WO2022199660A1 - 鼠李糖乳杆菌、调节皮肤微生态的发酵溶胞物、制法及其应用 - Google Patents

鼠李糖乳杆菌、调节皮肤微生态的发酵溶胞物、制法及其应用 Download PDF

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WO2022199660A1
WO2022199660A1 PCT/CN2022/082787 CN2022082787W WO2022199660A1 WO 2022199660 A1 WO2022199660 A1 WO 2022199660A1 CN 2022082787 W CN2022082787 W CN 2022082787W WO 2022199660 A1 WO2022199660 A1 WO 2022199660A1
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fermentation
lysate
strain
lactobacillus rhamnosus
supernatant
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PCT/CN2022/082787
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English (en)
French (fr)
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陆震
魏玉洁
孙元军
杜冉冉
赵岩
郭学平
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华熙生物科技股份有限公司
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Priority to EP22774314.3A priority Critical patent/EP4317411A1/en
Publication of WO2022199660A1 publication Critical patent/WO2022199660A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A61Q17/005—Antimicrobial 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the invention relates to the technical field of cosmetic raw materials, in particular to a Lactobacillus rhamnosus, a fermented lysate for regulating skin microecology, a preparation method and applications thereof.
  • Skin microecology is to study the relationship between the structure, function, and human skin itself of human skin microbiota, so as to explain the interaction between skin and microorganisms and the nature of abnormal changes in skin.
  • the skin microecology is mainly composed of various microorganisms such as bacteria, fungi, viruses, mites and arthropods, as well as the tissues, cells, various secretions and microenvironments on the skin surface.
  • the interaction of skin microbes, host and external environment constitutes the skin microecological balance.
  • the human skin microecology is directly related to human health. When the human microecology is abnormal, symptoms such as burning, stinging, itching, and tightness of sensitive skin will appear.
  • the skin barrier consists of four layers, the outermost microbial barrier, chemical barrier, physical barrier and immune barrier. Among them, the four types of barrier functions are in balance with each other. Under normal conditions, they have the function of quickly repairing the barrier and immune homeostasis. An imbalance at a certain level causes the skin's susceptibility to external pressure and skin diseases. Skin immunity and skin barrier are closely related, and the microbial barrier and immune barrier together constitute the skin immune function.
  • skin care products can increase the diversity of beneficial skin flora by adding certain ingredients, or inhibit the invasion and proliferation of pathogenic bacteria by enhancing the reproduction of probiotics.
  • This concept can be modeled on the human body. Intestinal probiotics, through the consumption of probiotic-containing foods can help to regulate intestinal health, which is to achieve the overall improvement of the human immune function by regulating the intestinal flora.
  • lactic acid bacteria can also play some of the functions of live bacteria preparations, and there is a certain dose correlation, so inactivated lactic acid bacteria preparations provide another relatively stable direction for the industrialization of microecological preparations.
  • products such as high temperature heating, strong acid and strong alkali are often obtained by violent cell crushing, so that although high-efficiency cell crushing can be achieved, the active ingredient will be severely affected by the crushing process.
  • the destruction of the bacteria leads to inactivation; or the bacteria are inactivated by a mild method, but the entire structure of the bacteria remains relatively intact, so that the relatively complete cell structure cannot be efficiently absorbed and utilized by human skin cells in cosmetics.
  • the really highly active substances are the oligosaccharides, oligopeptides, amino acids, etc. that make up the entire structure of the cell. Only small molecular substances can be absorbed and utilized by the skin cells. Therefore, looking for a way to mildly degrade the cell structure of probiotics can really be used. Prebiotics work.
  • lactic acid bacteria have different effects.
  • Bifidobacterium has repair and protection function
  • Lactobacillus plantarum usually has antioxidant effect
  • Lactobacillus rhamnosus has the effect of these lactic acid bacteria.
  • It is often used as a safe strain in food products, which can improve human immunity after use.
  • the source of strains screened in this patent is obtained from breast milk, which has a large number of antibodies in breast milk itself, and it is long-term colonized in this kind of bacteria.
  • Lactobacillus rhamnosus has undergone long-term domestication during its own growth in the environment, and has a higher function of metabolizing immune active substances.
  • the selected strain is prepared according to the patented process, and its fermentation product is found to be able to express antibacterial at a high level through efficacy experiments. At the same time, it has the ability to regulate the diversity of skin colonies and effectively inhibit the transformation process of the pathogenic properties of Candida albicans.
  • the patent application whose application number is 200810068138.X, the invention title is "inactivated lactic acid bacteria microecological preparation and preparation method thereof" discloses a preparation method of an inactivated lactic acid bacteria preparation, wherein the method of inactivation is heat inactivation , freezing inactivation and formaldehyde inactivation, etc., the chemical inactivation method described in the patent application is to use formaldehyde and other fungicides for inactivation, and the safety of the product is not guaranteed.
  • lactic acid bacteria have been used in a low pH environment for a long time, although they can be inactivated at low pH, the structure of lactic acid bacteria will not be greatly damaged. In this way, raw materials that are used in cosmetics to inactivate but maintain a complete cell structure are in the form of cells. As a functional ingredient, skin cells cannot be used and absorbed, and the inherent efficacy of lactic acid bacteria will not be exerted.
  • the Lactobacillus rhamnosus has a good immune regulation effect on the body, and the inhibitory effect on the expression of inflammatory factors, the promotion effect on the expression of anti-inflammatory factors and the promotion effect on lymphocyte proliferation are significantly better than the existing commercial Lactobacillus rhamnosus.
  • the strain has significantly better immune regulation function to the body than the existing Lactobacillus rhamnosus strains. During the experiment, the strains were all tested for efficacy by the application of live bacteria cells, and the extracellular products of the bacteria were discarded. As a result, this strain only has the activity of general Lactobacillus rhamnosus, such as the expression of antimicrobial peptide genes and protein levels, the regulation of pathogenic bacteria such as albicans, and the analysis and comparison of active substances.
  • the present invention provides a strain and a fermentation lysate obtained by fermentation of the strain.
  • the fermented lysate product is stable, has a high content of active substances, and is safe and healthy and meets the requirements of cosmetic raw materials.
  • the strain of the present invention is Lactobacillus rhamnosus 11-7 (Lactobacillus rhamnosus 11-7), which is isolated from the breast milk of newborns. 2021185.
  • the above-mentioned strains are used for fermentation and culture, and then the fermented lysate obtained by enzymatic hydrolysis can inhibit the reproduction and growth of pathogenic bacteria, promote some probiotic bacteria, and have a positive effect on the pathogenic forms of Candida albicans. It has a strong inhibitory effect. At the same time, during the fermentation process, it can not only produce antibacterial peptides, but also upregulate the expression of antibacterial peptides in skin cells at the gene and protein levels.
  • Lactobacillus rhamnosus 11-7 (Lactobacillus rhamnosus 11-7), it is preserved in China Type Culture Collection, and the preservation number is CCTCC NO:M 2021185.
  • Lactobacillus rhamnosus 11-7 whose deposit number is CCTCC NO: M 2021185 in the field of fermentation, preferably in the field of fermentation to produce fermentation lysate.
  • a fermentation lysate comprising protein, polysaccharide and amino acid, in terms of the mass percentage of the fermented lysate, the protein is 0.2-1%, preferably 0.6-0.9%, the polysaccharide is It is 0.3-0.8%, preferably 0.4-0.6%; the amino acid is 0.2-1%, preferably 0.4-0.5%.
  • the fermentation lysate is prepared by a method comprising the following steps:
  • the medium contains carbon source, nitrogen source and inorganic salt, preferably, the added amount of the carbon source is 1-5% (w/v), preferably 1-4% (w/v), and the nitrogen source is added in an amount of 1-5% (w/v).
  • the addition amount is 0.5-2wt%, and the addition amount of the inorganic salt is 0.1-1wt%;
  • the fermentation strain is subjected to enzymatic hydrolysis to obtain fermentation lysate.
  • centrifugation is used to obtain bacterial cell sediment, which is then suspended with sodium chloride solution, centrifuged to obtain bacterial cell debris, and then suspended with the second fermentation broth to obtain the fermentation lysate.
  • a method of preparing a fermentation lysate comprising the steps of:
  • Fermentation lysates were prepared using Lactobacillus rhamnosus 11-7 with deposit number CCTCC NO:M 2021185.
  • the medium contains carbon source, nitrogen source and inorganic salt, preferably, the added amount of the carbon source is 1-5% (w/v), preferably 1-4% (w/v), and the nitrogen source is added in an amount of 1-5% (w/v).
  • the addition amount is 0.5-2wt%, and the addition amount of the inorganic salt is 0.1-1wt%;
  • the fermented strain is obtained by enzymatic hydrolysis.
  • centrifugation is used to obtain bacterial cell sediment, which is then suspended with sodium chloride solution, centrifuged to obtain bacterial cell debris, and then suspended with the second fermentation broth to obtain the fermentation lysate.
  • the fermentation strain selected in the present invention is probiotics, and a strain of Lactobacillus rhamnosus is isolated and screened from breast milk. After fermentation, the prepared fermented liquid is subjected to the process of cell breaking, and an enzymatic hydrolysis and high The mild cell crushing method combined with osmotic solution inactivates and breaks the cells. After the cells are crushed, a variety of active ingredients such as amino acids, polysaccharides, polypeptides, etc., which are easily absorbed by small molecules, can be formed. In the form of easily absorbed small molecules.
  • the addition amount of the enzymatic hydrolysis solution can be reduced, the risk of skin allergy can be reduced, and the active components existing in the supernatant outside the cells can also be retained.
  • the whole process not only maintains the types and activities of all the functional substances in the fermentation, but also converts them into a form that is easy to absorb.
  • the cycle is short, the process is controllable, easy to scale up, the product is stable, and the active substance content is high, the effect is good, the absorption is easy, and the safety and health are in line with the characteristics of cosmetic raw materials.
  • the fermentation lysate prepared by this strain with the patented process has the following characteristics:
  • Fig. 1 is the electrophoresis result schematic diagram in embodiment 1;
  • Figure 2 is a schematic diagram of the growth of Candida albicans mycelium in Experimental Example 5, wherein Figure 2A is a schematic diagram of the growth of Candida albicans mycelium in GE medium, and Figure 2B is a schematic diagram of the growth of Candida albicans mycelium added with 5% S1 Schematic diagram, Figure 2C is a schematic diagram of the growth of C. albicans mycelium with 5% C1 added, Figure 2D is a schematic diagram of the growth of C. albicans mycelium with 5% C2 added, Figure 2E is a schematic diagram of the growth of C. Figure 2F is a schematic illustration of C. albicans hyphal growth with 5% C4 added.
  • Figure 3A is a schematic diagram of a blank control for HBD2 immunohistochemical detection.
  • Figure 3B is a schematic diagram of a positive control for HBD2 immunohistochemical detection.
  • Figure 3C is a schematic diagram of the S1 sample set for HBD2 immunohistochemical detection.
  • Figure 3D is a schematic diagram of a blank control for HBD3 immunohistochemical detection.
  • Figure 3E is a schematic diagram of a positive control for HBD3 immunohistochemical detection.
  • Figure 3F is a schematic diagram of the S1 sample set for HBD3 immunohistochemical detection.
  • the strain Lactobacillus rhamnosus 11-7 (Lactobacillus rhamnosus 11-7) used in the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on February 22, 2021, and the deposit number is CCTCC NO: M 2021185. Address: China. Wuhan. Wuhan University, Postal Code: 430072; Tel: (027)-68754052.
  • the invention provides a strain, the strain is Lactobacillus rhamnosus 11-7 (Lactobacillus rhamnosus 11-7), which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2021185.
  • the strain was isolated and purified from neonatal breast milk.
  • the present invention provides the application of the above-mentioned strain in the field of fermentation, preferably in the field of fermentation to produce fermentation lysate.
  • the present invention uses the above-mentioned strains for fermentation.
  • Lactobacillus rhamnosus as a safe strain, is widely used as an excellent probiotic in the fields of food, medicine and cosmetics, and breast milk is the first protective barrier for newborns.
  • the activity of probiotics is self-evident, so separating and screening the probiotics colonized in breast milk and then preparing the fermented lysate through this process can well retain all the active substances in the final product, so from the source, Its activity and efficacy are far superior to traditional probiotics in terms of strain type and preparation process.
  • the present invention provides a fermentation lysate, which includes protein, polysaccharide and amino acid, and in terms of the mass percentage in the fermentation lysate, the protein is 0.2-1%, preferably 0.6-0.9%, so The polysaccharide is 0.3-0.8%, preferably 0.4-0.6%; the amino acid is 0.2-1%, preferably 0.4-0.5%.
  • the protein is 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, etc. based on the mass percentage in the fermentation lysate;
  • the polysaccharide is 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, etc.;
  • the amino acids are 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% and the like.
  • the fermentation lysate is obtained by fermentation and enzymolysis using the Lactobacillus rhamnosus 11-7 strain with deposit number CCTCC NO:M 2021185, preferably, the fermentation lysate is obtained by comprising the following The method of the above steps is prepared:
  • the fermented strain is obtained by enzymatic hydrolysis.
  • the fermentation medium contains carbon sources, nitrogen sources and inorganic salts known to those skilled in the art, the carbon sources can be, for example, glucose, lactose, sucrose, maltose or mannitol, preferably glucose, and the added amount of the carbon sources It can be 1-5% (w/v), preferably 1-4% (w/v);
  • the nitrogen source can be, for example, beef extract, peptone, yeast powder, bean cake powder, etc., preferably peptone, and the nitrogen source can be added in an amount of 0.5-2wt%, preferably 1-2wt%;
  • the inorganic salt can be, for example, magnesium sulfate, dipotassium hydrogen phosphate, sodium chloride, manganese chloride, etc., preferably magnesium sulfate and dipotassium hydrogen phosphate, and the addition amount of magnesium sulfate can be 0.1-1wt%, preferably 0.5- 0.8wt%, the addition amount of dipotassium hydrogen phosphate can be 0.1-0.5wt%, preferably 0.1-0.2wt%.
  • the inoculum amount is 0.1-0.5%.
  • the enzymes used in the enzymatic hydrolysis are lysozyme, neutral protease and helicase, preferably the specific enzyme activity of the lysozyme is 2000-20000 IU/mg, and the specific enzyme activity of the neutral protease is 2000-20000 IU/mg.
  • the activity is 50000-100000IU/g.
  • the specific enzyme activity refers to the enzyme activity per milligram of the product.
  • the lysozyme also known as muramidase or N-acetylmuramide glycanohydrlase, is an alkaline enzyme capable of hydrolyzing mucopolysaccharides in bacteria. Lysozyme mainly breaks down the ⁇ -1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, so that the cell wall insoluble mucopolysaccharide is decomposed into soluble glycopeptides, resulting in the rupture of the cell wall and the escape of the contents. dissolve bacteria.
  • the neutral protease is obtained by fermentation and extraction of Bacillus subtilis, belongs to an endonuclease, and can be used for various protein hydrolysis treatments. At a certain temperature and pH value, macromolecular proteins can be hydrolyzed into amino acids and other products. It can be widely used in the enzymatic hydrolysis and refining process of animal, plant and microbial protein products. This product can act on the cell wall soluble polypeptide, and the compound lysozyme can completely degrade it.
  • the helicase is a mixed enzyme prepared from the snail's sac and digestive tract, and it contains more than 20 kinds of enzymes such as cellulase, pectinase, amylase, and protease.
  • Helicase is a valuable enzyme. It can be used to break the yeast cell wall, so it is widely used in the research of cell biology and genetic engineering; it can be used as a feed additive to improve the digestibility of the feed; it can also be used for clarification of fruit juice, decapsulation of oranges, and jam making Wait.
  • the specific enzyme activity of the lysozyme is 2000IU/mg, 3000IU/mg, 4000IU/mg, 5000IU/mg, 6000IU/mg, 7000IU/mg, 8000IU/mg, 9000IU/mg, 10000IU/mg, 11000IU/mg , 12000IU/mg, 13000IU/mg, 14000IU/mg, 15000IU/mg, 16000IU/mg, 17000IU/mg, 18000IU/mg, 19000IU/mg, 20000IU/mg, etc.;
  • the specific enzyme activity of the neutral protease is 5000IU/mg, 6000IU/mg, 7000IU/mg, 8000IU/mg, 9000IU/mg, 10000IU/mg and the like.
  • the wall-breaking rate refers to 10-20 mg of enzyme per gram of cells in isotonic sorbitol solution with pH 5.8-7.2 at 37°C for 1 hour. dissolve the cell wall.
  • the enzymatic hydrolysis time is 1-2 h, and the enzymatic hydrolysis temperature is 35-40°C.
  • the enzymolysis time is 1h, 1.5h, 2h, etc.
  • the enzymolysis temperature is 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.
  • the added lysozyme is 0.01-0.1 mg
  • the added helicase is 1-10 mg
  • the added neutral protease is 0.01-0.1 mg.
  • the added lysozyme can be 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, etc.;
  • the added helicase can be 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, etc.;
  • the added neutral protease can be 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, etc.;
  • the present invention uses the above-mentioned enzyme to enzymatically hydrolyze the fermented strain, which adopts a mild crushing method to inactivate and crush the fermented strain.
  • the cellular form that cannot be absorbed by human skin is transformed into a small molecule state that is easily absorbed.
  • the present invention does not limit the method of adding the above-mentioned enzymes.
  • three enzymes can be added after mixing, or they can be added one by one.
  • the order of adding one by one is not limited in the present invention. way to select.
  • the supernatant obtained after the centrifugation of the first fermentation broth is filtered through a filter membrane to obtain the second fermentation broth.
  • centrifugation is used to obtain bacterial cell sediment, which is then suspended with sodium chloride solution, centrifuged to obtain bacterial cell debris, and then suspended with the second fermentation broth to obtain the fermentation lysate.
  • the present invention uses sodium chloride to suspend, resulting in the degradation of the cell wall of the hypertonic environment after the enzyme treatment, and the remaining intracellular substances will be ruptured due to hypertonicity to form bacterial fragments, and the sodium chloride mainly causes a hypertonic environment.
  • the supernatant after the centrifugation of the first fermentation liquid that is, the second fermentation liquid
  • the second fermentation liquid contains extracellular active substances in the fermentation process
  • the fermentation lysate contains Extracellular active substances.
  • the present invention provides a method for preparing fermentation lysate, comprising the following steps:
  • Fermentation lysates were prepared using Lactobacillus rhamnosus 11-7 with deposit number CCTCC NO:M 2021185.
  • the fermentation lysate is obtained by subjecting the strain to fermentation and enzymatic hydrolysis.
  • the method comprises the steps of:
  • the inoculation amount is 0.1-0.5%.
  • the fermentation is carried out at a temperature of 35-38° C., and the fermentation time is 40-70 h. Within this time range, the first fermentation broth is obtained after the sugar is exhausted.
  • the present invention does not limit the preparation of strains in log phase, which can be prepared by using methods commonly used in the art.
  • the strains are inoculated into MRS liquid medium, and cultured at 37° C. for 15-20 hours to obtain logarithmic strains. period strains.
  • the enzymes used in the enzymatic hydrolysis are lysozyme, neutral protease and helicase, preferably the specific enzyme activity of the lysozyme is 2000-20000 IU/mg, and the specific enzyme activity of the neutral protease is 2000-20000 IU/mg.
  • the activity is 50000-100000IU/g; preferably, the enzymolysis time is 1-2h, and the enzymolysis temperature is 35-40°C;
  • the added lysozyme is 0.01-0.1 mg
  • the added helicase is 1-10 mg
  • the added neutral protease is 0.01-0.1 mg.
  • the supernatant obtained after centrifugation of the first fermentation broth is filtered through a filter membrane to obtain the second fermentation broth.
  • the added amount of the enzymatic hydrolyzate can be reduced, the risk of skin allergy can be reduced, and the active components existing in the supernatant outside the cells can also be retained.
  • the whole process not only maintains the types and activities of all the functional substances in the fermentation, but also converts them into a form that is easy to absorb.
  • the filter membrane is a 0.22 ⁇ m filter membrane.
  • centrifugation is used to obtain thalline sediment, and then sodium chloride solution is used to suspend, and the thalline fragments are obtained by centrifugation, and then the second fermentation broth is used to suspend to obtain the fermentation lysate.
  • the bacterial sediment refers to centrifugation after enzymatic hydrolysis, and the sediment obtained by removing the supernatant after centrifugation is the bacterial sediment.
  • the bacterial cell debris refers to the bacterial cell debris obtained by suspending the bacterial cell sediment in a sodium chloride solution, and then centrifuging to remove the supernatant.
  • the sodium chloride solution is a sterilized sodium chloride solution, and the concentration of the sodium chloride is 3-5wt%.
  • the mass volume percentage of the bacterial fragments in the second fermentation broth is 1-10%, preferably 5-8%.
  • the mass volume percentage of the bacterial fragments in the second fermentation broth may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% Wait.
  • the fermentation lysate comprises proteins, polysaccharides and amino acids, and the protein is 0.2-1%, preferably 0.6-0.9% by mass percentage in the fermentation lysate, so The polysaccharide is 0.3-0.8%, preferably 0.4-0.6%; the amino acid is 0.2-1%, preferably 0.4-0.5%.
  • the fermented lysate of the present invention can inhibit the reproduction and growth of pathogenic bacteria, promotes some probiotics, and has a strong inhibitory effect on the pathogenic form of Candida albicans hyphae, and at the same time, it can not only produce antibacterial peptides during the fermentation process.
  • the protein level up-regulates the expression of antibacterial peptides in skin cells, improves skin immunity, and can significantly regulate the skin micro-ecological environment as a cosmetic raw material.
  • the mild microbial cell crushing method combining the enzymatic hydrolysis of the enzyme preparation and the hypertonic solution is used to inactivate and crush the microbial cells. From a cellular form that cannot be absorbed by human skin to a small molecule state that is easily absorbed.
  • the addition amount of the enzymatic hydrolysis solution can be reduced, the risk of skin allergy can be reduced, and the extracellular active components in the supernatant can also be retained.
  • the whole process not only maintains the types and activities of all the functional substances in the fermentation, but also converts them into a form that is easy to absorb.
  • the cycle is short, the process is controllable, easy to scale up, the product is stable, and the active substance content is high, the effect is good, the absorption is easy, and the safety and health are in line with the characteristics of cosmetic raw materials.
  • the present invention provides the application of the above-mentioned fermented lysate or the fermented lysate prepared by the above-mentioned preparation method in the field of cosmetics.
  • the fermented lysate is used for regulating skin microecology.
  • the present invention generally and/or specifically describes the materials and test methods used in the test.
  • % represents wt%, that is, weight percentage.
  • the reagents or instruments used without the manufacturer's indication are all conventional reagent products that can be obtained from the market.
  • Collect neonatal breast milk dilute the breast milk 10 times and mix it with non-sterile purified water, take 100 ⁇ L and spread it on a solid MRS plate, incubate at 37°C for 48 hours, and then dilute and coat the single colony on a solid MRS plate for separation and screening. , 37 °C incubator for 48h, the single colony morphology and physiological and biochemical experiments are as follows.
  • the strains were spread on glass slides, stained with Gram stain, and the cell morphology of the bacteria was observed.
  • the colony spreader was coated on the MRS solid plate at 37°C overnight for 24 hours, and the single colony state of the bacteria on the plate was observed.
  • strains were obtained for the following physiological and biochemical experiments, fermentation gas production experiment, contact enzyme reaction, starch hydrolysis reaction, casein hydrolysis reaction, phenylalanine deaminase reaction, citrate reaction, indole reaction, gelatin reaction Liquefaction reaction, nitrate reduction reaction, litmus milk reaction.
  • the genome of this strain was identified by Shanghai Sangon Bioengineering (Shanghai) Co., Ltd.
  • the sequencing result of 16S rDNA is shown in SEQ ID NO: 3, and the comparison by Blast proves that the strain is Lactobacillus rhamnosus 11-7.
  • the nucleotide sequence is as follows:
  • the fermentation medium contains 2 wt % of glucose, 1.5 wt % of peptone, 0.1 wt % of dipotassium hydrogen phosphate and 0.5 wt % of magnesium sulfate, cultured at 37° C. for 50 hours without residual sugar, the fermentation is completed, and the first fermentation broth is obtained, Denoted as fermentation broth 1.
  • the fermentation medium contains 4 wt % of glucose, 2 wt % of peptone, 0.2 wt % of dipotassium hydrogen phosphate and 0.8 wt % of magnesium sulfate, cultured at 37° C. for 65 hours, and the fermentation is completed after no residual sugar, and a first fermentation broth is obtained, Recorded as fermentation broth 2.
  • the fermentation medium contains 4 wt % of glucose, 2 wt % of peptone, 0.2 wt % of dipotassium hydrogen phosphate and 0.8 wt % of magnesium sulfate, cultured at 37° C. for 65 hours, and the fermentation is completed after no residual sugar, and a first fermentation broth is obtained, Recorded as fermentation broth 3.
  • the fermentation medium contains 2 wt % of glucose, 1 wt % of peptone, 0.1 wt % of dipotassium hydrogen phosphate and 0.5 wt % of magnesium sulfate, cultured at 37° C. for 65 hours, and the fermentation is completed after no residual sugar, and a first fermentation broth is obtained, Denoted as fermentation broth 4.
  • the fermentation medium contains 1 wt % of glucose, 0.5 wt % of peptone, 0.1 wt % of dipotassium hydrogen phosphate and 0.1 wt % of magnesium sulfate, cultured at 37° C. for 65 hours, and the fermentation is completed without residual sugar, and the first fermentation broth is obtained. , denoted as fermentation broth 5.
  • the fermentation medium contains 5 wt % of glucose, 0.8 wt % of peptone, 0.5 wt % of dipotassium hydrogen phosphate and 1 wt % of magnesium sulfate, cultured at 37° C. for 65 hours, and the fermentation is completed after no residual sugar, and the first fermentation broth is obtained, Denoted as fermentation broth 6.
  • the fermentation medium contains 5 wt % of glucose, 0.8 wt % of peptone, 0.5 wt % of dipotassium hydrogen phosphate and 1 wt % of magnesium sulfate, cultured at 37° C. for 65 hours, and the fermentation is completed after no residual sugar, and the first fermentation broth is obtained, Recorded as fermentation broth 7.
  • the protein determination method is as follows: It uses the Coomassie brilliant blue method:
  • Reagent Acid staining solution Take Coomassie brilliant blue G250 0.1g, add 50ml of ethanol to dissolve, add 100ml of phosphoric acid, add water to dilute to 1000ml, and mix well. Filter, take the filtrate, that is. This reagent should be placed in a brown bottle. If there is a precipitate, it should be filtered before use.
  • test solution The preparation of the test solution should be prepared according to the method specified under each category.
  • the protein concentration should be basically the same as that of the reference solution.
  • Determination method Precisely measure 0.0ml, 0.01ml, 0.02ml, 0.04ml, 0.06ml, 0.08ml, 0.1ml of the reference solution (the amount of the reference solution can be adjusted appropriately within the measurement range of this method), and place plugs respectively.
  • the determination method of amino acid content is obtained by amino acid automatic analyzer (LA8080), and the content of amino acid is determined to be 0.5%;
  • Determination method of polysaccharide The content of polysaccharide was detected by the phenol-sulfuric acid method.
  • Standard solution Accurately weigh 100 mg of dry and constant weight glucose (analytical grade) into a volumetric flask, add water to make up to 250 mL, shake well and accurately draw 10 mL of the solution, add water to make up to 100 mL.
  • the supernatant was discarded by centrifugation to obtain bacterial fragments, and the bacterial fragments (13.0 g) were suspended with 130 mL of the second fermentation broth to obtain fermentation microcells (S4). Fang analyzed, among them, the protein was 0.3%, the amino acid was 0.3%, and the polysaccharide was 0.8%.
  • the fermentation strain (13.1 g) was suspended in 260 mL of purified water, 0.655 mg of lysozyme (specific enzyme activity was 5000 IU/mg), 0.131 mg neutral protease (specific enzyme activity was 60000 IU/mg), 78.6 mg Helicase, fully mixed, and centrifuged to discard the supernatant after enzymolysis at 37°C for 1 h to obtain the bacterial sediment; the bacterial sediment was suspended with 100 ml of 4% filtration-sterilized sodium chloride solution, and allowed to stand after suspension.
  • the supernatant was discarded by centrifugation to obtain bacterial fragments, and the bacterial fragments (3.6 g) were suspended with 60 mL of the second fermentation broth to obtain fermentation microcells (S7).
  • Fang analysis in which, the protein is 0.2%, the amino acid is 0.3%, and the polysaccharide is 0.3%.
  • Example 3-1 Take fermentation broth 7 (200 mL) and heat it in a 60°C water bath for 1 h to obtain a comparative sample of lactic acid bacteria lysate, denoted as C1, and analyzed according to the method described in Example 3-1, wherein the protein is 0.1% , 0.15% for amino acids and 0.2% for polysaccharides.
  • C2 A commercially available product of lactic acid bacteria lysate of difidus yeast was selected as a comparative sample, denoted as C2 (commercially available), wherein the protein was 0.1%, the amino acid was 0.07%, and the polysaccharide was 0.2%.
  • Fermentation broth 7 (200ml) was taken, centrifuged at 8000rpm to collect bacterial cells, and the supernatant was filtered through a 0.22-micron membrane for use.
  • the bacterial cells were suspended in 260 mL of purified water, 2.1 mg of lysozyme, 2.1 mg of neutral protease, and 63 mg of helicase were added, and the cells were mixed thoroughly. The solution was centrifuged for 1.5 h and the supernatant was discarded.
  • a total of 9.0 g of bacterial fragments after enzymatic hydrolysis of the fermentation broth were suspended with 4% sodium chloride solution sterilized by filtration, the suspension volume was 100 ml, left standing for 1 h, centrifuged to take the supernatant, and 130 mL of the sterilized fermentation broth 1 was used. The supernatant was suspended, and the sample was recorded as C3, and analyzed according to the method described in Example 3-1, wherein the protein was 0.2%, the amino acid was 0.1%, and the polysaccharide was 0.1%.
  • Comparative Example 4 uses commercially available Lactobacillus rhamnosus fermentation to obtain fermentation lysate (C4).
  • the analysis was carried out as described above, wherein the protein was 0.1%, the amino acid was 0.3%, and the polysaccharide was 0.2%.
  • MRS solid plate To prepare MRS solid plate, take the example and the comparative example to coat 100 ⁇ L of the stock solution with a coater respectively, dilute 10 times to coat 100 ⁇ L, dilute 100 times to coat 100 ⁇ L, and place it in a 37°C incubator for 48 hours of static culture, and count single colonies. The results are shown in Table 9.
  • Example 3-4 1 0 0
  • Example 3-5 1 0 0
  • Examples 3-6 0 1 0
  • Examples 3-7 1 0 0
  • Examples 3-8 1 0 0
  • Examples 3-9 0 1 0 Comparative Example 1 56 16 0
  • Comparative Example 2 34 4 0
  • Comparative Example 3 twenty one 3 0 Comparative Example 4 16 3 0
  • the amino acid content was measured with the samples of the examples and comparative examples, using an amino acid automatic analyzer, and the results are shown in Table 10.
  • test example The samples of the test example and the comparative example were diluted 10 times with purified water respectively, and the Oxford cup bacteriostatic test method was adopted. The inhibitory effects of the samples on pathogenic bacteria were compared, and the results are shown in Table 11.
  • the fermentation lysates prepared in the examples can inhibit the growth of pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and the like.
  • Candida albicans hyphae The pathogenicity of Candida albicans hyphae is related to morphological transformation.
  • Candida albicans can grow in three forms, namely yeast form, pseudohyphal form and mycelial form. Pseudohyphal state can produce germ tube-specific antigen, which can significantly enhance its point-attachment and prevent the phagocytosis and killing of neutrophils. After being swallowed, germ tubes can still be formed and hyphae grow in the swallowing cells, thereby penetrating and destroying the host cell membrane.
  • Candida albicans grown in the form of hyphae had stronger pathogenicity. The normal sojourn is yeast-like, which only exists in the surface layer of keratinocytes, and does not penetrate into it.
  • a germ tube When albicans is infected, a germ tube is first formed, and adheres to the surface of the host cell with the help of structures such as adhesin in the outermost layer of the cell wall. After that, the germ tube gradually transforms into a budding hyphae or hyphal phase, and penetrates into the cell. Growth, when showing mycelial shape indicates a strong pathogenicity.
  • Example 3-1 and Comparative Examples 1 to 4 were cultured for 6 hours. , the germination of Candida albicans hyphae was inhibited, and the growth results were shown in Figure 2A to Figure 2F, respectively.
  • the S1 sample can well inhibit the germination of the hyphae of C. albicans pathogenic bacteria.
  • Lactic acid bacteria fermentation lysate has immunomodulatory and chemotactic effects.
  • skin cell-related antimicrobial peptides such as defensins and other factors will be significantly up-regulated.
  • the expression of antimicrobial peptides in the skin was significantly reduced.
  • the fermentation samples of Examples and Comparative Examples were added to keratinocytes at 0.4% and were purchased from the ATCC10231 cell bank. The expression levels were then evaluated to evaluate the regulatory effect of the three samples on skin immunity-related genes.
  • the treated keratinocytes were treated with RNAiso Plus and then collected. According to the kit instructions, RNA extraction, reverse transcription and fluorescence quantitative PCR were performed. The results were analyzed by relative quantitative method. The detection steps were as follows.
  • RNAiso Plus 1-2 mL of RNAiso Plus to the 10cm 2 growing cultured cells and shake to ensure that the lysate is evenly distributed on the cell surface.
  • the cDNA template was synthesized by the reverse rate according to the reaction system shown in the reverse transcription kit, and then fluorescence quantitative PCR was carried out according to the reaction system shown in the real-time PCR kit (Shanghai Gema Pharmaceutical Technology Co., Ltd.), and the results as follows.
  • Example 3-5 1.686 1.654 Examples 3-6 1.990 1.342 Examples 3-7 1.643 1.872 Examples 3-8 1.701 1.545 Examples 3-9 1.797 1.411 Comparative Example 1 1.201 1.132 Comparative Example 2 0.995 1.010 Comparative Example 3 1.121 1.242 Comparative Example 4 1.337 1.122
  • the fermentation lysate obtained by using the strain of the present invention for fermentation and enzymatic hydrolysis by the method of the present invention has higher expression levels of HBD2 and HBD3, thus illustrating the present invention
  • the fermentation lysate has immunomodulatory and chemotactic effects.
  • the 3D skin model was used to evaluate its efficacy in the expression of antimicrobial peptides in the skin.
  • the S1 sample group was the lactic acid bacteria fermentation lysate obtained in Example 3-1 at a concentration of 39 ⁇ L. /cm 2 was added to the 3D skin model, the positive control group used 50mJ/cm 2 of UVB to continuously irradiate the 3D skin model for two days, which was used for the detection of HBD2 and HBD3 proteins, and the blank control was the 3D skin without treatment model, the process is as follows:
  • Sample group Take 25 ⁇ L of the prepared S1 sample group working solution and add it to the surface of the model;
  • Blank control Take 25 ⁇ L EpiGrowth (Guangdong Boxi Biological) culture medium and add it to the surface of the model;
  • Dewaxing and hydration Immerse the slices in xylene for 10 min, replace the xylene and then soak for 10 min, soak in absolute ethanol for 5 min, soak in 95% ethanol for 5 min, and soak in 75% ethanol for 5 min. Washed with PBS buffer 3 times, 5 min each time.
  • Antigen retrieval Put the paraffin sections into 0.01M sodium citrate antigen retrieval solution, use high pressure to repair, and take out the sections after cooling. Washed with PBS buffer solution 3 times, 5 min/time.
  • Block peroxidase (horseradish catalase DAB chromogenic kit (Shanghai Shenggong)): add 1 drop of 3% H 2 O 2 to each slice, and incubate at room temperature for 30 min to block internal derived peroxidase activity. Washed with PBS buffer solution 3 times, 5 min/time.
  • Serum blocking dropwise add serum homologous to the secondary antibody and block at 37°C for 60min without washing.
  • Primary antibody incubation drop the primary antibody working solution and incubate at 4°C overnight. Washed with PBS buffer 3 times, 5 min/time, using mouse-derived or sheep-derived recombinant Anti-HBD2 (Abcam)/Anti-HBD3 (Abcam) as the primary antibody, and using 3% BSA (bovine serum albumin) as the primary antibody. Dilute with PBS buffer at a dilution concentration of 1:100 to 1:500 to prepare primary antibody working solution.
  • BSA bovine serum albumin
  • Secondary antibody incubation drop the secondary antibody working solution and incubate at room temperature for 1 h. Washing with PBS buffer 3 times, 5 min/time, wherein, goat anti-mouse or rabbit anti-goat antibody is used as the secondary antibody, which is diluted with PBS buffer at 1:20-1:50 to prepare the secondary antibody working solution.
  • ABC complex incubation dropwise add ABC complex solution ( ABC-Peroxidase Kits), incubated at room temperature for 30min. Washed with PBS buffer 3 times, 5 min/time.
  • DAB staining add 1 drop of freshly prepared DAB solution to each section (specific parts will be stained brown), observe under microscope for 5-30s.
  • Figure 3A is a schematic diagram of a blank control for HBD2 immunohistochemical detection
  • Figure 3B is a schematic diagram of a positive control for HBD2 immunohistochemical detection
  • Figure 3C is HBD2 immunohistochemical detection
  • Figure 3D is a schematic diagram of a blank control for HBD3 immunohistochemical detection
  • Figure 3E is a schematic diagram of a positive control for HBD3 immunohistochemical detection
  • Figure 3F is a schematic diagram of the S1 sample group for HBD3 immunohistochemical detection .
  • the HBD2 protein of the lactic acid bacteria fermentation lysate sample group can be significantly increased by 39% in the skin 3D model, which is higher than the level of the positive control, while the effect of HBD3 protein is not obvious. .
  • the Lactobacillus rhamnosus of the present invention is used for fermentation and enzymolysis, and the obtained fermented lysate can strongly inhibit the reproduction and growth of pathogenic bacteria, and has a promoting effect on some probiotics, especially for The pathogenic form of Candida albicans hyphae has a strong inhibitory effect; and in the fermentation process, it can not only produce antimicrobial peptides, but also up-regulate the expression of antimicrobial peptides in skin cells at the gene and protein levels, and improve skin immunity.
  • the raw material can significantly regulate the skin micro-ecological environment.

Abstract

公开了一种保藏编号为CCTCC NO:M 2021185的鼠李糖乳杆菌、由其制备的调节皮肤微生态的发酵溶胞物及其制法。该发酵溶胞物包括蛋白质、多糖和氨基酸，以在发酵溶胞物中所占的质量百分比计，蛋白质为0.2-1％，多糖为0.3-0.8％，氨基酸为0.2-1％。使用该菌株进行发酵所得到的发酵溶胞物能够强力抑制致病菌的繁殖生长，对部分益生菌有促进作用，对白色念珠菌致病形态菌丝状有强烈抑制作用，并且在发酵过程中不但自身能够产生抗菌肽，还在基因水平，蛋白水平上调了肌肤细胞抗菌肽的表达量，提高皮肤免疫力。

Description

鼠李糖乳杆菌、调节皮肤微生态的发酵溶胞物、制法及其应用 技术领域
本发明涉及化妆品原料技术领域，尤其涉及一种鼠李糖乳杆菌、调节皮肤微生态的发酵溶胞物、制法及其应用。
背景技术
皮肤微生态是研究人体皮肤微生物群结构、功能、与人体皮肤本身之间的相互关系，从而阐述皮肤与微生物的相互作用关系和皮肤发生异常变化的本质。皮肤微生态主要由细菌、真菌、病毒、螨虫和节肢动物等各种微生物与皮肤表面的组织、细胞及各种分泌物、微环境等共同组成的生态系统。皮肤微生物、宿主及外环境三者相互作用构成了皮肤微生态平衡。人体皮肤微生态与人的健康有着直接的关系，当人体微生态出现异常诸如敏感肌肤类的灼热、刺痛、瘙痒、紧绷等症状就会出现。
皮肤屏障由四个层面构成，最外层的微生物屏障，化学屏障，物理屏障以及免疫屏障。其中，四大类屏障功能之间相互平衡，正常情况具备快速修复屏障与免疫稳态的功能，某一层次的失衡，造成皮肤对外界压力的易感性与皮肤疾病。皮肤免疫和皮肤屏障有着密切的关系，微生物屏障和免疫屏障共同构成了皮肤免疫功能。
皮肤免疫功能低下往往最先表现在角质层屏障，位于其上的微生态屏障，即和皮肤共生的微生物菌群特别容易被忽视。人体皮肤的基本特性是干燥、凉爽和弱酸性的，这些条件并不是微生物赖以生存的理想环境，但由于皮肤上大量褶皱，毛囊汗腺等的分布，在进化的过程中，皮肤上定植了种类多样的微生物，包括细菌、真菌和寄生虫等。据预测，每平方厘米的皮肤上可能定植最高数以十亿以上的微生物。
通过对人体表皮微生态的分析，护肤品中可以通过添加某些成分增加皮肤有益菌群的多样性，或者通过增强益生菌的繁殖来抑制致病菌的入侵和增殖，这一概念可以仿照人体的肠道益生菌，通过食用含益生菌类食品可以有助于调节肠道健康，这是通过对肠道菌群调节实现了对人体免疫功能整体状 况的提升。借此理论，人们通过筛选活性益生菌，人为外源性在各类的护肤品中添加含有益生菌的发酵产物，其中益生菌发酵产生的活性蛋白可以作为抗菌肽对有害的暂住菌(指的是人类在社会自然活动中与外界环境发生一系列行为时而无意识接触从而生长在人体身上的微生物)生长实现抑制作用，同时益生菌经过裂解释放的活性成分对人体肌肤起到供给营养作用。所以如何将益生菌发酵过程所获得的所有活性物不受破坏的温和完整的提取出来显得尤为重要，在化妆品法规中，活菌是无法直接作为原料添加到产品当中的，已有大量研究表明，乳酸菌的死菌体也能发挥活菌制剂的部分功能，而且还存在一定的剂量相关性，因此灭活乳酸菌制剂为微生态制剂的产业化提供另外一个相对稳定的方向。在现有的产品中，常常是通过剧烈的菌体破碎方式来获得产物如加热高温、强酸强碱等，这样虽然可以实现高效破碎菌体但是其中活性成分会因破碎工艺的影响而受到较为严重的破坏导致失活；或者是通过温和的方式使得菌体失去活性，而菌体整个结构保持的相对完整，这样相对完整的细胞结构在化妆品中使用人体皮肤细胞无法高效吸收利用，发挥功效作用，真正具有高活性的物质是构成细胞整个结构中的寡糖，寡肽，氨基酸成分等，只有小分子物质皮肤细胞才能吸收利用，所以寻找一种将益生菌的细胞结构温和降解的方式才能真正作为益生元发挥作用。
另外在益生菌种的选择上同样十分重要，不同的乳酸菌作用有所不同，如双歧杆菌具有修复防护功能，植物乳杆菌通常具有抗氧化作用，而鼠李糖乳杆菌与这几种乳酸菌作用不尽相同，它常常作为安全菌种应用到食品产品中，使用后能够提高人体免疫力，本专利所筛选菌种来源是从母乳中获得，在母乳本身具有大量的抗体，长期定植在这种环境中鼠李糖乳杆菌自身生长过程中经历了长期驯化，具有了更高代谢免疫活性物的功能，所筛选的菌种按照本专利工艺制备，其发酵产物经功效实验发现可以高水平表达抗菌肽，同时具备调节皮肤菌落多样性强效抑制白色念珠菌致病性态的转变过程。
(1)申请号为200810068138.X、发明名称为“灭活的乳酸菌微生态制剂及其制备方法”的专利申请公开了一种灭活乳酸菌制剂的制备方法，其中灭活的方法为热灭活、冷冻灭活和甲醛灭活等，专利申请中所述的化学灭活方法是采用甲醛等杀菌剂进行灭活，产品的安全性没有保证。
(2)韩国细胞生物技术公司的专利申请(申请号为：201180004722.9)中也是采用热灭活的处理方式，日本好侍健康食品株式会社的专利申请(申请 号为：200910206343.2)也采用热灭活工艺。
(3)申请号为201510259592.3发明名称为“一种灭活的乳酸菌液体制剂及其制备方法”的专利申请公开了一种灭活的乳酸菌液体制剂及其制备方法，提供一种灭活乳酸菌液体制剂，选用甜菜碱盐酸盐、氨基葡萄糖盐酸盐、γ-氨基丁酸等酸性添加剂作为灭活剂。其整个灭活工艺中均是添加盐酸盐类物质，主要依靠低pH实现灭活菌体，低pH下多种活性物同样存在失活可能，特别是一些碱性氨基酸类如赖氨酸、精氨酸、组氨酸在强酸下容易失活。另外由于乳酸菌长期适用低pH环境，虽在低pH下可以失活，但是乳酸菌细胞的结构不会有较大破坏，这样在化妆品中使用以灭活但保持完整细胞结构的原料产品，以细胞形态作为功效物质成分皮肤细胞无法利用吸收，不会发挥乳酸菌固有功效作用。
(4)申请号为CN 110122877 A的发明名称为“鼠李糖乳杆菌及其用途”的专利申请公开了一株鼠李糖乳杆菌及其用途，提出了鼠李糖乳杆菌(Lactobacillus rhamnosus)在调节免疫力方面的用途，所述鼠李糖乳杆菌于2017年8月10日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.14511，分类命名为：鼠李糖乳杆菌Lactobacillus rhamnosus。该鼠李糖乳杆菌对机体具有良好的免疫调节功效，对炎症因子表达的抑制作用、对抗炎因子表达的促进作用以及对淋巴细胞增殖的促进作用显著优于现有商业鼠李糖乳杆菌菌株，其对机体的免疫调节功能显著优于现有鼠李糖乳杆菌菌株。该菌种在进行实验过程中均是将活菌菌体细胞应用进行功效测试，菌体的胞外产物都被弃掉，同时没有破壁工艺胞内的活性物不能充分释放，活性大大降低，致使该菌种只具有一般鼠李糖乳杆菌的活性作用，如抗菌肽基因及蛋白水平的表达，白念等致病菌的调节作用及活性物的分析比较均有所欠缺。
发明内容
鉴于上述问题，本发明提供了一种菌株以及使用菌株发酵得到发酵溶胞物，所述的发酵溶胞物产品稳定，且活性物质含量高，安全健康符合化妆品原料的要求。
本发明所述的菌株为鼠李糖乳杆菌11-7(Lactobacillus rhamnosus 11-7)，是从新生儿母乳中分离得到，该菌株保藏于中国典型培养物保藏中心，保藏 编号为CCTCC NO:M 2021185。
本发明使用上述所述的菌株进行发酵培养，然后进行酶解所得到的发酵溶胞物能够抑制致病菌的繁殖生长，对部分益生菌促进作用，对白色念珠菌致病形态菌丝状有强烈抑制作用，同时发酵过程中不但自身能够产生抗菌肽，还在基因水平，蛋白水平上调了肌肤细胞抗菌肽的表达量，提高皮肤免疫力，作为化妆品原料能够显著调节皮肤微生态环境。
本发明具体技术方案如下：
1.一种鼠李糖乳杆菌11-7(Lactobacillus rhamnosus 11-7)，其保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 2021185。
2.保藏编号为CCTCC NO:M 2021185的鼠李糖乳杆菌11-7在发酵领域中的应用，优选在发酵生产发酵溶胞物领域中的应用。
3.一种发酵溶胞物，其包括蛋白质、多糖和氨基酸，以在发酵溶胞物中所占的质量百分比计，所述蛋白质为0.2-1％，优选为0.6-0.9％，所述多糖为0.3-0.8％，优选为0.4-0.6％；所述氨基酸为0.2-1％，优选为0.4-0.5％。
4.根据项3所述的发酵溶胞物，其中，所述发酵溶胞物通过使用保藏编号为CCTCC NO:M 2021185的鼠李糖乳杆菌菌株进行发酵、酶解而得到，
优选所述发酵溶胞物通过包含下述步骤的方法制备得到：
将处于对数期的所述菌株接种到发酵培养基中进行发酵，发酵至无残糖得到第一发酵液，对第一发酵液进行离心后去上清得到发酵菌株；优选的，所述发酵培养基包含碳源、氮源和无机盐，优选的，所述碳源的添加量为1-5％(w/v)，优选为1-4％(w/v)，所述氮源的添加量为0.5-2wt％，所述无机盐的添加量为0.1-1wt％；
将发酵菌株进行酶解从而得到发酵溶胞物。
5.根据项4所述的发酵溶胞物，其中，所述酶解所使用的酶为溶菌酶、中性蛋白酶和蜗牛酶，优选的所述溶菌酶的比酶活力为2000-20000IU/mg，所述中性蛋白酶的比酶活力为50000-100000IU/g。
6.根据项4或5所述的发酵溶胞物，其中，酶解时间为1-2h，酶解温度为35-40℃。
7.根据项5-6中任一项所述的发酵溶胞物，其中，针对1g的发酵菌株，添加的所述溶菌酶为0.01-0.1mg，添加的所述蜗牛酶为1-10mg，添加的所述中性蛋白酶为0.01-0.1mg。
8.根据项4-7中任一项所述的发酵溶胞物，其中，离心后得到的上清液用滤膜过滤得到第二发酵液。
9.根据项8所述的发酵溶胞物，其中，在酶解之后，在得到所述发酵溶胞物之前，还包括下述步骤:
酶解结束后离心得到菌体沉淀物，然后使用氯化钠溶液悬浮，离心得到菌体碎片物，接着使用第二发酵液悬浮得到所述发酵溶胞物。
10.根据项9所述的发酵溶胞物，其中，所述氯化钠的浓度为3-5wt％。
11.根据项9或10所述的发酵溶胞物，其中，所述菌体碎片物在所述第二发酵液的质量体积百分比为1-10％，优选为5-8％。
12.一种制备发酵溶胞物的方法，其包括下述步骤：
使用保藏编号为CCTCC NO:M 2021185的鼠李糖乳杆菌11-7来制备发酵溶胞物。
13.根据项12所述的方法，其中，将所述菌株进行发酵、酶解得到所述发酵溶胞物。
14.根据项13所述的方法，其中，所述方法包括下述步骤：
将处于对数期的所述菌株接种到发酵培养基上进行发酵，发酵至无残糖得到第一发酵液，对第一发酵液进行离心后去上清得到发酵菌株；优选的，所述发酵培养基包含碳源、氮源和无机盐，优选的，所述碳源的添加量为1-5％(w/v)，优选为1-4％(w/v)，所述氮源的添加量为0.5-2wt％，所述无机盐的添加量为0.1-1wt％；
将发酵菌株进行酶解得到。
15.根据项13或14所述的方法，其中，所述酶解所使用的酶为溶菌酶、中性蛋白酶和蜗牛酶，优选的所述溶菌酶的比酶活力为2000-20000IU/mg，所述中性蛋白酶的比酶活力为50000-100000IU/g。
16.根据项14或15所述的方法，其中，酶解时间为1-2h，酶解温度为35-40℃。
17.根据项15或16所述的方法，其中，以发酵菌株为1g计，所述溶菌酶为0.01-0.1mg，所述蜗牛酶为1-10mg，所述中性蛋白酶为0.01-0.1mg。
18.根据项14-17中任一项所述的方法，其中，离心后得到的上清液用滤膜过滤得到第二发酵液。
19.根据项18所述的方法，其中，在酶解之后，在得到所述发酵溶胞物 之前，还包括下述步骤:
酶解结束后离心得到菌体沉淀物，然后使用氯化钠溶液悬浮，离心得到菌体碎片物，接着使用第二发酵液悬浮得到所述发酵溶胞物。
20.根据项19所述的方法，其中，所述氯化钠的浓度为3-5wt％。
21.根据项19或20所述的方法，其中，所述菌体碎片物在所述第二发酵液的质量体积百分比为1-10％，优选为5-8％。
22.项3-11中任一项所述的发酵溶胞物或者项12-21中任一项所述的制备方法制备得到的发酵溶胞物在化妆品领域中的应用。
23.根据项22所述的应用，其中，所述发酵溶胞物用于调节皮肤微生态。
发明的效果
本发明所选用发酵菌种为益生菌，从母乳中分离筛选得到的一株鼠李糖乳杆菌，经发酵后，所制备的发酵液经菌体破碎工艺，探索出了一种酶解与高渗溶液相结合的温和菌体破碎方式将菌体灭活破碎，菌体破碎后可以形成多种小分子易于吸收的活性成分如氨基酸、多糖、多肽等，这样从人体皮肤无法吸收的细胞形态转化为易于吸收的小分子状态。并且，通过将菌体和发酵上清液分别处理，既可减少酶解液的添加量，降低皮肤过敏的风险，又使得胞外存在于上清液的活性成分也得以保留。整个工艺过程不仅保持了全部发酵中的功效物质种类及活性，还将其转化为易于吸收的形式。周期短、过程可控、易放大、产品稳定，且活性物质含量高、功效好、易吸收、安全健康符合化妆品原料的特点。同时该菌种以专利工艺所制备的发酵溶胞物具有以下特点：
1.强力抑制致病菌的繁殖生长，对部分益生菌有促进作用，尤其对白色念珠菌致病形态菌丝状有强烈抑制作用。
2.发酵过程中不但自身能够产生抗菌肽，还在基因水平，蛋白水平上调了肌肤细胞抗菌肽的表达量，提高皮肤免疫力，作为化妆品原料能够显著调节皮肤微生态环境。
附图说明
图1是实施例1中的电泳结果示意图；
图2是实验例5中的白色念珠菌菌丝生长的示意图，其中，图2A是GE 培养基的白色念珠菌菌丝生长的示意图，图2B是添加5％S1的白色念珠菌菌丝生长的示意图，图2C是添加5％C1的白色念珠菌菌丝生长的示意图，图2D是添加5％C2的白色念珠菌菌丝生长的示意图，图2E是添加5％C3的白色念珠菌菌丝生长的示意图，图2F是添加5％C4的白色念珠菌菌丝生长的示意图。
图3A是HBD2免疫组化检测的空白对照的示意图。
图3B是HBD2免疫组化检测的阳性对照的示意图。
图3C是HBD2免疫组化检测的S1样品组的示意图。
图3D是HBD3免疫组化检测的空白对照的示意图。
图3E是HBD3免疫组化检测的阳性对照的示意图。
图3F是HBD3免疫组化检测的S1样品组的示意图。
菌株保藏信息
本发明用的菌株鼠李糖乳杆菌11-7(Lactobacillus rhamnosus 11-7)，于2021年2月22日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:M 2021185，保藏地址：中国.武汉.武汉大学，邮政编码：430072；电话：(027)-68754052。
具体实施方式
下面结合附图所描述的实施方式对本发明做以详细说明，其中所有附图中相同的数字表示相同的特征。虽然附图中显示了本发明的具体实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
需要说明的是，在说明书及权利要求当中使用了某些词汇来指称特定组件。本领域技术人员应可以理解，技术人员可能会用不同名词来称呼同一个组件。本说明书及权利要求并不以名词的差异作为区分组件的方式，而是以组件在功能上的差异作为区分的准则。如在通篇说明书及权利要求当中所提及的“包含”或“包括”为开放式用语，故应解释成“包含但不限定于”。说明书后续描述为实施本发明的较佳实施方式，然而所述描述乃以说明书的一般原则为目的，并非用以限定本发明的范围。本发明的保护范围当视所附权利要 求所界定者为准。
本发明提供了一种菌株，所述菌株为鼠李糖乳杆11-7(Lactobacillus rhamnosus 11-7)，其保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 2021185。
所述菌株是从新生儿母乳中进行分离纯化得到。
本发明提供了上述所述的菌株在发酵领域中的应用，优选在发酵生产发酵溶胞物领域中的应用。
本发明使用上述所述的菌株进行发酵，鼠李糖乳杆菌作为一种安全菌种广泛作为优良的益生菌应用于食品、医药、化妆品领域中，而母乳作为新生儿的第一道防护屏障其中益生菌的活性不言而喻，所以将定植在母乳中的益生菌分离筛选后再通过本工艺来制备发酵溶胞物可以很好的将活性物质全部保留在最终产物中，所以从来源上、菌种类型上、制备工艺上其活性功效都要远远优于传统益生菌。
本发明提供了一种发酵溶胞物，其包括蛋白质、多糖和氨基酸，以在发酵溶胞物中所占的质量百分比计，所述蛋白质为0.2-1％，优选为0.6-0.9％，所述多糖为0.3-0.8％，优选为0.4-0.6％；所述氨基酸为0.2-1％，优选为0.4-0.5％。
例如，以在发酵溶胞物中所占的质量百分比计，所述蛋白质为0.2％、0.3％、0.4％、0.5％、0.6％、0.7％、0.8％、0.9％、1.0％等；
所述多糖为0.3％、0.4％、0.5％、0.6％、0.7％、0.8％等；
所述氨基酸为0.2％、0.3％、0.4％、0.5％、0.6％、0.7％、0.8％、0.9％、1.0％等。
在一个实施方案中，所述发酵溶胞物通过使用保藏编号为CCTCC NO:M 2021185的鼠李糖乳杆菌11-7菌株发酵、酶解得到，优选的，所述发酵溶胞物通过包含下述步骤的方法制备得到：
将处于对数期的上述所述菌株接种到发酵培养基上进行发酵，发酵至无残糖得到第一发酵液，对第一发酵液进行离心后去上清得到发酵菌株；
将发酵菌株进行酶解得到。
所述发酵培养基含有本领域技术人员公知的碳源、氮源和无机盐，所述碳源例如可以为葡萄糖、乳糖、蔗糖、麦芽糖或甘露醇，优选为葡萄糖，所述碳源的添加量可以为1-5％(w/v)，优选为1-4％(w/v)；
所述氮源例如可以为牛肉膏、蛋白胨、酵母粉、豆饼粉等，优选为蛋白胨，所述氮源的添加量可以为0.5-2wt％，优选为1-2wt％；
所述无机盐例如可以为硫酸镁、磷酸氢二钾、氯化钠、氯化锰等，优选为硫酸镁和磷酸氢二钾，硫酸镁的添加量可以为0.1-1wt％，优选为0.5-0.8wt％，磷酸氢二钾的添加量可以为0.1-0.5wt％，优选为0.1-0.2wt％。
所述接种量为0.1-0.5％。
在一个实施方案中，所述酶解所使用的酶为溶菌酶、中性蛋白酶和蜗牛酶，优选的所述溶菌酶的比酶活力为2000-20000IU/mg，所述中性蛋白酶的比酶活力为50000-100000IU/g。
所述比酶活力指的每毫克产品所具有的酶活。
所述溶菌酶又称胞壁质酶(muramidase)或N-乙酰胞壁质聚糖水解酶(N-acetylmuramide glycanohydrlase)，是一种能水解细菌中黏多糖的碱性酶。溶菌酶主要通过破坏细胞壁中的N-乙酰胞壁酸和N-乙酰氨基葡萄糖之间的β-1,4糖苷键，使细胞壁不溶性黏多糖分解成可溶性糖肽，导致细胞壁破裂内容物逸出而使细菌溶解。
所述中性蛋白酶是由枯草芽孢杆菌经发酵提取而得的，属于一种内切酶，可用于各种蛋白质水解处理。在一定温度、pH值下，能将大分子蛋白质水解为氨基酸等产物。可广泛应用于动植物、微生物蛋白产品的酶解，精制工艺过程，本品可作用于细胞壁可溶性多肽，复合溶菌酶使用可使其彻底降解。
所述蜗牛酶是从蜗牛的嗦囊和消化道中制备的混合酶，它含有纤维素酶，果胶酶，淀粉酶，蛋白酶等20多种酶。蜗牛酶是很有价值的一种酶。它可以用于酵母细胞壁的破碎，因此广泛用于细胞生物学和基因工程学的研究；它可做饲料添加剂，从而提高饲料的消化率；也可用作果汁澄清，橘子脱囊衣，果酱制作等。
例如，所述溶菌酶的比酶活力为2000IU/mg、3000IU/mg、4000IU/mg、5000IU/mg、6000IU/mg、7000IU/mg、8000IU/mg、9000IU/mg、10000IU/mg、11000IU/mg、12000IU/mg、13000IU/mg、14000IU/mg、15000IU/mg、16000IU/mg、17000IU/mg、18000IU/mg、19000IU/mg、20000IU/mg等；
所述中性蛋白酶的比酶活力为5000IU/mg、6000IU/mg、7000IU/mg、8000IU/mg、9000IU/mg、10000IU/mg等。
对于蜗牛酶，其破壁率在95％，所述破壁率指的是pH5.8-7.2等渗的山 梨醇溶液中，每克细胞经10～20mg酶在37℃保湿1小时，即可溶解细胞壁。在一个实施方案中，酶解时间为1-2h，酶解温度为35-40℃。
例如，酶解时间为1h、1.5h、2h等，酶解温度为35℃、36℃、37℃、38℃、39℃、40℃等。
在一个实施方案中，针对1g的发酵菌株，添加的所述溶菌酶为0.01-0.1mg，添加的所述蜗牛酶为1-10mg，添加的所述中性蛋白酶为0.01-0.1mg。
例如，针对1g的发酵菌株，添加的所述溶菌酶可以为0.01mg、0.02mg、0.03mg、0.04mg、0.05mg、0.06mg、0.07mg、0.08mg、0.09mg、0.1mg等；
添加的所述蜗牛酶可以为1mg、2mg、3mg、4mg、5mg、6mg、7mg、8mg、9mg、10mg等；
添加的所述中性蛋白酶可以为0.01mg、0.02mg、0.03mg、0.04mg、0.05mg、0.06mg、0.07mg、0.08mg、0.09mg、0.1mg等；
本发明使用上述所述的酶来酶解发酵菌株，其采用温和的破碎方式将发酵菌株灭活破碎，破碎后可以形成多种小分子易于吸收的活性成分如氨基酸、多糖、多肽等，这样从人体皮肤无法吸收的细胞形态转化为易于吸收的小分子状态。
对于上述所述酶的添加方式，本发明不作任何限制，例如，可以将三种酶混合之后添加，也可以逐一添加，对于逐一添加的顺序，本发明不作任何限定，其可以按照本领域技术公知的方式进行选择。
在一个实施方案中，第一发酵液离心后得到的上清液用滤膜过滤后得到第二发酵液。
在一个实施方案中，在酶解之后，在得到发酵溶胞物之前，还包括下述步骤:
酶解结束后离心得到菌体沉淀物，然后使用氯化钠溶液悬浮，离心得到菌体碎片物，接着使用第二发酵液悬浮得到所述发酵溶胞物。
本发明使用氯化钠进行悬浮，造成高渗环境在用酶处理后细胞壁降解，剩余的胞内物质会因为高渗而破裂，形成菌体岁碎片，氯化钠主要是造成一个高渗环境。
本发明采用第一发酵液离心后的上清液即第二发酵液进行悬浮，由于第二发酵液中含有发酵过程中的胞外活性物质，这样在进行悬浮时所述发酵溶 胞物包含了胞外活性物质。
本发明提供了一种制备发酵溶胞物的方法，其包括下述步骤：
使用保藏编号为CCTCC NO:M 2021185的鼠李糖乳杆菌11-7来制备发酵溶胞物。
在一个实施方案中，其中，将所述菌株进行发酵、酶解得到所述发酵溶胞物。
在一个实施方案中，所述方法包括下述步骤：
将处于对数期的上述所述菌株接种到发酵培养基上进行发酵，发酵至无残糖得到第一发酵液，对第一发酵液进行离心后去上清得到发酵菌株；
优选的，接种量为0.1-0.5％。
优选的，在温度为35-38℃下进行发酵，发酵时间为40-70h，在该时间范围内，糖耗尽后得到第一发酵液。
对于对数期的菌株的制备，本发明不作限制，其可以使用本领域的常用方法进行制备，例如，将菌株接种到MRS液体培养基中，在37℃下培养15-20h即得到处于对数期的菌株。
在一个实施方案中，所述酶解所使用的酶为溶菌酶、中性蛋白酶和蜗牛酶，优选的所述溶菌酶的比酶活力为2000-20000IU/mg，所述中性蛋白酶的比酶活力为50000-100000IU/g；优选的，酶解时间为1-2h，酶解温度为35-40℃；
优选的，针对1g的发酵菌株，添加的所述溶菌酶为0.01-0.1mg，添加的所述蜗牛酶为1-10mg，添加的所述中性蛋白酶为0.01-0.1mg。
在一个实施方案中，将第一发酵液离心后得到的上清液用滤膜过滤得到第二发酵液。
在本发明中，通过将菌体和发酵上清液分别处理，既可减少酶解液的添加量，降低皮肤过敏的风险，又使得胞外存在于上清液的活性成分也得以保留。整个工艺过程不仅保持了全部发酵中的功效物质种类及活性，还将其转化为易于吸收的形式。
所述滤膜为0.22μm的滤膜。
在一个实施方案中，在酶解之后，在得到所述发酵溶胞物之前，还包括下述步骤:
酶解结束后离心得到菌体沉淀物，然后使用氯化钠溶液悬浮，离心得到 菌体碎片物，接着使用第二发酵液悬浮得到所述发酵溶胞物。
所述菌体沉淀物指的是酶解结束之后进行离心，离心后去除上清所得到的沉淀即为菌体沉淀物。
所述菌体碎片物指的是将菌体沉淀物悬浮于氯化钠溶液中，然后进行离心去除上清后所得到的沉淀即为菌体碎片物。
所述氯化钠溶液为已过虑除菌的氯化钠溶液，所述氯化钠的浓度为3-5wt％。
在一个实施方案中，所述菌体碎片物在所述第二发酵液的质量体积百分比为1-10％，优选为5-8％。
例如，所述菌体碎片物在所述第二发酵液的质量体积百分比可以为1％、2％、3％、4％、5％、6％、7％、8％、9％、10％等。
在一个实施方案中，所述发酵溶胞物包含蛋白质、多糖和氨基酸，以在发酵溶胞物中所占的质量百分比计，所述蛋白质为0.2-1％，优选为0.6-0.9％，所述多糖为0.3-0.8％，优选为0.4-0.6％；所述氨基酸为0.2-1％，优选为0.4-0.5％。
本发明所述的发酵溶胞物能够抑制致病菌的繁殖生长，对部分益生菌促进作用，对白色念珠菌致病形态菌丝状有强烈抑制作用，同时发酵过程中不但自身能够产生抗菌肽，还在基因水平，蛋白水平上调了肌肤细胞抗菌肽的表达量，提高皮肤免疫力，作为化妆品原料能够显著调节皮肤微生态环境。
本发明使用酶制剂酶解和高渗溶液相结合的温和菌体破碎方式将菌体灭活破碎，菌体破碎后可以形成多种小分子易于吸收的活性成分如氨基酸、多糖、多肽等，这样从人体皮肤无法吸收的细胞形态转化为易于吸收的小分子状态。此外，通过将菌体和发酵上清液分别处理，既可减少酶解液的添加量，降低皮肤过敏的风险，又使得胞外存在于上清液的活性成分也得以保留。整个工艺过程不仅保持了全部发酵中的功效物质种类及活性，还将其转化为易于吸收的形式。周期短、过程可控、易放大、产品稳定，且活性物质含量高、功效好、易吸收、安全健康符合化妆品原料的特点。
本发明提供了上述所述的发酵溶胞物或者上述所述的制备方法制备得到的发酵溶胞物在化妆品领域中的应用。
优选的，所述发酵溶胞物用于调节皮肤微生态。
实施例
本发明对试验中所用到的材料以及试验方法进行一般性和/或具体的描述，在下面的实施例中，如果无其他特别的说明，％表示wt％，即重量百分数。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。
实施例1菌株的获得
收集新生儿母乳，将母乳稀释10倍混匀无灭菌的纯化水中，取100μL涂布于固体MRS平板中，37℃培养箱培养48h，生长的单菌落再次进行稀释涂布固体MRS平板分离筛选，37℃培养箱培养48h，所述单菌落形态及生理生化实验如下。
1.1形态学观察
该菌种涂布于载玻片中，革兰氏染色进行染色，观察菌体细胞形态。该菌落涂布器涂布于MRS固体平板上37℃过夜培养24h，观察平板上菌体单菌落状态。
1.2生理生化实验
经分离纯化后得到菌株进行一下生理生化实验，发酵产气实验、接触酶反应、淀粉水解反应、酪素水解反应、苯丙氨酸脱氨酶反应、柠檬酸盐的反应、吲哚反应、明胶液化反应、硝酸盐还原反应、石蕊牛乳反应。
表1.显微镜下观察菌落形态
革兰氏染色 菌体形态 芽孢形态 鞭毛
阳性 短杆转 无 无
表2.平板上观察菌落形态
菌落形状 边缘形态 表面 颜色
规则圆形 齐整 湿润、光滑、有凸起 白色
表3.生理生化实验
生理生化项目 实验结果
发酵产气反应 -
接触酶反应 -
淀粉水解反应 -
酪素水解反应 -
苯丙氨酸脱氨酶反应 -
柠檬酸盐的反应 -
吲哚反应 -
明胶液化反应 -
硝酸盐还原反应 -
石蕊牛乳反应 +
从上述鉴定结果看可以初步判定为属于乳杆菌属。
1.3菌种鉴定
1主要材料与试剂如表4所示：
表4
试剂名称 供应商 编号
Ezup柱式细菌基因组DNA抽提试剂盒 生工生物 B518259
SanPrep柱式DNA胶回收试剂盒 生工生物 B518131
Taq Plus DNA聚合酶 BBI B600090
琼脂糖B BBI A600014
4S Red Plus核酸染色剂(10,000X水溶液) BBI A606695
GeneRuler DNA Ladder Mix Thermo Scientific B300721
2主要仪器如表5所示：
表5
名称 厂家 型号
洁净工作台 江苏苏洁净化设备厂 SW-CJ-1D
PCR反应扩增仪 BBI PCR-96
高速微量离心机，100～14800rpm 生工生物 G508009
电泳仪 北京六一 DYY-6C
凝胶成像系统 上海复日科技有限公司 FR980
微量分光光度计 Merinton Instrument,Inc SMA4000
测序仪 ABI,Foster,CA,USA 3730XL
3实验步骤
3.1引物序列分别如SEQ ID NO:1和SEQ ID NO:2所示，其中，核苷酸序列如下：
27F:AGAGTTTGATCMTGGCTCAG(SEQ ID NO:1)
1492R:GGTTACCTTGTTACGACTT(SEQ ID NO:2)
3.3 DNA抽提
操作步骤详见Ezup柱式细菌基因组DNA抽提试剂盒(B518259)。
3.4 PCR扩增
3.4.1 PCR反应体系如表6所示：
表6
反应成分 体积(μl)
10 X PCR Buffer  
dNTP(each 10mM)  
Taq Plus DNA Polymerase(5U/μl)  
50mM MgSO4 共12.5
引物F(10μM) 1
引物R(10μM) 1
Template(DNA) 1
ddH 2O 9.5
Total 25
3.4.2 PCR反应条件如表7所示
表7
Figure PCTCN2022082787-appb-000001
Figure PCTCN2022082787-appb-000002
3.4.3 PCR电泳
1.5％琼脂糖凝胶，1x TAE，150V，100mA，20min电泳观察，结果如图1所示。
该菌株由上海生工生物工程(上海)股份有限公司对其基因组进行测序鉴定，测序结果16S rDNA如SEQ ID NO:3所示，并经Blast进行比对，证明该菌株为鼠李糖乳杆菌11-7。其中，核苷酸序列如下：
Figure PCTCN2022082787-appb-000003
实施例2-1
从试管挑取鼠李糖乳杆菌菌种接种于500mL含MRS的无菌种子培养液中，静置，37℃下培养18h后，至对数生长期，按照0.5％接种量再次接种到5L发酵培养基中，发酵培养基中含有葡萄糖2wt％、蛋白胨1.5wt％、磷酸氢二钾0.1wt％和硫酸镁0.5wt％，37℃下培养50h无残糖，发酵结束，得第一发酵液，记为发酵液1。
实施例2-2
从试管挑取鼠李糖乳杆菌菌种接种于100mL含MRS的无菌种子培养液中，静置，37℃下培养20h后，至对数生长期，按照0.3％接种量再次接种到3L发酵培养基中，发酵培养基中含有葡萄糖4wt％、蛋白胨2wt％、磷酸氢二钾0.2wt％和硫酸镁0.8wt％，37℃下培养65h，无残糖后发酵结束，得第一发酵液，记为发酵液2。
实施例2-3
从试管挑取鼠李糖乳杆菌菌种接种于100mL含MRS的无菌种子培养液中，静置，37℃下培养15h后，至对数生长期，按照0.5％接种量再次接种到3L发酵培养基中，发酵培养基中含有葡萄糖4wt％、蛋白胨2wt％、磷酸氢二钾0.2wt％和硫酸镁0.8wt％，37℃下培养65h，无残糖后发酵结束，得第一发酵液，记为发酵液3。
实施例2-4
从试管挑取鼠李糖乳杆菌菌种接种于100mL含MRS的无菌种子培养液中，静置，37℃下培养20h后，至对数生长期，按照0.5％接种量再次接种到3L发酵培养基中，发酵培养基中含有葡萄糖2wt％、蛋白胨1wt％、磷酸氢二钾0.1wt％和硫酸镁0.5wt％，37℃下培养65h，无残糖后发酵结束，得第一发酵液，记为发酵液4。
实施例2-5
从试管挑取鼠李糖乳杆菌菌种接种于100mL含MRS的无菌种子培养液中，静置，37℃下培养20h后，至对数生长期，按照0.2％接种量再次接种到3L发酵培养基中，发酵培养基中含有葡萄糖1wt％、蛋白胨0.5wt％、磷酸氢二钾0.1wt％和硫酸镁0.1wt％，37℃下培养65h，无残糖后发酵结束，得第一发酵液，记为发酵液5。
实施例2-6
从试管挑取鼠李糖乳杆菌菌种接种于100mL含MRS的无菌种子培养液中，静置，37℃下培养20h后，至对数生长期，按照0.5％接种量再次接种到3L发酵培养基中，发酵培养基中含有葡萄糖5wt％、蛋白胨0.8wt％、磷 酸氢二钾0.5wt％和硫酸镁1wt％，37℃下培养65h，无残糖后发酵结束，得第一发酵液，记为发酵液6。
实施例2-7
从试管挑取鼠李糖乳杆菌菌种接种于100mL含MRS的无菌种子培养液中，静置，37℃下培养20h后，至对数生长期，按照0.5％接种量再次接种到3L发酵培养基中，发酵培养基中含有葡萄糖5wt％、蛋白胨0.8wt％、磷酸氢二钾0.5wt％和硫酸镁1wt％，37℃下培养65h，无残糖后发酵结束，得第一发酵液，记为发酵液7。
实施例3-1
(1)取发酵液7(200mL)，以8000rpm离心收集发酵后的菌株，得12.2g发酵菌株，其中上清液经0.22微米滤膜过滤得到第二发酵液；
(2)将发酵菌株(12.2g)悬浮于1.2L纯化水中，添加0.122mg溶菌酶(比酶活力为5000IU/mg)，0.122mg中性蛋白酶(比酶活力为60000IU/mg)，73.2mg蜗牛酶，充分混匀，37℃酶解1h后离心弃上清得到菌体沉淀物；将所述菌体沉淀物使用100ml的4％过滤除菌的氯化钠溶液进行悬浮，悬浮后静置1h后，离心弃上清得到菌体碎片物，将菌体碎片物(9.4g)用160mL的第二发酵液进行悬浮得到发酵微胞物(S1)，将S1进行组分分析，其中，
蛋白质的测定方法如下：其采用考马斯亮蓝法：
试剂酸性染色液取考马斯亮蓝G250 0.lg，加乙醇50ml溶解后，加磷酸100ml，加水稀释至1000ml,混匀。滤过，取滤液，即得。本试剂应置棕色瓶内，如有沉淀产生，使用前需经滤过。
对照品溶液的制备除另有规定外，取血清白蛋白(牛)对照品或蛋白质含量测定国家标准品，加水溶解并制成每lml中含lmg的溶液。
供试品溶液的制备照各品种项下规定的方法制备
(蛋白质浓度应与对照品溶液基本一致)。
测定法精密量取对照品溶液0.0ml、0.01ml、0.02ml,0.04ml、0.06ml、0.08ml、0.1ml(对照品溶液取用量可在本法测定范围内进行适当调整)，分别置具塞试管中，各加水至0.1ml，再分别加人酸性染色液5.0ml，立即混匀，照紫外-可见分光光度法(通则0401)，立即在595nm的波长处测定吸光 度；同时以0号管作为空白。
以对照品溶液浓度与其相对应的吸光度计算线性回归方程。另精密量取供试品溶液适量，同法测定，从线性回归方程计算供试品溶液中的蛋白质浓度，并乘以稀释倍数，即得，测得蛋白质的含量为0.9％；
氨基酸含量的测定方法采用氨基酸自动分析仪(LA8080)得到，测定氨基酸的含量为0.5％；
多糖的测定方法：采用苯酚硫酸法检测多糖含量。
(1)仪器：可见-紫外分光光度计、分析天平(精度0.0001g)、漩涡混合器(2)试剂：
2.1标准溶液：精确称取干燥恒重的葡萄糖(分析纯)100mg至容量瓶中，加水定容至250mL，摇匀后精确吸取10mL该溶液，加水定容至100mL。
2.2 80％苯酚液的配制：准确移取重蒸酚80mL，加蒸馏水定容至100mL，即得80％苯酚液，棕色瓶中避光保存。
2.3 6％苯酚液的配制：将80％苯酚液加水稀释至6％，临用现配。
2.4浓硫酸(优级纯)
(3)检测：
3.1制作标准曲线：分别吸取葡萄糖标准液0.0，0.4，0.8，1.2，1.6，2.0mL于具塞试管中，各加水补至2.0mL。分别加入6％苯酚溶液1mL，混合均匀后快速加入浓硫酸5.0mL(浓硫酸时悬空加入，不能贴壁)，即刻摇匀，室温反应20min后于490nm测吸光度，以0管做空白对照，纵坐标为多糖浓度，横坐标为吸光度，得标准曲线。
3.2样品制备：分别称取实施例5～8中的样品S1-S4各0.2ml待置于50mL容量瓶中，加适量水，完全溶解后加水定容至刻度作为贮备液，摇匀。用前量取贮备液5mL，置50mL容量瓶中，加水至刻度，再以相同方法稀释10倍。取2mL于具塞试管中，按上述“加入6％重蒸酚1.0mL”起，同法操作，由标准曲线得待测样品多糖浓度，根据稀释倍数计算多糖含量，得到多糖的含量为0.6％。
实施例3-2
(1)取发酵液7(200mL)，以8000rpm离心收集发酵后的菌株，得15.8g发酵菌株，其中上清液经0.22微米滤膜过滤得到第二发酵液；
(2)将发酵菌株(15.8g)悬浮于316mL纯化水中，添加0.79mg溶菌酶(比酶活力为15000IU/mg)，0.79mg中性蛋白酶(比酶活力为80000IU/mg)，79mg蜗牛酶，充分混匀，38℃酶解1h后离心弃上清得到菌体沉淀物；将所述菌体沉淀物使用120ml的4％过滤除菌的氯化钠溶液进行悬浮，悬浮后静置1h后，离心弃上清得到菌体碎片物，将菌体碎片物(13.9g)用200mL的第二发酵液进行悬浮得到发酵微胞物(S2)，按照与实施例3-1所述的方进行分析，其中，按照与实施例3-1所述的方法进行分析，测得蛋白质为0.6％，氨基酸为0.4％，多糖为0.4％。
实施例3-3
(1)取发酵液7(200mL)，以8000rpm离心收集发酵后的菌株，得13.7g发酵菌株，其中上清液经0.22微米滤膜过滤得到第二发酵液；
(2)将所述发酵菌株(13.7g)悬浮于137mL纯化水中，添加1.37mg溶菌酶(比酶活力为14000IU/mg)，1.37mg中性蛋白酶(比酶活力为80000IU/mg)，137mg蜗牛酶，充分混匀，38℃酶解1h后离心弃上清得到菌体沉淀物；将所述菌体沉淀物使用150ml的3％过滤除菌的氯化钠溶液进行悬浮，悬浮后静置1h后，离心弃上清得到菌体碎片物，将菌体碎片物(11.5g)用150mL的第二发酵液进行悬浮得到发酵微胞物(S3)，按照与实施例3-1所述的方进行分析，其中，蛋白质为0.7％，氨基酸为0.5％，多糖为0.6％。
实施例3-4
(1)取发酵液7(200mL)，以8000rpm离心收集发酵后的菌株，得16.1g发酵菌株，其中上清液经0.22微米滤膜过滤得到第二发酵液；
(2)将所述发酵菌株(16.1g)悬浮于330mL纯化水中，添加0.161mg溶菌酶(比酶活力为8000IU/mg)，1.61mg中性蛋白酶(比酶活力为90000IU/mg)和80.5mg蜗牛酶，充分混匀，38℃酶解1h后离心弃上清得到菌体沉淀物；将所述菌体沉淀物使用200ml的4％过滤除菌的氯化钠溶液进行悬浮，悬浮后静置1h后，离心弃上清得到菌体碎片物，将菌体碎片物(13.0g)用130mL的第二发酵液进行悬浮得到发酵微胞物(S4)，按照与实施例3-1所述的方进行分析，其中，蛋白质为0.3％，氨基酸为0.3％，多糖为0.8％。
实施例3-5
(1)取发酵液7(200mL)，以8000rpm离心收集发酵后的菌株，得11.4g发酵菌株，其中上清液经0.22微米滤膜过滤得到第二发酵液；
(2)将所述发酵菌株(11.4g)悬浮于380mL纯化水中，添加0.114mg溶菌酶(比酶活力为5000IU/mg)，0.114mg中性蛋白酶(比酶活力为60000IU/mg)，114mg蜗牛酶，充分混匀，37℃酶解1h后离心弃上清得到菌体沉淀物；将所述菌体沉淀物使用100ml的4％过滤除菌的氯化钠溶液进行悬浮，悬浮后静置1h后，离心弃上清得到菌体碎片物，将菌体碎片物(8.5g)用150mL的第二发酵液进行悬浮得到发酵微胞物(S5)，按照与实施例3-1所述的方进行分析，其中，蛋白质为0.9％，氨基酸为0.3％，多糖为0.9％。
实施例3-6
(1)取发酵液7(200mL)，以8000rpm离心收集发酵后的菌株，得13.1g发酵菌株，其中上清液经0.22微米滤膜过滤得到第二发酵液；
(2)将所述发酵菌株(13.1g)悬浮于260mL纯化水中，添加0.655mg溶菌酶(比酶活力为5000IU/mg)，0.131mg中性蛋白酶(比酶活力为60000IU/mg)，78.6mg蜗牛酶，充分混匀，37℃酶解1h后离心弃上清得到菌体沉淀物；将所述菌体沉淀物使用100ml的4％过滤除菌的氯化钠溶液进行悬浮，悬浮后静置1h后，离心弃上清得到菌体碎片物，将菌体碎片物(10.2g)用170mL的第二发酵液进行悬浮得到发酵微胞物(S6)，按照与实施例3-1所述的方进行分析，其中，蛋白质为0.3％，氨基酸为0.8％，多糖为0.7％。
实施例3-7
(1)取发酵液7(100mL)，以8000rpm离心收集发酵后的菌株，得5.4g发酵菌株，其中上清液经0.22微米滤膜过滤得到第二发酵液；
(2)将所述发酵菌株(5.4g)悬浮于108mL纯化水中，添加0.54mg溶菌酶(比酶活力为5000IU/mg)，0.054mg中性蛋白酶(比酶活力为60000IU/mg)，32.4mg蜗牛酶，充分混匀，37℃酶解1h后离心弃上清得到菌体沉淀物；将所述菌体沉淀物使用100ml的4％过滤除菌的氯化钠溶液进行悬浮，悬浮后静置1h后，离心弃上清得到菌体碎片物，将菌体碎片物(3.6g)用60mL的第二发酵液进行悬浮得到发酵微胞物(S7)，按照与实施例3-1 所述的方进行分析，其中，蛋白质为0.2％，氨基酸为0.3％，多糖为0.3％。
实施例3-8
(1)取发酵液7(300mL)，以8000rpm离心收集发酵后的菌株，得21.0g发酵菌株，其中上清液经0.22微米滤膜过滤得到第二发酵液；
(2)将所述发酵菌株(21.0g)悬浮于420mL纯化水中，添加0.21mg溶菌酶(比酶活力为5000IU/mg)，0.21mg中性蛋白酶(比酶活力为60000IU/mg)，105mg蜗牛酶，充分混匀，37℃酶解1h后离心弃上清得到菌体沉淀物；将所述菌体沉淀物使用100ml的4％过滤除菌的氯化钠溶液进行悬浮，悬浮后静置1h后，离心弃上清得到菌体碎片物，将菌体碎片物(15.5g)用250mL的第二发酵液进行悬浮得到发酵微胞物(S8)，按照与实施例3-1所述的方进行分析，其中，蛋白质为0.5％，氨基酸为0.2％，多糖为0.7％。
实施例3-9
(1)取发酵液7(500mL)，以8000rpm离心收集发酵后的菌株，得32.4g发酵菌株，其中上清液经0.22微米滤膜过滤得到第二发酵液；
(2)将所述发酵菌株(32.4g)悬浮于650mL纯化水中，添加3.24mg溶菌酶(比酶活力为10000IU/mg)，1.62mg中性蛋白酶(比酶活力为90000IU/mg)，324mg蜗牛酶，充分混匀，38℃酶解1h后离心弃上清得到菌体沉淀物；将所述菌体沉淀物使用600ml的5％过滤除菌的氯化钠溶液进行悬浮，悬浮后静置1h后，离心弃上清得到菌体碎片物，将菌体碎片物(19.5g)用1950mL的第二发酵液进行悬浮得到发酵微胞物(S9)，按照与实施例3-1所述的方进行分析，其中，蛋白质为0.4％，氨基酸为0.6％，多糖为0.3％。
表8实施例的工艺条件
Figure PCTCN2022082787-appb-000004
对比例1
取发酵液7(200mL)接置于60℃水浴锅中加热1h，得乳酸菌溶胞物对比样品，记为C1，按照与实施例3-1所述的方进行分析，其中，蛋白质为0.1％，氨基酸为0.15％，多糖为0.2％。
对比例2
选取市售二裂酵母乳酸菌溶胞物的产品作为对比样品，记为C2(市售)，其中，蛋白质为0.1％，氨基酸为0.07％，多糖为0.2％。
对比例3
取发酵液7(200ml)，以8000rpm离心收集菌体，上清液经0.22微米滤膜过滤后待用。菌体悬浮于260mL纯化水中，添加2.1mg溶菌酶，2.1mg中性蛋白酶，63mg蜗牛酶，充分混匀，发酵液7菌体湿重为10.5g，用100ml酶解液进行悬浮，37℃酶解1.5h离心弃上清。发酵液酶解后的菌体碎片共计9.0g，再以4％过滤除菌的氯化钠溶液进行悬浮，悬浮体积100ml，静置1h，离心取上清，用130mL发酵液1除菌后的上清液进行悬浮，得样品记为C3，按照与实施例3-1所述的方进行分析，其中，蛋白质为0.2％，氨基酸为0.1％，多糖为0.1％。
对比例4
对比例4和实施例3-7的工艺条件相同，其区别在于，对比例4使用的是市售鼠李糖乳杆菌发酵制备得到发酵溶胞物(C4)，按照与实施例2-1所述的方进行分析，其中，蛋白质为0.1％，氨基酸为0.3％，多糖为0.2％。
试验例1
配制MRS固体平板，取实施例和对比例分别用涂布器涂布原液100μL，稀释10倍涂布100μL，稀释100倍涂布100μL，置于37℃培养箱中静置培养48h，计数单菌落数目，其结果如表9所示。
表9单菌落数目
  原液菌落数量(个) 稀释10倍菌落数量(个) 稀释100倍菌落数量(个)
实施例3-1 0 0 0
实施例3-2 0 0 0
实施例3-3 0 0 0
实施例3-4 1 0 0
实施例3-5 1 0 0
实施例3-6 0 1 0
实施例3-7 1 0 0
实施例3-8 1 0 0
实施例3-9 0 1 0
对比例1 56 16 0
对比例2 34 4 0
对比例3 21 3 0
对比例4 16 3 0
从实验结果看出，采用对比例所述的方式无法将菌体破碎灭活，菌体生长不受影响，而实施例中效果较为理想，说明采用特定的工艺以及使用特定的菌株，其效果较好。
实验例2
以实施例和对比例的样品，进行氨基酸含量测定，采用氨基酸自动分析仪进行，结果如表10所示。
表10氨基酸结果
Figure PCTCN2022082787-appb-000005
实验例3
取待测实施例和对比例样品分别用纯化水稀释10倍，采用牛津杯抑菌实验法，指示菌选取金黄色葡萄球菌、铜绿假单胞菌、大肠杆菌三种菌作为病原菌的指示菌，比较样品对病原菌的抑制作用，其结果如表11所示。
表11不同样品的抑菌圈直径
Figure PCTCN2022082787-appb-000006
Figure PCTCN2022082787-appb-000007
从表11可以看出，实施例所制备得到的发酵溶胞物能够抑制致病菌如金黄色葡萄球菌、铜绿假单胞菌、大肠杆菌等的生长。
实验例4
白色念珠菌菌丝白念的致病力与形态转换有关。白色念珠菌能以三种形态生长，即酵母形态、假菌丝形态及菌丝形态。假菌丝状态可产生芽管特异性抗原,此抗原能明显增强其點附性，并能阻止中性粒细胞的吞唾杀伤作用。白念被吞唾后，在吞唆细胞内仍能生成芽管并长出菌丝，从而穿透、破坏宿主细胞膜。实验表明，以菌丝形态生长的白色念珠菌具有更强的致病性。正常寄居的白念呈酵母相，只存在于角质细胞表层,并不穿入其中。白念感染时，首先形成芽管，并借助于胞壁最外层的粘附素等结构粘附于宿主细胞表面，之后芽管逐渐向芽生菌丝或菌丝相转变,并穿入细胞内生长，当呈现菌丝状时说明具有强烈的致病性。
用GE培养基(市售)来诱导白色念珠菌购自ATCC10231菌种保藏库菌丝生长，分别向白色念珠菌培养基中添加5％浓度实施例3-1和对比例1～4样品培养6h，比较抑制白色念珠菌菌丝的萌发，其生长结果分别如图2A至图2F所示。
从图中可以看出，S1样品可以很好的抑制白色念珠菌致病菌菌丝的萌发。
实验例5
乳酸菌发酵溶胞物具有免疫调节作用和趋化作用，当皮肤受到外界的刺激时，皮肤细胞相关抗菌肽如防御素等因子会显著性上调表达，而随着年龄的增加，皮肤趋向衰老后，皮肤中的抗菌肽表达量显著性降低，将发酵样品实施例和对比例以0.4％的添加量作用于角质细胞购自ATCC10231细胞保藏 库，检测胶质细胞皮肤免疫相关抗菌肽基因HBD2、HBD3的表达量，进而评价三种样品对皮肤免疫相关基因的调节作用。
对作用后的角质形成细胞进行RNAiso Plus处理后收样，依据试剂盒说明书，进行RNA提取、反转录及荧光定量PCR操作，采用相对定量法分析结果，检测步骤如下。
1.倒出培养液，1*PBS清洗一次。
2. 10cm 2生长的培养细胞中加入1-2mL的RNAiso Plus,晃动，确保使其裂解液均匀分布于细胞表面。
3.将内涵细胞的裂解液移至离心管中，用移液枪反复吹吸直至裂解液中无明显沉淀。
4.室温静置5min，然后从蛋白中分离RNA。
5.向上述溶液中加入氯仿，盖紧离心盖，混合至溶液为乳白色，静置5min。
6. 12000g 4℃离心15min，从离心机中小心取出离心管，此时无色上清液含有RNA。
7.吸取上清至新离心管中。
8.吸上清夜中加入0.5-1倍的RNAiso Plus体积的异丙醇，上下颠倒离心管充分混匀后，室温下静置10min。
9. 12000g 4℃离心10min，小心弃去上清，残留少量异丙醇，加入与RNAiso Plus等量的75％乙醇，轻轻上下颠倒洗涤离心管管壁，15000g 4℃离心5min后小心弃去上清，切勿触及沉淀。
10.打开离心管，室温干燥沉淀几分钟，沉淀干燥后，加入适量的RNase-free水溶解沉淀即可。
11.提成RNA后按照反转录试剂盒所示反应体系进行反转率合成cDNA模板，再按照real-timePCR试剂盒(上海吉玛制药技术有限公司)所示反应体系进行荧光定量PCR，其结果如下。
检测结果变化趋势如表12所示。
表12 HBD2和HBD3的表达量
基因名称 HBD2 HBD3
BC 1.025 1.025
实施例3-1 2.241 2.241
实施例3-2 2.161 2.443
实施例3-3 2.282 2.519
实施例3-4 1.875 1.507
实施例3-5 1.686 1.654
实施例3-6 1.990 1.342
实施例3-7 1.643 1.872
实施例3-8 1.701 1.545
实施例3-9 1.797 1.411
对比例1 1.201 1.132
对比例2 0.995 1.010
对比例3 1.121 1.242
对比例4 1.337 1.122
从表12可以看出，采用本发明所述的菌株进行发酵，并采用本发明所述的方法进行酶解所得到的发酵溶胞物对HBD2和HBD3的表达量较高，从而说明了本发明所述的发酵溶胞物具有免疫调节作用和趋化作用。
实验例6
为进一步验证乳酸菌发酵溶胞物的活性，在3D皮肤模型上来评价其在皮肤抗菌肽表达方面的功效效果，S1样品组是将实施例3-1所得到的乳酸菌发酵溶胞物以浓度为39μL/cm 2加入到3D皮肤模型中，阳性对照组是使用50mJ/cm 2的UVB连续照射3D皮肤模型两天，其是用于HBD2蛋白和HBD3蛋白的检测，空白对照为不做处理的3D皮肤模型，过程如下：
1.将3D皮肤模型(
Figure PCTCN2022082787-appb-000008
由广东博溪生物科技有限公司生产)转移到6孔板中(提前添加0.9mL模型培养液)，在6孔板上标注测试组编号。
2.样品组：取配置好的S1样品组工作液25μL加于模型表面；
空白对照：取25μL EpiGrowth(广东博溪生物)培养液加于模型表面；
阳性对照：取25μL EpiGrowth(广东博溪生物)培养液加于模型表面后，用50mJ/cm 2的UVB连续照射皮肤模型48h；
轻轻抖动模型，使样品均匀分布于模型表面，置于CO 2培养箱(37℃、5％CO 2)中孵育24h。
3.孵育结束后，用装有无菌PBS溶液的洗瓶清洗模型表面残留的受试物，用无菌棉签轻轻拭去模型内、外残留液体。
4.将模型环切取下：用于免疫组化检测的模型，进行固定、石蜡包埋、切片。
5.烤片脱蜡：石蜡切片置于70℃烤片机中，烤片4小时。
6.脱蜡水化：将切片置于二甲苯中浸泡10min，更换二甲苯后再浸泡10min，无水乙醇中浸泡5min，95％乙醇中浸泡5min，75％乙醇中浸泡5min。PBS缓冲液清洗3次，每次5min。
7.抗原修复：将石蜡切片放入0.01M柠檬酸钠抗原修复溶液，采用高压修 复，冷却后取出切片。PBS缓冲溶液清洗3次，5min/次。
8.阻断过氧化物酶(辣根过氧化氢酶DAB显色试剂盒(上海生工))：每张切片加1滴3％的H 2O 2，室温下孵育30min，以阻断内源性过氧化物酶的活性。PBS缓冲溶液清洗3次，5min/次。
9.血清封闭：滴加与二抗同源的血清37℃封闭60min，无需冲洗。
10.一抗孵育：滴加一抗工作液，4℃孵育过夜。PBS缓冲液清洗3次，5min/次，其中分别采用鼠源或羊源重组Anti-HBD2(Abcam)/Anti-HBD3(Abcam)为一抗抗体，用含有3％BSA(牛血清白蛋白)的PBS缓冲液进行稀释，稀释浓度为1:100～1:500，制备得到一抗工作液。
11.二抗孵育：滴加二抗工作液，室温孵育1h。PBS缓冲液清洗3次，5min/次，其中，选用羊抗鼠或兔抗羊抗体为二抗抗体，使用PBS缓冲液以1:20～1:50进行稀释，制备得到二抗工作液。
12.ABC复合物孵育：滴加ABC复合液(
Figure PCTCN2022082787-appb-000009
ABC-Peroxidase Kits)，室温孵育30min。PBS缓冲液清洗3次，5min/次。
13.DAB染色：每张切片加1滴新鲜配置的DAB液(特异性部位会染成棕色)，显微镜观察5～30s。
14.复染：苏木素复染30s。
15.脱水：切片经梯度酒精脱水(75％，95％，100％，100％)各5min，再将切片置于二甲苯中浸泡10min，更换二甲苯后再浸泡10min，干燥，中性树脂封固，晒干，观察图3A-3F的结果,其中，图3A是HBD2免疫组化检测的空白对照的示意图，图3B是HBD2免疫组化检测的阳性对照的示意图，图3C是HBD2免疫组化检测的S1样品组的示意图，图3D是HBD3免疫组化检测的空白对照的示意图，图3E是HBD3免疫组化检测的阳性对照的示意图，图3F是HBD3免疫组化检测的S1样品组的示意图。
并使用ImageJ通过灰度比较法，比较HBD2、HBD3积分光密度(IOD)值，对图进行相对定量分析，其结果分别如表13和表14所示。
表13 HBD2积分光密度(IOD)值分析结果
样品名称 相对IOD平均值
空白对照 1.00
阳性对照 1.38
S1样品组 1.39
表14 HBD3积分光密度(IOD)值分析结果
样品名称 相对IOD平均值
空白对照 1.00
阳性对照 1.22
S1样品组 1.02
从表13和表14可以看出，与空白组相比，乳酸菌发酵溶胞产物样品组的HBD2蛋白在皮肤3D模型中可以显著提高39％，高于阳性对照水平，而HBD3蛋白作用效果不明显。
综上所述，采用本发明所述的鼠李糖乳杆菌进行发酵，并进行酶解，所得到的发酵溶胞物能够强力抑制致病菌的繁殖生长，对部分益生菌促进作用，尤其对白色念珠菌致病形态菌丝状有强烈抑制作用；并且在发酵过程中不但自身能够产生抗菌肽，还在基因水平，蛋白水平上调了肌肤细胞抗菌肽的表达量，提高皮肤免疫力，作为化妆品原料能够显著调节皮肤微生态环境。
以上所述，仅是本发明的较佳实施例而已，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。

Claims (23)

  1. 一种鼠李糖乳杆菌11-7(Lactobacillus rhamnosus 11-7)，其保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 2021185。
  2. 保藏编号为CCTCC NO:M 2021185的鼠李糖乳杆菌11-7在发酵领域中的应用，优选在发酵生产发酵溶胞物领域中的应用。
  3. 一种发酵溶胞物，其包括蛋白质、多糖和氨基酸，以在发酵溶胞物中所占的质量百分比计，所述蛋白质为0.2-1％，优选为0.6-0.9％，所述多糖为0.3-0.8％，优选为0.4-0.6％；所述氨基酸为0.2-1％，优选为0.4-0.5％。
  4. 根据权利要求3所述的发酵溶胞物，其中，所述发酵溶胞物通过使用保藏编号为CCTCC NO:M 2021185的鼠李糖乳杆菌菌株进行发酵、酶解而得到，
    优选所述发酵溶胞物通过包含下述步骤的方法制备得到：
    将处于对数期的所述菌株接种到发酵培养基中进行发酵，发酵至无残糖得到第一发酵液，对第一发酵液进行离心后去上清得到发酵菌株；优选的，所述发酵培养基包含碳源、氮源和无机盐，优选的，所述碳源的添加量为1-5％(w/v)，优选为1-4％(w/v)，所述氮源的添加量为0.5-2wt％，所述无机盐的添加量为0.1-1wt％；
    将发酵菌株进行酶解从而得到发酵溶胞物。
  5. 根据权利要求4所述的发酵溶胞物，其中，所述酶解所使用的酶为溶菌酶、中性蛋白酶和蜗牛酶，优选的所述溶菌酶的比酶活力为2000-20000IU/mg，所述中性蛋白酶的比酶活力为50000-100000IU/g。
  6. 根据权利要求4或5所述的发酵溶胞物，其中，酶解时间为1-2h，酶解温度为35-40℃。
  7. 根据权利要求5-6中任一项所述的发酵溶胞物，其中，针对1g的发酵菌株，添加的所述溶菌酶为0.01-0.1mg，添加的所述蜗牛酶为1-10mg，添加的所述中性蛋白酶为0.01-0.1mg。
  8. 根据权利要求4-7中任一项所述的发酵溶胞物，其中，离心后得到的上清液用滤膜过滤得到第二发酵液。
  9. 根据权利要求8所述的发酵溶胞物，其中，在酶解之后，在得到所述 发酵溶胞物之前，还包括下述步骤:
    酶解结束后离心得到菌体沉淀物，然后使用氯化钠溶液悬浮，离心得到菌体碎片物，接着使用第二发酵液悬浮得到所述发酵溶胞物。
  10. 根据权利要求9所述的发酵溶胞物，其中，所述氯化钠的浓度为3-5wt％。
  11. 根据权利要求9或10所述的发酵溶胞物，其中，所述菌体碎片物在所述第二发酵液的质量体积百分比为1-10％，优选为5-8％。
  12. 一种制备发酵溶胞物的方法，其包括下述步骤：
    使用保藏编号为CCTCC NO:M 2021185的鼠李糖乳杆菌11-7来制备发酵溶胞物。
  13. 根据权利要求12所述的方法，其中，将所述菌株进行发酵、酶解得到所述发酵溶胞物。
  14. 根据权利要求13所述的方法，其中，所述方法包括下述步骤：
    将处于对数期的所述菌株接种到发酵培养基上进行发酵，发酵至无残糖得到第一发酵液，对第一发酵液进行离心后去上清得到发酵菌株；优选的，所述发酵培养基包含碳源、氮源和无机盐，优选的，所述碳源的添加量为1-5％(w/v)，优选为1-4％(w/v)，所述氮源的添加量为0.5-2wt％，所述无机盐的添加量为0.1-1wt％；
    将发酵菌株进行酶解得到。
  15. 根据权利要求13或14所述的方法，其中，所述酶解所使用的酶为溶菌酶、中性蛋白酶和蜗牛酶，优选的所述溶菌酶的比酶活力为2000-20000IU/mg，所述中性蛋白酶的比酶活力为50000-100000IU/g。
  16. 根据权利要求14或15所述的方法，其中，酶解时间为1-2h，酶解温度为35-40℃。
  17. 根据权利要求15或16所述的方法，其中，以发酵菌株为1g计，所述溶菌酶为0.01-0.1mg，所述蜗牛酶为1-10mg，所述中性蛋白酶为0.01-0.1mg。
  18. 根据权利要求14-17中任一项所述的方法，其中，离心后得到的上清液用滤膜过滤得到第二发酵液。
  19. 根据权利要求18所述的方法，其中，在酶解之后，在得到所述发酵溶胞物之前，还包括下述步骤:
    酶解结束后离心得到菌体沉淀物，然后使用氯化钠溶液悬浮，离心得到菌体碎片物，接着使用第二发酵液悬浮得到所述发酵溶胞物。
  20. 根据权利要求19所述的方法，其中，所述氯化钠的浓度为3-5wt％。
  21. 根据权利要求19或20所述的方法，其中，所述菌体碎片物在所述第二发酵液的质量体积百分比为1-10％，优选为5-8％。
  22. 权利要求3-11中任一项所述的发酵溶胞物或者权利要求12-21中任一项所述的制备方法制备得到的发酵溶胞物在化妆品领域中的应用。
  23. 根据权利要求22所述的应用，其中，所述发酵溶胞物用于调节皮肤微生态。
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