VL-2菌液，5000rpm/min离心5min，用PBS缓冲液洗2次，再用同体积PBS缓冲液重悬菌体后稀释50倍，作为菌悬液。
2.2.1盐度耐受性试验
在无菌条件下，向96孔板中分别加入190μL盐浓度为1％、2％、3％、4％、5％、6％、7％、8％的BSM液体培养基，每个盐浓度做3个平行，然后再加入10μL菌悬液，不接菌的孔作为对照。每孔加入50μL高压灭菌过的石蜡油以防止培养过程中水分蒸发。置于37℃恒温培养，观察培养基是否变浑浊。
结果显示，VL-2菌株在1％～8％盐浓度下均可生长，其最大耐受盐浓度高于8％。
2.2.2过氧化氢酶实验
取新鲜菌液，滴一滴于干净的载玻片上，然后在其上滴加一滴3％过氧化氢溶液，观察到VL-2菌株不产生气泡，是阴性反应。
2.2.3碳源代谢试验
本实验中所用的基础培养基配方如下：
蛋白胨1.5g；酵母提取物0.6g；吐温80 0.1g；盐溶液0.5mL；酚红 18mg；蒸馏水100mL；pH7.4±0.2。盐溶液成分：MgSO 4·7H 2O 11.5g，MnSO 4·4H 2O 2.8g，蒸馏水100mL。
配制10g/100mL的糖、醇和苷类碳水化合物溶液，并用0.22μm的无菌过滤器进行过滤。在无菌条件下，向96孔板中加入20μL除菌后的碳水化合物溶液，每种碳水化合物4个平行，然后加入170μL灭菌后含酚红的基础培养基，再加入10μL菌悬液，不接菌的孔作为对照。每孔加入50μL液体石蜡以防止培养过程中水分蒸发。37℃厌氧培养，以酚红为指示剂，观察培养基颜色变化。具体结果见表1。
表1 VL-2菌株碳源代谢结果
Figure PCTCN2022090283-appb-000001
注：“+”阳性反应；“-”阴性反应。
2.3分子生物学鉴定
2.3.1 16s rDNA基因序列分析
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
Figure PCTCN2022090283-appb-000002
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VL-2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，初步确定VL-2菌株为鼠李糖乳杆菌(Lactobacillus rhamnosus)。序列如下所示：
Figure PCTCN2022090283-appb-000003
Figure PCTCN2022090283-appb-000004
2.3.2 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，得到细菌鉴定结果。鉴定结果显示，VL-2菌株为鼠李糖乳杆菌(Lactobacillus rhamnosus)，其Riboprinter指纹图谱结果见图2。
2.3.3 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 PCTCN2022090283-appb-000005
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。VL-2菌株的RAPD指纹图谱如图3所示。
2、rep-PCR指纹图谱
1)rep-PCR引物
Figure PCTCN2022090283-appb-000006
2)rep-PCR的反应体系
表4 rep-PCR的反应体系
Figure PCTCN2022090283-appb-000007
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。VL-2菌株的的rep-PCR指纹图谱如图4所示。
综上，将VL-2菌株的菌落形态以及生理生化特性结果上传至网站 http://www.tgw1916.net/bacteria_logare_desktop.html，同时结合文献De Clerck E，et al.Systematic and applied microbiology,2004，27(1)50公布的结果，进行比对。综合分子生物学的鉴定结果，可以得出结论，VL-2菌株为一株新的鼠李糖乳杆菌，将其命名为鼠李糖乳杆菌VHProbi E06(Lactobacillus rhamnosus VHProbi E06)，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：M2021588。
实施例3 鼠李糖乳杆菌VHProbi E06的溶血性及抗生素耐受性实验
3.1溶血性实验
(1)接种液制备：将冷冻保存的鼠李糖乳杆菌VHProbi E06菌株划线接种于MRS琼脂培养基中，在温度37℃培养24～48h，再经MRS液体培养基传代培养1次后，以5％的接种量把鼠李糖乳杆菌VHProbi E06接种到新鲜的MRS液体培养基中37℃培养24～48h，获得新鲜的菌液，作为接种液。
(2)血细胞培养基准备：称取TBS基础培养基的各种组分，溶解，121℃高压灭菌15min，等培养基冷却到50℃的时候加入5％的无菌脱纤维绵羊血，混匀，倒平板。
(3)划线培养：将测试菌株划线接种于准备好的血细胞平板，37℃培养箱培养，24～48h观察测试菌是否有溶血现象。
结果显示：鼠李糖乳杆菌VHProbi E06不能生长，血细胞平板没有变化，说明鼠李糖乳杆菌VHProbi E06不产生溶血素，不能够溶解血细胞。
3.2抗生素耐受性实验
(1)抗生素配制：氨苄青霉素、红霉素、庆大霉素、链霉素、四环素均配制成2048μg/mL的贮存液，-20℃保存备用。使用时将贮存液用BSM液体培养基进行2倍系列梯度稀释成使用液，梯度稀释浓度为1～1024μg/mL共11个梯度。
(2)接种液制备：取适量新鲜菌液(24h，37℃培养)，5000rpm离心5min，用无菌生理盐水洗一次，再用同体积生理盐水重悬菌体后稀释50倍，作为接种液。
(3)微量肉汤稀释法测定抗生素对鼠李糖乳杆菌VHProbi E06的最小抑菌浓度MIC值
a.96孔板第1列次加入不含抗生素的MRS液体培养基，作为阴性对照，向第2～12列依次加入190μL含不同浓度抗生素的MRS液体培养基，然后分别接种10μL上述接种液，做3个平行孔，并以1个孔不加菌液作为空白。
b.加入50μL石蜡油覆盖防止水分蒸发。
c.将96孔板于37℃培养24h后取出，测定OD 600值，用24h的结果统计抗生素对菌株的MIC值，具体结果见表5。
表5 鼠李糖乳杆菌VHProbi E06的抗生素MIC值
Figure PCTCN2022090283-appb-000008
MIC单位μg/mL
从表5的结果可以看出，本发明提供的鼠李糖乳杆菌VHProbi E06对红霉素、四环素等常见抗生素敏感，生物安全性良好。
实施例4 鼠李糖乳杆菌VHProbi E06清除DPPH自由基能力的测定
4.1 PBS菌悬液制备
将生长状态优良的单菌落接种于3mLMRS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。
4.2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样本，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％。
以副干酪乳杆菌(Lactobacillus paracasei)IMC-4菌株为阳性对照，结果见表6。
表6 DPPH自由基清除率
Figure PCTCN2022090283-appb-000009
从表6的数据可以看出，本发明提供的鼠李糖乳杆菌VHProbi E06能有效清除DPPH自由基，清除率达到38.22％，显著高于副干酪乳杆菌(L.paracasei)IMC-4菌株。
实施例5 鼠李糖乳杆菌VHProbi E06在促进细胞增殖中的应用
5.1热灭活菌体制备：
鼠李糖乳杆菌VHProbi E06使用MRS液体培养基培养至稳定期，使用无菌PBS洗涤3次，并用无菌PBS将菌体重悬至5×10 7CFU/mL，于70℃水浴中20分钟热灭活备用。
5.2细胞准备：
人永生化角质形成细胞HaCat使用高糖DMEM(10％FBS)培养液培养至所需用量，胰酶消化计数，细胞悬液加入24孔板中，每个细胞培养孔中的细胞个数为2×10 5，每孔培养液的添加量为0.6ml。置于二氧化碳培养箱(5％CO2，37℃)中培养24小时后，进行后续实验。
5.3实验方法：
将鼠李糖乳杆菌VHProbi E06热灭活菌体按MOI(Multiplicity of Infection，感染复数)值为10和100的比例分别加入HACAT中，并设置不加菌的对照组，继续培养24h后，待检测的每个细胞培养孔中加入终浓度为0.3mg/ml的MTT溶液，置于37℃5％二氧化碳培养箱中孵育3h。小心弃掉上清，每孔加入500ul的DMSO，37℃下孵育30min，使紫色结晶充分溶解，酶标仪490nm下检测吸光度值。每组设3个平行样本检测。 分别计算各组的细胞活力。
细胞活力(％)＝(检测样品吸光度-空白吸光度)/(对照组吸光度-空白吸光度)×100。
5.4实验结果
检测结果如图5所示：与对照组相比，添加鼠李糖乳杆菌VHProbi E06热灭活菌体的两个益生菌处理组HaCat细胞的活力得到显著提高。从而说明本发明提供的鼠李糖乳杆菌VHProbi E06对HaCat细胞的增殖有明显的促进作用(MOI:10，p<0.05；MOI:100，p<0.05)。
实施例6 鼠李糖乳杆菌VHProbi E06在降低金黄色葡萄球菌感染细胞损伤中的应用
6.1热灭活菌体制备：
方法同实施例5。
6.2 HaCat细胞准备：
方法同实施例5。
6.3实验方法：
将HaCaT细胞原培养液更换为无抗性无血清DMEM培养液。
实验设置对照组和益生菌处理组，其中：对照组每孔细胞中接种金黄色葡萄球菌菌液1μL；益生菌处理组每孔中接种金黄色葡萄球菌菌液1μL，同时按MOI(Multiplicity of Infection，感染复数)值为10的比例加入鼠李糖乳杆菌VHProbi E06热灭活菌体。
将细胞培养板置于37℃、5％二氧化碳培养箱中继续培养16h；细胞培养上清液离心去杂质；按照乳酸脱氢酶(LDH)细胞毒性检测试剂盒(碧云天C0016)产品说明进行检测操作并计算细胞毒性或死亡率(％)。
6.4实验结果：
从图6的结果可知，被金黄色葡萄球菌侵染的对照组HaCaT细胞死亡率高达56.6％，而同时加入鼠李糖乳杆菌VHProbi E06热灭活菌体的益生菌处理组HaCaT细胞的死亡率下降至41.3％，效果非常显著(P<0.005)。从而说明，本发明提供的鼠李糖乳杆菌VHProbi E06能有效降低金黄色葡萄球菌感染造成的细胞损伤。
实施例7 鼠李糖乳杆菌VHProbi E06在降低细胞氧化损伤中的应用
7.1热灭活菌体制备：
方法同实施例5。
7.2 HaCat细胞准备：
方法同实施例5。
7.3实验方法：
实验设置对照组、过氧化氢损伤组、益生菌处理组。其中：对照组：细胞正常培养；过氧化氢损伤组：加入终浓度为0.5mM的过氧化氢溶液；益生菌处理组：先按MOI值为10的比例将鼠李糖乳杆菌VHProbi E06灭活菌体加入HaCat细胞中，置于37℃、5％二氧化碳培养箱中培养3h，然后加入终浓度为0.5mM的过氧化氢溶液。
以上各组细胞均置于37℃、5％二氧化碳培养箱中继续培养1h。
各组细胞弃掉原培养液，并使用PBS润洗两次，每孔再加入0.6mL的新鲜培养液。置于37℃、5％二氧化碳培养箱中继续培养16h。将待检测的每个细胞培养孔中加入终浓度为0.3mg/ml的MTT溶液，置于37℃、5％二氧化碳培养箱中孵育3h。小心弃掉上清，每个24孔板细胞培养孔中加入500ul的DMSO，37℃孵育30min，使紫色结晶充分溶解，酶标仪490nm下检测吸光度值。每组设3个平行样本检测。分别计算各组的细胞活力。
细胞活力％＝(检测样品吸光度-空白吸光度)/(对照组吸光度-空白吸光度)×100。
7.4实验结果：
从图7的结果可知，与对照组相比，过氧化氢损伤组HaCat细胞的活力大幅下降，仅为49.7％；而经过鼠李糖乳杆菌VHProbi E06灭活菌体预处理的益生菌处理组细胞活力达到59.1％，提高明显(P<0.005)。从而说明，本发明提供的鼠李糖乳杆菌VHProbi E06能有效降低过氧化氢造成的细胞损伤。
实施例8 鼠李糖乳杆菌VHProbi E06在降低细胞光损伤中的应用
8.1热灭活菌体制备：
方法同实施例5。
8.2 HaCat细胞准备：
方法同实施例5。
8.3实验方法：
实验设置对照组、UVB损伤组、益生菌处理组，其中：
(1)对照组：细胞正常培养，不加益生菌，未经UVB照射；
(2)UVB损伤组：UVB紫外灯下进行光照，光剂量为60mJ/cm2；
(3)益生菌处理组：先将鼠李糖乳杆菌VHProbi E06灭活菌体按MOI值为100的比例加入HaCat细胞中，置于37℃、5％二氧化碳培养箱中培养3h；然后UVB紫外灯下进行光照，光剂量为60mJ/cm2；
照射结束后，将细胞置于37℃、5％二氧化碳培养箱中继续培养16h。
待检测的每个细胞培养孔中加入MTT溶液，终浓度为0.3mg/ml，置于37℃、5％二氧化碳培养箱中孵育3h。小心弃掉上清，每个24孔板细胞培养孔中加入500ul的DMSO，37℃下孵育30min，使紫色结晶充分溶解，酶标仪490nm下检测吸光度值。每组设3个平行样本检测。分别计算各组的细胞活力。
细胞活力％＝(检测样品吸光度-空白吸光度)/(对照组吸光度-空白吸光度)×100。
8.4实验结果：
从图8的结果可知，与对照组相比，UVB损伤组HaCat细胞的活力大幅下降，仅为50.2％，而经鼠李糖乳杆菌VHProbi E06灭活菌体预处理的益生菌处理组细胞活力高达70.1％(P<0.05)。从而说明，本发明提供的鼠李糖乳杆菌VHProbi E06能有效降低紫外线UVB照射造成的皮肤细胞损伤。
实施例9 鼠李糖乳杆菌VHProbi E06在缓解皮肤UVB光损伤中的应用
9.1热灭活菌体制备：
方法同实施例5。
9.2 3D重建人体皮肤模型：
3D重建人体皮肤模型EpiSkin TM购于上海斯安肤诺生物科技有限公 司，是一种正常人类角质形成细胞在胶原基质上经过气液培养而成的体外重建人类表皮模型。该模型在组织结构上能够模拟正常人类表皮的分层结构，包含具有屏障功能的角质层。
9.3实验方法：
EpiSkin TM3D皮肤模型分为对照组、UVB损伤组、益生菌处理组，其中：
(1)对照组：在皮肤模型表面均匀涂抹无菌PBS，未经UVB照射；
(2)UVB损伤组：在皮肤模型表面均匀涂抹无菌PBS，37℃，5％二氧化碳培养箱中孵育3h后，置于UVB紫外灯下进行光照刺激，光剂量为200mJ/cm 2；
(3)益生菌处理组：先在皮肤模型表面均匀涂抹鼠李糖乳杆菌VHProbi E06热灭活菌体，37℃，5％二氧化碳培养箱中孵育3h后，置于UVB紫外灯下进行光照刺激，光剂量为200mJ/cm 2；
照射结束后，将皮肤模型置于37℃、5％二氧化碳培养箱中继续培养72h。
培养结束后，分别进行促炎症反应细胞因子和细胞活力检测，每组设3个平行样本检测。
细胞因子检测方法：
吸取下层培养液，使用ELISA方法对培养液中的IL-1α、IL-1β及IL-8含量进行测定。结果如图9所示。
皮肤细胞活力检测方法：
使用MTT法，即将3D皮肤组织在含有终浓度为0.3mg/mlMTT的检测培养基中孵育3h，使用打孔器将皮肤组织从支架中切割分离，置于1.5mlEP管中，加入1ml DMSO，在避光条件下将皮肤组织浸泡2h，并涡旋震荡，使紫色结晶充分溶解，酶标仪490nm下检测吸光度值。计算各组皮肤细胞的活力，如图10所示。
细胞活力(％)＝(检测样品吸光度-空白吸光度)/(对照组吸光度-空白吸光度)×100。
9.4实验结果：
从图9的结果可知，与对照组相比，经过UVB光照刺激后，皮肤细胞对IL-1α、IL-1β和IL-8三种促炎症反应细胞因子的分泌水平均有显著升高；而光照刺激前，先经鼠李糖乳杆菌VHProbi E06灭活菌体预处理后的益生菌处理组皮肤细胞对IL-1α、IL-1β及IL-8的分泌水平明显回落(p<0.005)。从而表明，本发明提供的鼠李糖乳杆菌VHProbi E06对紫外线UVB照射造成的皮肤损伤具有显著的抗炎和免疫调节作用。
从图10的结果可知，与正常对照组相比，经过UVB光照刺激后，皮肤细胞活力大幅降低，仅有50.1％，而光照刺激前，先经鼠李糖乳杆菌VHProbi E06灭活菌体预处理后的益生菌处理组细胞的活力能达到70.4％(P<0.05)。从而说明，本发明提供的鼠李糖乳杆菌VHProbi E06能有效缓解紫外线UVB对皮肤造成的损伤。
实施例10 鼠李糖乳杆菌VHProbi E06在缓解TritonX-100造成的皮肤损伤中的应用
10.1热灭活菌体制备：
方法同实施例5。
10.2 3D重建人体皮肤模型
同实施例9。
10.3实验方法：
TritonX-100(聚乙二醇辛基苯基醚)，是一种非离子表面活性剂，能破坏脂质双分子层，除去细胞的质膜和内膜系统，破坏分子间微弱结合键的大部分蛋白质抗原。0.1％的TritonX-100处理皮肤样本，会对皮肤细胞造成损伤，而损伤后的皮肤跨膜电阻值会显著下降。
EpiSkin TM3D皮肤模型分为对照组、TritonX-100损伤组、益生菌处理组，其中：
(1)对照组：在皮肤模型表面上均匀涂抹无菌PBS；
(2)TritonX-100损伤组：在皮肤模型表面均匀涂抹0.1％的TritonX-100溶液；
(3)益生菌处理组：先在皮肤模型表面均匀涂抹鼠李糖乳杆菌VHProbi E06热灭活菌体，37℃，5％二氧化碳培养箱中孵育3h后，再涂 抹0.1％的TritonX-100溶液；
将各组皮肤模型置于37℃、5％二氧化碳培养箱中孵育1h；然后使用无菌PBS缓冲液反复冲洗皮肤模型表面至少10次，擦干表面液体，置于37℃、5％二氧化碳培养箱中继续培养24h后，分别检测皮肤模型的跨膜电阻值。每组设3个平行样本检测。
跨膜电阻值检测方法为：将EVOM2跨膜电阻仪的功能开关调至OHMs，连接电极，将电极放入预热至37℃的Hanks平衡盐溶液(HBSS)中平衡20min。移除皮肤模型培养板中的培养基，加入预热的HBSS，上层每孔加0.5ml，下层每孔加1.5ml，37℃平衡20min，移除HBSS，重新加入37℃预热的HBSS，测定跨膜电阻值。用一个未接种皮肤细胞的空白载体重复上述步骤以获得空白值。计算各组皮肤模型的跨膜电阻值。
跨膜电阻值(TEER)＝(测定电阻值—空白值)*皮肤模型表面积(cm 2)。
10.4实验结果：
从图11的结果可知，与对照组相比，表面涂抹0.1％TritonX-100溶液的皮肤模型的跨膜电阻值大幅下降了60.2％，而经鼠李糖乳杆菌VHProbi E06灭活菌体预处理后的益生菌处理组皮肤模型的跨膜电阻值的下降程度有所缓解，仅比对照组降低了50.6％(P<0.05)。从而说明，本发明提供的鼠李糖乳杆菌VHProbi E06能有效降低TritonX-100对皮肤造成的损伤，效果显著。
综上所述，本发明提供的鼠李糖乳杆菌VHProbi E06耐酸性强，不产生溶血素，不能溶解血细胞，对红霉素、四环素等常见抗生素敏感，生物安全性良好。该菌株具有很强的抗氧化能力，能有效清除DPPH自由基。该菌株对HaCat细胞的增殖有明显的促进作用；并且能有效降低金黄色葡萄球菌感染、过氧化氢氧化、紫外线UVB照射以及TritonX-100刺激造成的各种皮肤损伤，效果非常显著。本发明提供的鼠李糖乳杆菌VHProbi E06及其发酵产物可广泛应用于化妆品或药品中，应用前景广阔。
以上对本发明所提供的一株对皮肤损伤具有保护作用的鼠李糖乳杆 菌及其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 (6)

  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：M2021588。
  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，所述制品为化妆品。
  4. 如权利要求2所述的应用，其特征在于，所述制品为药品。
  5. 一种具有防晒或抗衰老功效的化妆品，其特征在于，所述的化妆品包含有权利要求1所述鼠李糖乳杆菌和/或权利要求1所述鼠李糖乳杆菌的发酵产物。
  6. 一种用于缓解皮肤损伤的药品，其特征在于，所述的药品包含有权利要求1所述鼠李糖乳杆菌和/或权利要求1所述鼠李糖乳杆菌的发酵产物。
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