WO2022222259A1 - 一株具有改善衰老皮肤和增强毛发健康作用的罗伊氏乳杆菌及其应用 - Google Patents

一株具有改善衰老皮肤和增强毛发健康作用的罗伊氏乳杆菌及其应用 Download PDF

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WO2022222259A1
WO2022222259A1 PCT/CN2021/101274 CN2021101274W WO2022222259A1 WO 2022222259 A1 WO2022222259 A1 WO 2022222259A1 CN 2021101274 W CN2021101274 W CN 2021101274W WO 2022222259 A1 WO2022222259 A1 WO 2022222259A1
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lactobacillus reuteri
month
old
group
skin
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PCT/CN2021/101274
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French (fr)
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段治
张陆霞
崔洪昌
张景燕
郭超群
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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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Publication of WO2022222259A1 publication Critical patent/WO2022222259A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • 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
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A61Q5/12—Preparations containing hair conditioners
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/173—Reuteri

Definitions

  • the invention belongs to the technical field of screening and application of probiotics, in particular to a strain of Lactobacillus reuteri with the effects of improving aging skin and enhancing hair health and application thereof.
  • probiotics play an important role in delaying human aging. More and more evidence shows that the health effects of probiotics on the human body are not only limited to the gut, but also have a wider range of effects, such as endocrine balance regulation, immune balance regulation, nervous system regulation, and respiratory system regulation. Therefore, taking probiotics to delay aging and improve skin health has become a new treatment idea.
  • the purpose of the present invention is to provide a new strain of Lactobacillus reuteri and its application; the provided Lactobacillus reuteri is isolated from the fresh feces of centenarians, which can increase the content of skin collagen and improve skin relaxation , Improve hair follicle health, protect hair, improve skin aging status, and have anti-aging effect.
  • the Lactobacillus reuteri provided by the present invention is Lactobacillus reuteri VHPribo E18 (Lactobacillus reuteri VHPribo E18), which has been deposited in the China Center for Type Culture Collection on January 25, 2021, and its deposit number is CCTCC NO: M2021153 .
  • the Lactobacillus reuteri VHPribo E18 strain provided by the present invention has its Riboprinter fingerprint as shown in Figure 2; its RAPD fingerprint as shown in Figure 3, and its rep-PCR fingerprint as shown in Figure 4;
  • Lactobacillus reuteri VHPribo E18 strain provided by the present invention is used to prepare a product with antioxidant function;
  • Lactobacillus reuteri VHPribo E18 strain provided by the invention can also be used to prepare a product with the effect of delaying and repairing skin aging.
  • the product is preferably a functional food.
  • the said product is preferably a cosmetic.
  • the Lactobacillus reuteri VHProbi E18 provided by the invention has strong tolerance to artificial gastrointestinal juice, and can germinate in artificial gastrointestinal juice; the strain is sensitive to common antibiotics such as erythromycin and ampicillin, and does not produce hemolysis It cannot dissolve blood cells and has good biological safety;
  • the strain has strong anti-oxidative ability, the inhibition rate of anti-lipid peroxidation of the cell is 6.76%, the supernatant is 9.93%, and the intracellular extract is 10.68%; the DPPH clearance rate reaches 32.8%, and the HRS clearance rate reaches 4.32 %.
  • the strain can also degrade cholesterol, and the degradation rate reaches 16.24%; in addition, the cell surface hydrophobicity of this strain is 61.7%, and the adhesion ability is 4.7.
  • the Lactobacillus reuteri VHProbi E18 provided by the present invention has no toxic effect on the body, and can be added to food to prepare functional food with anti-aging effect, and can also be added to cosmetics for delaying skin aging. application prospects.
  • Fig. 1 is the carbon source metabolism map of E18 strain API 50CHL;
  • Fig. 2 is the fingerprint of E18 strain Riboprinter
  • Fig. 3 is the RAPD fingerprint of E18 strain
  • Fig. 4 is the rep-PCR fingerprint of E18 strain
  • Figure 5 shows the results of sensory evaluation of mouse fur in each group
  • Fig. 6 is the measurement result of the skin moisture content of each group of mice.
  • Fig. 7 is the measurement result of MDA content in the skin of mice of each group.
  • Fig. 8 is the determination result of the hydroxyproline content of the mouse skin of each group
  • Figure 9 shows the results of HE staining of mouse skin in each group, in which (A) 3-month-old control group; (B) 12-month-old control group; (C) 12-month-old smear group; (D) 12-month-old gavage group Group;
  • Figure 10 shows the results of measuring the number of hair follicles, epidermal thickness, dermal thickness and collagen fiber content in the skin of mice in each group, in which (A) 3-month-old control group; (B) 12-month-old control group; (C) 12-month-old control group Smear group; (D) 12-month-old gavage group*: compared with 3-month-old control group, p ⁇ 0.05; **: compared with 12-month-old control group, p ⁇ 0.05.
  • the Lactobacillus reuteri VHProbi E18 provided by the invention complies with the requirements of regulations, can be used as a source of food raw materials, and has no side effects and risks of overdose when taken for a long time. Lactobacillus reuteri VPHrobi E18 was identified as a newly discovered strain by multiphase taxonomy. The Lactobacillus reuteri VHProbi E18 provided by the present invention has an anti-aging effect, and the use of the strain alone without compounding with prebiotics and/or other probiotics can improve skin aging and other age-related aging problems, It has important application value.
  • the applicant deposited the Lactobacillus reuteri VHProbi E18 in the China Center for Type Culture Collection of Wuhan University on January 25, 2021, and its deposit number is CCTCC NO: M2021153.
  • the screening method of the present invention is not limited to those described in the examples, and any known methods that can achieve the purpose of screening can be used.
  • the screening descriptions in the examples are only for the description of the present invention, and do not limit the protection scope of the present invention. Modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all belong to the scope of the present invention.
  • MRS agar medium was prepared to adjust pH to 6.2-6.5, and autoclaved at 121°C for 15min.
  • the 20 strains of Lactobacillus E01, E02,..., E18, E19, and E20 obtained by screening were inoculated into the above acid-resistant medium according to the inoculum of 6%. Bacterial count.
  • the E18 strain had the largest amount of viable bacteria after being re-screened by the acid-resistant medium, and the logarithm value was as high as 8.01Log CFU/mL. Therefore, the E18 strain has the highest acid resistance.
  • the E18 strain was inoculated on the MRS agar medium, and after anaerobic culture at 37°C for 24 hours, the single colony of E18 was milky white, the colony diameter was about 1.5-2mm, the surface was smooth, and the end was rounded under the microscope. Campylobacter.
  • the preparation of the inoculum in this example is as follows: under aseptic conditions, take an appropriate amount of fresh E18 bacterial solution, centrifuge at 5000 rpm/min for 5 min, wash twice with PBS buffer, and then reconstitute the bacteria with the same volume of PBS buffer and dilute to 50 times as an inoculum.
  • the carbon source metabolism experiment was carried out on the E18 strain using the API 50CHL reagent strip.
  • the carbon source metabolism results of E18 strain API 50CHL are shown in Figure 1.
  • the 16s rDNA sequence SEQ ID NO: 1 of the E18 strain was obtained by sequencing, and the sequence was compared in the NCBI database, and the E18 strain was preliminarily determined to be Lactobacillus reuteri.
  • Primer sequence CTACGGCAAGGCGACGCTGACG.
  • the rep-PCR fingerprint of E18 strain is shown in FIG. 4 .
  • E18 strain was determined to be a new Lactobacillus reuteri and named as Lactobacillus reuteri VHProbi E18.
  • the viable count method was used to measure the bacterial count according to the national standard "GB4789.35-2016 - Food Microbiological Inspection Lactic Acid Bacteria Inspection", and the viable bacterial count (Log CFU/mL) of this strain after being digested by artificial intestinal juice is shown in Table 1.
  • TBS basal medium dissolve, sterilize by autoclaving at 121 °C for 15 min, add 5% sterile defibrillated sheep blood when the medium cools to 50 °C, mix well, and pour into a plate.
  • the test strains were streak-inoculated on the prepared blood cell plates, cultured in a 37°C incubator, and observed for 24-48h to observe whether the test bacteria had hemolysis.
  • the minimum inhibitory concentration (MIC) value of antibiotics against Lactobacillus reuteri VHProbi E18 was determined by the micro-broth dilution method. The specific results are shown in Table 2.
  • the Lactobacillus reuteri VHProbi E18 provided by the present invention is sensitive to common antibiotics such as erythromycin and ampicillin, and has good biological safety.
  • Clearance % (A sample -A control )/(A blank -A control ) ⁇ 100%, wherein A control is deionized water substitute sample, A blank is deionized water substitute sample and H 2 O 2 , the results are shown in the table 4.
  • Preparation of cholesterol micelle solution Accurately weigh 1 g of cholesterol, dissolve it in absolute ethanol, and dilute to 100 mL, and filter and sterilize it with a 0.22 ⁇ m microporous membrane under sterile conditions.
  • Preparation of the bacterial solution to be tested Pick the purified Lactobacillus reuteri VHProbi E18 colony and inoculate it in the newly prepared MRS liquid medium, and cultivate at 37°C for 24-48h. The inoculum of 1% (V/V) was then transferred to MRS liquid medium at 37°C for 24-48 hours, and then centrifuged at 6000 ⁇ g for 10 min. The bacteria were resuspended in 1mL solution of 0.1M KNO 3 as the bacteria solution to be tested.
  • C57 mice were SPF grade, male, with 6 mice aged 3 months and 18 mice aged 12 months, weighing 19-25 g.
  • Environmental conditions for the feeding and management of experimental animals room temperature is 20-26°C, daily temperature difference is ⁇ 4°C, relative humidity is 40-70%, and light-dark alternation time is 12/12h.
  • Animals were housed in standard mouse cages, 6 per cage.
  • Animal feed, drinking water free access to food and water. The feed is the growth and breeding feed for SPF rats and mice. Drinking water is city tap water that has been pasteurized at high temperatures.
  • mice were randomly divided into 3-month-old control group, 12-month-old control group, 12-month-old smear group, and 12-month-old gavage group after 7 days of adaptive feeding, with 6 mice in each group.
  • the 12-month-old gavage group was given 0.2 mL/10 g of probiotics solution by gavage
  • the 12-month-old smear group was given the same amount of probiotics solution as the gavage group
  • the 3-month-old control group and the 12-month-old control group were given the same amount of probiotics by gavage.
  • Probiotics equal volume of normal saline. 70 days in total.
  • mice At the end, the back fur of the mice was scored sensory, and the scoring rules are shown in Table 6. Changes of water content, SOD activity and MDA content in mouse skin were detected, and hydroxyproline content in mouse skin and tail tendon was detected.
  • the back skin tissue was taken, fixed with 4% paraformaldehyde, sampled, dehydrated, embedded in paraffin, sectioned, and stained with HE to detect the number of hair follicles, dermal thickness and sebaceous gland cells.
  • the 3-month-old mice had thick hair and neat luster; the 12-month-old mice in the control group had dry hair, more hair loss and gray hair; the 12-month-old mice in the smear group had shiny hair and no white hair; 12 months The hair of mice in the gavage group was thick, shiny and black.
  • the comparison results of the sensory scores of the back fur of the mice in each group are shown in Figure 5.
  • the epidermal structure of the 3-month-old control mice was complete, with clear cell stratification, with obvious epidermal processes and dermal papillae, no inflammatory cell infiltration in the visual field, sebaceous gland hyperplasia, and dermal collagen fibers in the visual field.
  • the 12-month-old mice in the control group had obvious thinning of the epidermis, visible stratum corneum shedding, lack of structural integrity, decreased number of cells, irregular arrangement, significantly reduced collagen fiber layer, and fiber breakage and uneven distribution.
  • 12-month-old mice in the gavage group had hyperplasia of epithelial cells, with occasional loose and disordered arrangement, occasional proliferation of hair follicles and sebaceous glands, occasional inflammatory cells, loose distribution of collagen fibers, and visible fractures; 12-month-old mice In the smear group, the epithelial cells proliferated obviously, the stratum corneum fell off, the hair follicles and sebaceous glands proliferated, inflammatory cells were visible, the skin was loose, the collagen fiber layer was loosely arranged and distributed, and the fracture was obvious.
  • Typical HE section results of mouse skin are shown in Figure 9.
  • the skin hair follicle density of the 12-month-old control group decreased, and there was a significant difference (P ⁇ 0.05).
  • the hair follicle density increased in the smear group and the 12-month-old gavage group, and there was a significant difference (P ⁇ 0.05).
  • the skin epidermal thickness of the 12-month-old control group decreased, but there was no significant difference; there was no difference in the skin and epidermal thickness of the 12-month-old mice.
  • the dermal thickness of the 12-month-old control group decreased, and there was a significant difference (P ⁇ 0.05).
  • the thickness of the skin and dermis in the month-old gavage group increased, and there was a significant difference (P ⁇ 0.05).
  • the 12-month-old gavage group and the 12-month-old smear group have improved fur gloss, no white hair, and reduced hair loss.
  • the skin moisture content of the mice in the 12-month-old gavage group and the 12-month-old smear group increased, while the MDA content decreased.
  • Hydroxyproline is a rich and stable amino acid in the dermis, and its content can directly reflect the changes in the content of collagen in the dermis, which is one of the indicators to detect skin aging.
  • the Lactobacillus reuteri VHProbi E18 provided by the present invention has a strong tolerance to simulated artificial gastrointestinal fluid, which lays a foundation for the probiotic strain to successfully pass through the gastrointestinal tract and colonize the colon to exert a probiotic function.
  • Antibiotic resistance test confirmed that Lactobacillus reuteri VHProbi E18 is sensitive to common antibiotics, does not produce hemolysin, and has good biological safety.
  • Lactobacillus reuteri VHProbi E18 can scavenge DPPH and HRS free radicals, inhibit lipid peroxidation, has a certain antioxidant activity, can degrade cholesterol, and has probiotic properties of lowering serum cholesterol.
  • Lactobacillus reuteri VHProbi E18 has anti-aging effects, can increase skin collagen content, improve hair follicle health, and improve skin health.
  • the Lactobacillus reuteri VHProbi E18 can be used to prepare functional foods or cosmetics with the effect of delaying and repairing skin aging, and has broad application prospects.

Abstract

提供一株新的罗伊氏乳杆菌，为罗伊氏乳杆菌VHPribo E18株(Lactobacillus reuteriVHPribo E18)，已于2021年1月25日保藏于中国典型培养物保藏中心，其保藏号为CCTCC NO：M2021153。提供的罗伊氏乳杆菌VHProbi E18对人工肠胃液具有很强的耐受性，在人工肠胃液中能进行萌发；该菌株对红霉素和氨苄西林等常见的抗生素敏感，不产生溶血素，不能够溶解血细胞，具有良好的生物安全性。提供的罗伊氏乳杆菌VHPribo E18株可用于制备具有延缓修复皮肤衰老效果的功能性食品或化妆品，具有广阔的应用前景。

Description

一株具有改善衰老皮肤和增强毛发健康作用的罗伊氏乳杆菌及其应用
本申请要求于2021年4月18日提交中国专利局、申请号为202110415247.X、发明名称为“一株具有改善衰老皮肤和增强毛发健康作用的罗伊氏乳杆菌及其应用”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
本发明属于益生菌筛选与应用技术领域，具体涉及一株具有改善衰老皮肤和增强毛发健康作用的罗伊氏乳杆菌及其应用。
背景技术
随着我国逐渐进入老龄化社会，老年人口所占人口比重越来越大，与人体衰老相关的各种健康问题如、皮肤松弛、脱发等逐渐显现并困扰人们的日常生活，生活质量受到影响。在改善皮肤健康、防治脱发方面，现有药物不仅需要长期服用，而且还会有严重的副作用问题。寻找开发抗衰老活性物质已经成为应对人口老龄化问题的研究热点。
研究发现，益生菌在延缓人体衰老方面具有重要作用。越来越多的证据表明，益生菌对于人体的健康影响不仅局限于肠道，还有更广泛的作用范围，如内分泌平衡调节、免疫平衡调节、神经系统调节、呼吸系统调节等。因此，通过服用益生菌来延缓衰老、提高皮肤健康状况成为一种新的治疗思路。
发明内容
本发明的目的是提供一株新的罗伊氏乳杆菌(Lactobacillus reuteri)及其应用；所提供的罗伊氏乳杆菌分离自百岁老人的新鲜粪便，能够提高皮肤胶原蛋白含量，改善皮肤松弛、改善毛囊健康，保护毛发改善皮肤衰老状态， 具有抗衰老作用。
本发明所提供的罗伊氏乳杆菌，为罗伊氏乳杆菌VHPribo E18(Lactobacillus reuteriVHPribo E18)，已于2021年1月25日保藏于中国典型培养物保藏中心，其保藏号为CCTCC NO：M2021153。
本发明所提供的罗伊氏乳杆菌VHPribo E18株，其Riboprinter指纹图谱如图2所示；其RAPD指纹图谱如图3所示，rep-PCR指纹图谱如图4所示；
本发明所提供的罗伊氏乳杆菌VHPribo E18株用于制备具有抗氧化功能的制品；
本发明提供的罗伊氏乳杆菌VHPribo E18株还可用于制备具有延缓修复皮肤衰老效果的制品。
所述的制品，优选为功能性食品。
所述的制品，优选为化妆品。
本发明提供的罗伊氏乳杆菌VHProbi E18对人工肠胃液具有很强的耐受性，在人工肠胃液中能进行萌发；该菌株对红霉素和氨苄西林等常见的抗生素敏感，不产生溶血素，不能够溶解血细胞，具有良好的生物安全性；
该菌株抗氧化能力较强，菌体抗脂质过氧化抑制率菌体为6.76％、上清液为9.93％、胞内提取物为10.68％；DPPH清除率达到32.8％,HRS清除率达到4.32％。该菌株还能降解胆固醇，降解率达到16.24％；另外，该菌株细胞表面疏水性为61.7％，黏附能力为4.7。
在皮肤抗衰老功效研究中。与12月龄对照组小鼠相比，12月龄灌胃组和12月龄涂抹组小鼠皮毛光泽度均有改善，没有出现白毛，脱毛现象减少。12月龄灌胃组和12月龄涂抹组小鼠皮肤含水率上升，MDA含量下降。皮肤和尾腱 中羟脯氨酸含量上升，涂抹和灌胃罗伊氏乳杆菌VHProbi E18可以提高真皮内胶原蛋白的含量。从小鼠皮肤组织学检查看，与12月龄对照组小鼠相比，12月龄灌胃组和12月龄涂抹组小鼠表皮结构完整度改善，炎性细胞减少，成纤维细胞增多，并且灌胃组效果好于涂抹组。此外，12月龄灌胃组和12月龄涂抹组小鼠皮肤毛囊数量上升，真皮厚度增加，胶原纤维含量上升。结果表明，罗伊氏乳杆菌VHProbi E18无论是灌胃还是涂抹均可以改善衰老小鼠皮肤指征，达到延缓修复皮肤衰老效果。
本发明提供的罗伊氏乳杆菌VHProbi E18，对机体无毒害作用，可添加在食品中，制备成具有抗衰老功效的功能性食品，也可以添加在化妆品中，用于延缓皮肤衰老，具有广阔的应用前景。
附图说明
图1为E18菌株API 50CHL碳源代谢图谱；
图2为E18菌株Riboprinter指纹图谱；
图3为E18菌株的RAPD指纹图谱；
图4为E18菌株的rep-PCR指纹图谱；
图5为各组别小鼠皮毛感官评分结果；
图6为各组别小鼠皮肤含水量测定结果；
图7为各组别小鼠皮肤MDA含量测定结果；
图8为各组别小鼠皮肤羟脯氨酸含量测定结果；
图9为各组别小鼠皮肤HE染色结果图，其中(A)3月龄对照组；(B)12月龄对照组；(C)12月龄涂抹组；(D)12月龄灌胃组；
图10为各组别小鼠皮肤毛囊数量、表皮厚度、真皮厚度和胶原纤维含量 测定结果图，其中(A)3月龄对照组；(B)12月龄对照组；(C)12月龄涂抹组；(D)12月龄灌胃组*:与3月龄对照组，p<0.05；**:与12月龄对照组，p<0.05。
具体实施方式
本发明提供的罗伊氏乳杆菌VHProbi E18符合法规要求，可以作为一种食品原料来源使用，长期服用不会有副作用及过量的风险。经多相分类学鉴定，罗伊氏乳杆菌VPHrobi E18为一株新发现的菌株。本发明提供的罗伊氏乳杆菌VHProbi E18具有抗衰老功效，单独使用该菌株且无需与益生元和/或其它益生菌复配即可对皮肤衰老等与年龄相关的衰老问题起到改善功效，具有重要的应用价值。
申请人于2021年1月25日将所述罗伊氏乳杆菌VHProbi E18保藏于武汉大学的中国典型培养物保藏中心，其保藏号为CCTCC NO：M2021153。
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详细的描述。
实施例1 罗伊氏乳杆菌VHProbi E18的分离筛选
1、初筛
配制MRS琼脂培养基调pH 6.2-6.5，121℃高压灭菌15min。
取1g百岁老人的新鲜粪便样本(取样过程符合生物取样的伦理标准)，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℃厌氧培养48h，待平板长出单菌 落镜检。根据镜检结果，申请人共筛选出20株潜在乳酸杆菌，分别命名为E01、E02、……、E18、E19、E20。
2、复筛
配制1L的MRS液体培养基121℃高压灭菌15min，待培养基冷却后加入3.2g猪粘膜胃蛋白酶，摇匀溶解，置37℃水浴摇床中水浴1h制成耐酸性培养基。
将筛选得到的20株乳酸杆菌E01、E02、……、E18、E19、E20按6％接种量分别接种于上述耐酸性培养基中，37℃条件下厌氧静置培养48h，取发酵液进行菌量计数。
结果显示，在所述20株乳酸杆菌发酵液中活菌量的对数值中，E18菌株经耐酸性培养基复筛后活菌量最多，对数值高达8.01Log CFU/mL。从而说明E18菌株耐酸能力最高。
实施例2 菌株鉴定
1、菌落形态鉴定
将E18菌株接种于MRS琼脂培养基上，37℃厌氧培养24h后，可见E18单菌落呈乳白色，菌落直径在1.5-2mm左右，表面光滑，显微镜下末端呈圆形的弯曲杆菌。
2、生理生化特性鉴定
本实施例中接种液的准备如下：在无菌条件下，取适量新鲜E18菌液，5000rpm/min离心5min，用PBS缓冲液洗2次，再用同体积PBS缓冲液重菌体后稀释50倍，作为接种液。
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接种液，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E18菌株最大耐受盐浓度为5％。
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E18菌株不产生气泡，是阴性反应。
2.3、碳源代谢试验
利用API 50CHL试剂条对E18菌株进行碳源代谢实验，实验方法及结果判读具体参见API 50CHL试剂盒说明书。E18菌株鉴定结果为：％ID＝93且T值＝0.74，API结果为发酵乳杆菌，鉴定评语为好的鉴定(目前API可鉴定种属中没有罗伊氏乳杆菌，故鉴定结果为发酵乳杆菌)。E18菌株API 50CHL碳源代谢结果如图1所示。
3、分子生物学鉴定
3.1 16s rDNA基因序列分析
3.1.1、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。
3.1.2、16s rDNA基因扩增
引物序列：
27F：AGAGTTTGATCCTGGCTCA；
1492R：GGTTACCTTGTTACGACTT。
通过测序获得E18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E18菌株为罗伊氏乳杆菌。
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，E18菌株为罗伊氏乳杆菌，其Riboprinter指纹图谱结果见图2。
3.3 RAPD和rep-PCR指纹图谱鉴定
3.3.1、RAPD指纹图谱鉴定
引物序列：GAGGGTGGCGGTTCT。
E18菌株的RAPD指纹图谱如图3所示。
3.3.2、rep-PCR指纹图谱
引物序列：CTACGGCAAGGCGACGCTGACG。
E18菌株的的rep-PCR指纹图谱如图4所示。
将E18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，确定E18菌株为一株新的罗伊氏乳杆菌，将其命名为罗伊氏乳杆菌VHProbi E18。
实施例3 罗伊氏乳杆菌VHProbi E18对人工胃液和人工肠液的耐受性试验
1、人工胃液的配制
分别称取蛋白胨5g、酵母提取物2.5g、葡萄糖1g和NaCl 2g，加入1000mL蒸馏水，用稀盐酸调pH3.0，然后115℃灭菌20min。然后使用前加入3.2g猪粘膜胃蛋白酶，摇匀溶解，置37℃水浴摇床中温水浴1h，以模拟人体温度。
2、人工肠液的配制
分别称取蛋白胨5g、酵母提取物2.5g、葡萄糖1g、KH 2PO 4 6.8g和牛胆盐3.0g，加入77mL的0.2mol/L的NaOH溶液，定容至1000mL，用稀盐酸或者氢氧化钠溶液调pH6.8±0.1，115℃灭菌20min。然后使用前加入1g胰酶，摇匀溶解，置37℃水浴摇床中温水浴1h，以模拟人体温度。
3、试验方法
取2mL新鲜菌液，5000rpm/min离心5min收集菌体，菌体用生理盐水洗涤3次，再用2mL生理盐水重悬，作为接种液。取1mL接种液，加入到24mL人工肠液中，置于37℃水浴摇床(200rpm/min)3h，取样1mL，检测活菌量。
活菌计数方法按照国标《GB4789.35-2016-食品微生物检验乳酸菌检验》测定菌量，该菌株经过人工肠液消化后的活菌量(Log CFU/mL)见表1。
表1.人工胃肠液消化后的活菌量
Figure PCTCN2021101274-appb-000001
从表1可知，本发明筛选到的罗伊氏乳杆菌VHProbi E18经人工胃液和人工肠液消化后，活菌量上升。从而说明该菌株能够耐受人工胃液和人工肠液，而且还可以进行一定的萌发。
实施例4 罗伊氏乳杆菌VHProbi E18的溶血性及抗生素耐受性实验
1、溶血性实验
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。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：罗伊氏乳杆菌VHProbi E18不能生长，血细胞平板没有变化，说明罗伊氏乳杆菌VHProbi E18不产生溶血素，不能够溶解血细胞。
2、抗生素耐受性实验
微量肉汤稀释法测定抗生素对罗伊氏乳杆菌VHProbi E18的最小抑菌浓度MIC值具体结果见表2。
表2.罗伊氏乳杆菌VHProbi E18的抗生素MIC值
Figure PCTCN2021101274-appb-000002
MIC单位μg/mL
从表2的结果可以看出，本发明提供的罗伊氏乳杆菌VHProbi E18对红霉素和氨苄西林等常见抗生素敏感，生物安全性良好。
实施例5 罗伊氏乳杆菌VHProbi E18抗氧化功能测定
1、菌株清除DPPH(1，1-二苯基-2-三硝基苯肼)和羟基自由基(HRS)能力测定
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％。结果见表3。
表3.DPPH自由基清除率表
Figure PCTCN2021101274-appb-000003
3)菌株清除HRS能力的测定
将100μL 5mM的水杨酸钠-乙醇溶液，100μL 5mM的硫酸亚铁，500μL去离子水和200μL乳酸菌PBS菌悬液混匀后加入100μL过氧化氢溶液(3mM)，37℃水浴15min后在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 控制为去离子水替代样品，A 空白为去离子水替代样品和H 2O 2，结果见表4。
表4.HRS自由基清除率表
Figure PCTCN2021101274-appb-000004
2、菌株抗脂质过氧化实验鉴定
亚油酸乳化液的制备：0.1mL亚油酸，0.2mL Tween 20，19.7mL去离子水。
0.5mL的PBS溶液(pH 7.4)中加入1mL亚油酸的乳化液，1mLFeSO 4(1％)，再加入0.5mL样品，37℃水浴1.5h，混合液加入0.2mL TCA(4％)，2mL TBA (0.8％)，100℃水浴30min，迅速冷却，4000rpm/min离心15min，收集上清液在532nm下测吸光度即为A；对照组以0.5mL蒸馏水代替样品即为A 0。抑制率/％＝(A 0－A)/A 0×100％
注：A为样品组吸光度；A 0为对照组吸光度，结果见表5。
表5.抗脂质过氧化抑制率表
Figure PCTCN2021101274-appb-000005
实施例6 罗伊氏乳杆菌VHProbi E18体外胆固醇降解实验
1、胆固醇胶束溶液的配制：准确称取1g胆固醇，溶于无水乙醇中，并定容至100mL，在无菌条件下用0.22μm微孔滤膜过滤除菌。
2、称取蛋白胨10.0g牛肉膏10.0g酵母膏5.0g柠檬酸氢二铵2.0g葡萄糖20.0g，吐温80 1.0mL，乙酸钠5.0硫酸镁0.1硫酸锰0.05，磷酸氢二钾2.0g,胆盐1g，蒸馏水1000mL调节pH值7.3，115℃灭菌30min，然后加入胆固醇溶液使胆固醇终浓度为0.1％。
按照0.1％的接种量接种新鲜菌液，37℃静止培养48h，然后取0.2mL菌液，加入1.8mL无水乙醇，混匀，静止10分钟，3000转离心5分钟，取上清液用于测定胆固醇含量。胆固醇测定方法按照GB/T 5009.128-2003<食品中胆固醇的测定>。
结果显示：本发明提供的罗伊氏乳杆菌VHProbi E18对胆固醇的降解率达到16.24％(此为不含胆盐的数据)。
实施例7 罗伊氏乳杆菌VHProbi E18的疏水性细胞表面测试
1、待测菌液制备：挑取纯化好的罗伊氏乳杆菌VHProbi E18菌落接种于新配制的MRS液体培养基中，于37℃培养24～48h。再按1％(V/V)的接种量接至MRS液体培养基中于37℃继续培养24～48h后6000×g离心10min，收集菌体后用无菌生理盐水冲洗2次，再用灭菌0.1M KNO 3 1mL溶液重悬菌体，作为待测菌液。
2、表面疏水性测定：吸取50μL上述菌悬液加入2450μL的0.1M KNO 3并记录OD600为A 0,取1.5mL菌悬液与500μL二甲苯混匀后在室温下静置10min(此时形成两相体系)。将两相体系涡旋振荡2min后再静置20min,重新形成水相和有机相。小心吸取水相(不要吸到有机相)在600nm处测量吸光度A 1。细胞疏水性按公式Hydrophobicity％＝(A 0-A 1)/A 1×％计算，测三次实验取平均值。
结果显示：本发明提供的罗伊氏乳杆菌VHProbi E18细胞表面疏水性为61.76％，标准差为0.27％。
实施例8 罗伊氏乳杆菌VHProbi E18的细胞粘附性测试
Caco-2细胞，进行复苏、传代培养，培养细胞数量扩增至所需用量。加入胰酶后将细胞放回培养箱，肉眼观察到细胞完全脱落后使其尽量成为单细胞状态。使用血球计数板进行细胞计数，使用PBS适量稀释细胞悬液。使用MRS培养基将菌体重悬备用。将待测菌悬液与Caco-2细胞共同孵育2h后再用PBS洗去未黏附的细菌。加入胰酶消化后再加入细胞培养液终止消化，收集液体涂布计数。粘附能力(CFU/cells)＝每个培养孔内粘附的细菌总数/每个培养孔的总细胞数。
经检测，罗伊氏乳杆菌VHProbi E18黏附能力为4.7。
实施例9 罗伊氏乳杆菌VHProbi E18在改善衰老小鼠皮肤中的应用
1、实验动物
C57小鼠SPF级，雄性，3月龄小鼠6只，12月龄小鼠18只，体重19～25g。实验动物饲养管理的环境条件：室温20～26℃，日温差≤4℃，相对湿度40～70％，明暗交替时间为12/12h。动物饲养于标准小鼠笼具中，每笼6只。动物饲料、饮水：自由摄食、饮水。饲料为SPF级大小鼠生长繁育饲料。饮用水是经过高温消毒的城市自来水。
2、实验方法
小鼠适应性饲养7天后随机分为3月龄对照组、12月龄对照组组、12月龄涂抹组、12月龄灌胃组，每组6只小鼠。12月龄灌胃组按照0.2mL/10g灌胃给予益生菌菌液，12月龄涂抹组涂抹与灌胃组等量的益生菌液，3月龄对照组和12月龄对照组灌胃与益生菌等量的生理盐水。共持续70天。
3、检测指标
终末时，对小鼠背部皮毛感官评分，评分规则如表6所示。检测小鼠皮肤含水量、SOD活性和MDA含量变化，检测小鼠皮肤和尾腱中羟脯氨酸含量。
表6.小鼠背部皮毛感官评分量表
Figure PCTCN2021101274-appb-000006
试验结束后取背部皮肤组织，4％多聚甲醛固定，取材，脱水，石蜡包埋，切片，HE染色，检测毛囊数量、真皮厚度和皮脂腺细胞。
所有实验数据以均数±标准差表示，用Microsoft EXCEL进行数据统计和作图，两组数据间比较采用t检验，以P<0.05判定为有显著性差异。
4、实验结果
4.1小鼠背部皮毛感官评定
终末期时，3月龄小鼠毛发浓密，光泽整齐；12月龄对照组小鼠毛发干枯，脱毛较多并且有花白毛发；12月龄涂抹组小鼠毛发有光泽，无白色毛发；12月龄灌胃组小鼠毛发浓密有光泽，且呈现黑色。各组小鼠背部皮毛感官评分比较结果如图5所示。
4.2小鼠皮肤含水量、MDA和羟脯氨酸含量变化
12月龄各组别在终末约14～15月龄相对比3月龄对照组终末约5～6月龄，皮肤含水量均显著降低(p<0.05)，下降率约为15.95％；12月龄灌胃组相比12月龄对照组皮肤含水率上升，且具有显著性差异(p<0.05)。各组小鼠皮肤含水率比较如图6所示。
与12月龄对照组相比，12月龄灌胃组皮肤MDA含量显著降低，且具有显著性差异(p<0.05)，12月龄涂抹组皮肤MDA含量显著降低，也具有显著性差异(p<0.05)，12月龄涂抹组与12月龄灌胃组终末状态皮肤MDA含量相比差异不具有显著性(p>0.05)。各组小鼠皮肤MDA含量比较如图7所示。
与12月龄对照组相比，12月龄灌胃组和涂抹组皮肤和尾腱中的羟脯氨酸含量升高，且具有显著性差异(p<0.05)，12月龄涂抹组与12月龄灌胃组终末状态皮肤羟脯氨酸含量相比差异不具有显著性(p>0.05)。各组小鼠皮肤和 尾腱中羟脯氨酸含量比较如图8所示。
4.3小鼠背部皮肤组织学检查
光学显微镜下可见，3月龄对照组小鼠表皮结构完整，细胞分层清晰，具有明显的表皮突和真皮乳头，视野内未见炎性细胞浸润，可见皮脂腺增生，视野内可见真皮层胶原纤维，排列分布均匀成带状；12月龄对照组小鼠表皮变薄明显，角质层脱落可见，结构完整度欠缺，细胞数量减少，排列不规则，胶原纤维层明显减少，纤维断裂、分布不均匀、稀疏，成纤维细胞减少；12月龄灌胃组小鼠上皮细胞增生，排列偶有疏松紊乱，毛囊皮脂腺时有增生，炎性细胞偶有出现，胶原纤维分布松散，断裂可见；12月龄涂抹组上皮细胞增生明显，角质层脱落可见，毛囊和皮脂腺增生明显，炎性细胞可见，皮肤松散可见，胶原纤维层排列分布松散，断裂现象明显。小鼠皮肤典型HE切片结果如图9。
终末时，与3月龄对照组相比，12月龄对照组小鼠皮肤毛囊密度下降，且具有显著性差异(P<0.05)；与12月龄对照组小鼠相比，12月龄涂抹组和12月龄灌胃组毛囊密度上升，且具有显著性差异(P<0.05)。
与3月龄对照组相比，12月龄对照组小鼠皮肤表皮厚度有下降，但没有显著性差异；12月龄各组小鼠肤表皮厚度没有差异。
与3月龄对照组相比，12月龄对照组小鼠皮肤真皮厚度下降，且具有显著性差异(P<0.05)；与12月龄对照组小鼠相比，12月龄涂抹组和12月龄灌胃组皮肤真皮厚度上升，且具有显著性差异(P<0.05)。
与3月龄对照组相比，12月龄对照组小鼠皮肤胶原纤维面积下降，且具有显著性差异(P<0.05)；与12月龄对照组小鼠相比，12月龄涂抹组和12月龄灌胃组皮肤胶原纤维面积上升，且具有显著性差异(P<0.05)。各组别小鼠皮 肤衰老程度对比结果如图10所示。
由上述结果可知，与12月龄对照组小鼠相比，12月龄灌胃组和12月龄涂抹组小鼠皮毛光泽度均有改善，没有出现白毛，脱毛现象减少。12月龄灌胃组和12月龄涂抹组小鼠皮肤含水率上升，MDA含量下降。羟脯氨酸作为真皮内含量丰富稳定的氨基酸，其含量可以直接反映出真皮内胶原蛋白的含量变化，是检测皮肤衰老的指标之一。12月龄灌胃组和12月龄涂抹组小鼠皮肤和尾腱中羟脯氨酸含量上升，涂抹和灌胃罗伊氏乳杆菌VHProbi E18可以提高真皮内胶原蛋白的含量。从小鼠皮肤组织学检查看，与12月龄对照组小鼠相比，12月龄灌胃组和12月龄涂抹组小鼠表皮结构完整度改善，炎性细胞减少，成纤维细胞增多，并且灌胃组效果好于涂抹组。此外，12月龄灌胃组和12月龄涂抹组小鼠皮肤毛囊数量上升，真皮厚度增加，胶原纤维含量上升。结果表明罗伊氏乳杆菌VHProbi E18无论是灌胃还是涂抹均可以改善衰老小鼠皮肤指征达到延缓修复皮肤衰老效果。
综上所述，本发明提供的罗伊氏乳杆菌VHProbi E18对模拟人工肠胃液具有很强的耐受能力，这对于益生菌株顺利经过胃肠道在结肠处定植下来发挥益生功能奠定了基础。抗生素抗性试验证实罗伊氏乳杆菌VHProbi E18对常见抗生素敏感，不产溶血素，生物安全性良好。同时，罗伊氏乳杆菌VHProbi E18能够清除DPPH和HRS自由基，抑制脂质过氧化，具有一定的抗氧化功能活性，能够降解胆固醇，具有降低血清胆固醇的益生特性。经动物实验证实，罗伊氏乳杆菌VHProbi E18具有抗衰老作用，能够增加皮肤胶原蛋白含量，改善毛囊健康，提升皮肤健康状态。所述罗伊氏乳杆菌VHProbi E18可用于制备延缓修复皮肤衰老效果的功能性食品或化妆品，应用前景广阔。

Claims (8)

  1. 一种罗伊氏乳杆菌，其特征在于，所述的罗伊氏乳杆菌的保藏编号为CCTCC NO：M2021153。
  2. 如权利要求1所述的罗伊氏乳杆菌，其特征在于，所述的罗伊氏乳杆菌的指纹图谱如图2所示。
  3. 如权利要求1所述的罗伊氏乳杆菌，其特征在于，所述的罗伊氏乳杆菌的RAPD指纹图谱如图3所示。
  4. 如权利要求1所述的罗伊氏乳杆菌，其特征在于，所述的罗伊氏乳杆菌的rep-PCR指纹图谱如图4所示。
  5. 如权利要求1所述的罗伊氏乳杆菌，其特征在于，所述的罗伊氏乳杆菌的16s rDNA序列为SEQ ID NO:1。
  6. 权利要求1所述的罗伊氏乳杆菌在制备具有抗氧化功能的制品中的应用。
  7. 权利要求1所述的罗伊氏乳杆菌在制备具有延缓修复皮肤衰老效果的制品中的应用。
  8. 如权利要求6或权利要求7所述的应用，其特征在于，所述的制品为功能性食品。
PCT/CN2021/101274 2021-04-18 2021-06-21 一株具有改善衰老皮肤和增强毛发健康作用的罗伊氏乳杆菌及其应用 WO2022222259A1 (zh)

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