CN115287221A - Lactobacillus oralis and application thereof - Google Patents
Lactobacillus oralis and application thereof Download PDFInfo
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- CN115287221A CN115287221A CN202210660717.3A CN202210660717A CN115287221A CN 115287221 A CN115287221 A CN 115287221A CN 202210660717 A CN202210660717 A CN 202210660717A CN 115287221 A CN115287221 A CN 115287221A
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- lactobacillus buchneri
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- 241000186679 Lactobacillus buchneri Species 0.000 claims abstract description 12
- 238000004321 preservation Methods 0.000 claims abstract description 9
- 239000002537 cosmetic Substances 0.000 claims abstract description 6
- 239000003814 drug Substances 0.000 claims abstract description 6
- 230000001737 promoting effect Effects 0.000 claims abstract description 6
- 230000001580 bacterial effect Effects 0.000 claims description 13
- 241000186784 Lactobacillus oris Species 0.000 claims description 6
- 206010061218 Inflammation Diseases 0.000 claims description 5
- 235000013305 food Nutrition 0.000 claims description 4
- 239000006166 lysate Substances 0.000 claims description 4
- 239000002207 metabolite Substances 0.000 claims description 4
- 229930010796 primary metabolite Natural products 0.000 claims description 3
- 229930000044 secondary metabolite Natural products 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 2
- 230000004054 inflammatory process Effects 0.000 claims 2
- 201000004624 Dermatitis Diseases 0.000 abstract description 4
- 244000005700 microbiome Species 0.000 abstract description 3
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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Classifications
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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
- 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
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- 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
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- 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
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- 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
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Abstract
The invention mainly relates to the technical field of microorganisms, and particularly relates to lactobacillus buchneri and application thereof. The strain ProfMIC-203 is deposited in China center for type culture Collection with the preservation number of CCTCC NO: M20211438. The strain ProfMIC-203 has the functions of maintaining and repairing skin barrier, promoting cell repair, relieving skin inflammation, etc., and can be used in medicine, cosmetics, etc.
Description
Technical Field
The invention mainly relates to the technical field of microorganisms, and particularly relates to lactobacillus buchneri and application thereof.
Background
The skin is the largest organ in the human body, and the total weight accounts for about 16% of the weight of an individual, so that the body is kept stable on one hand, and the skin is the first defense line for resisting the invasion of external adverse factors on the other hand. Studies have shown that skin diseases are induced if the external environment causes abnormalities in the relevant genes in the skin barrier.
The skin barrier is a structural barrier formed by the epidermal keratinocytes of the stratum corneum and the lipids between the cutin. The skin barrier prevents the release of excess water from the human body and prevents harmful substances such as chemicals or microorganisms from entering our body. The corneocyte cortex, which constitutes the surface of dead keratinocytes, plays an important role in the stability of intercellular lipids. Skin barrier damage can cause skin dryness, skin aging, atopic dermatitis, eczema, psoriasis, ichthyosis, skin sensitivity such as solar rotatory optical dermatitis, irritant dermatitis, and hormone dependent dermatitis, and seborrheic diseases such as acne, rosacea, and seborrheic dermatitis.
The content of the keratinaceous structure lipid ceramide gradually increases in the process of differentiation from the basal layer to the cutin, and the stratum corneum is discharged to the intercellular space, so that a barrier for preventing water loss is formed. The water content in the keratinocytes is high, the shape of the keratinocytes gradually becomes flat as the cells are metabolically differentiated upwards, and the cell nucleus and the organelles begin to degenerate and shrink, so that dead cells without the cell nucleus and the organelles are formed in the horny layer. The stratum corneum generally contains 10 to 30% of water due to its own hydrophilicity and barrier function, and the natural moisturizing factors contained in the stratum corneum, i.e., amino acids, lactate, and saccharides, and this environment becomes a cradle for the growth of microbial colonies of the skin itself. However, the water content of the stratum corneum gradually decreases with age, and various problems of the skin are caused when the water content is less than 10%.
The microecological preparation can be used in medicine and cosmetics for balancing epidermal flora and repairing skin barrier. As reported in the literature, the external application of probiotics can treat atopic dermatitis, that is, inhibit the proliferation of other bacteria, balance the microbial flora on the skin surface and promote the repair of the skin barrier. Meanwhile, the microecological preparation plays an important role in maintaining the steady state of a host organism, activating an immune system, maintaining the immune balance of the organism and the like, researches find that the probiotics can prevent and treat various inflammatory diseases by regulating the immune function and phagocytic capacity of macrophages, and control the immune state of the whole body, including the release of regulatory cytokines.
The invention aims to develop related microbial strains and realize corresponding functions.
Disclosure of Invention
In order to realize the technical purpose, the lactobacillus lactis ProfMIC-203 separated and identified from the skin of a healthy boy has the functions of skin barrier repair and anti-inflammation, and has great application potential in the fields of food, medicines, cosmetics and the like.
The above purpose is realized by the following technical scheme:
the invention firstly provides a strain of Lactobacillus oralis (Lactobacillus oris) which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M20211438.
The Lactobacillus was derived from facial skin of healthy boys, and was identified as Lactobacillus (Lactobacillus oris) by 16S rRNA, which was assigned the number ProfMIC-203. The strain is gram-positive and rod-shaped under a microscope; the bacterial colony grows on the MRS plate, and a round bacterial colony with a smooth and opaque surface can be formed, is white and has a neat edge; the strain grows uniformly and turbulently in MRS liquid culture medium, and the strain is white precipitate after long-term storage, and the optimal growth temperature is 37 ℃.
The invention further provides application of the lactobacillus buchneri, which is used for skin barrier repair, anti-inflammation and the like.
According to the function of the lactobacillus, the invention further provides another application of the lactobacillus, which can be used for preparing food, medicine, cosmetics and other products related to skin barrier repair, anti-inflammation and the like.
Further, the lactobacillus may be a bacterial body thereof or/and a derivative thereof or/and a metabolite thereof when used for preparing related foods, pharmaceuticals, cosmetics, and the like.
Wherein, the thallus can be live thallus or/and inactivated thallus;
the derivative is lysate or/and extract of the lactobacillus buchneri, including but not limited to cell wall, intracellular protein, intracellular polysaccharide and the like;
the metabolite is understood to be the primary or secondary metabolite of the aforementioned lactobacillus lactis, such as probiotics, compounds containing immunogenic components, exopolysaccharides, etc., and may also be the supernatant of the fermentation broth.
The ProfMIC-203 has the following beneficial effects:
in-vitro cell experiments show that the lactobacillus oralis ProfMIC-203 has the effect of promoting epidermal cell repair, and the ProfMIC-203 improves the survival rate of HaCaT cells damaged by Sodium Dodecyl Sulfate (SDS) to 112.11-157.93%.
In vitro cell experiments show that the lactobacillus oralis ProfMIC-203 has the function of up-regulating the expression of cell repair related factors, namely, the filaggrin gene FLG and the lorir of the loricrin gene, and the gene expression level is up-regulated by 1.34 to 6.94 times.
In vitro cell experiments show that the lactobacillus bucillus ProfMIC-203 has the function of reducing the release of inflammatory factors and can reduce the generation amount of Nitric Oxide (NO) of mouse macrophage Raw264.7 induced by Lipopolysaccharide (LPS) by 24.70-28.11%.
In vitro cell experiments show that the lactobacillus lactis ProfMIC-203 has the function of reducing HaCaT cell inflammatory factor related gene expression induced by Staphylococcus aureus (Staphylococcus aureus), and the gene expression level is reduced by 30-66%.
Preservation information
Preservation time: 18 months 11/2021;
the name of the depository: china center for type culture Collection;
the preservation number is as follows: CCTCC NO: M20211438;
the address of the depository: wuhan, wuhan university;
and (3) classification and naming: lactobacillus kefir ProfMIC-203 (Lactobacillus oris ProfMIC-203).
Detailed Description
The present invention will be described in further detail with reference to the following examples, but it should not be construed that the scope of the present invention is limited to the examples. The techniques realized based on the above contents of the present invention all belong to the scope of the present invention, and the following embodiments are all completed by using the conventional prior art except for the specific description.
In the following examples, profMIC-203 refers to Lactobacillus oralis (Lactobacillus oris) deposited in China center for type culture Collection with the preservation number of CCTCC NO: M20211438.
The first embodiment is as follows: isolation of ProfMIC-203
Facial skin samples were taken from healthy boys at age 2. Properly processing a sample, uniformly mixing the sample in normal saline by shaking, taking a supernatant, streaking the supernatant on an MRS solid plate, culturing the MRS solid plate at a constant temperature of 37 ℃ for 24-48 hours, and selecting a white colony for repeated inoculation and screening until a uniform single colony is obtained, wherein the single colony is named as ProfMIC-203.
Gram staining microscopy: the strain ProfMIC-203 is gram-positive and rod-shaped under a microscope; growing on an MRS plate to form white round microcolonies with smooth, mellow and opaque surfaces and regular edges; the strain grows in MRS liquid culture medium in a uniform turbid way, and the strain is white and precipitated after being placed for a long time.
The second embodiment: nucleic acid identification of ProfMIC-203
1. 16s rRNA Gene sequence analysis:
picking single colony in MRS liquid culture medium, culturing overnight at 37 ℃, centrifuging and collecting thallus, and operating according to the steps of DNA extraction kit. The PCR amplification system is a 50 mu L system with the primers of bacterial universal primers 27F and 1492R and is pre-denatured at 95 ℃ for 5min;94 ℃ 15s,57 ℃ 15s,72 ℃ 40s,35 cycles; extension at 72 ℃ for 10min.
2. Results
The homology comparison (BLASTN) of the PCR product sequencing results with the published standard sequences in GenBank gave that the strain ProfMIC-203 was Lactobacillus (Lactobacillus oris).
Example three: profMIC-203 experiment for promoting SDS-induced HaCaT cell damage repair
1. Preparing ProfMIC-203 bacterial liquid:
culturing the activated lactobacillus oralis ProfMIC-203 bacterial liquid in MRS liquid culture medium in an incubator at 37 ℃ for standing culture for 16-18 h, detecting and adjusting OD (optical density) by PBS (phosphate buffer solution) 600 =2.0, after centrifugation, the supernatant was filtered through 0.22 μm filter, the cells were washed twice with PBS and resuspended to OD with PBS 600 =0.2, and the supernatant and the thallus sample are finally inactivated by autoclaving at 121 ℃ for 30 min.
2. Experiment for promoting HaCaT cell operation and repair
Inoculation with HaCaT cells (5X 10) 4 cell/well) to 96-well plates and cultured overnight until cells adhere. Preparing 50 mu g/ml SDS, adding 100 mu l of SDS into each hole, incubating for 8h, adding 5 percent (V/V, corresponding to the control group 1 being PBS with the same volume) of the strain supernatant into the experimental group 1, adding 10 percent (V/V, corresponding to the control group 2 being PBS with the same volume) of inactivated bacteria into the experimental group 2, setting 3 parallel groups, and incubating for 24h. Adding 10 μ l CCK-8 solution, incubating for 4h, and detecting light absorption at 450 nm.
Cell survival = a Experiment of /A Control ×100％。
Wherein A is Experiment of For detection of the absorbance at 450nm in the experimental group, A Control The absorbance was measured at 450nm for the control.
As a result:
ProfMIC-203 promotion of HaCaT cell repair
The inactivated supernatant and the inactivated thallus of ProfMIC-203 have a repairing effect on SDS damage of HaCaT cells.
Example four: profMIC-203 promotion HaCaT barrier repair related gene expression experiment
1. ProfMIC-203 was prepared (same as example three).
2. Inoculation of human immortalized keratinocytes HaCaT (5X 10) 5 cell/well) to 6-well plates and cultured overnight until cells adhere. 5% of strain supernatant (V/V, the corresponding control group is PBS with the same volume) is added into an experimental group 1, 10% of inactivated thallus (V/V, the corresponding control group is PBS with the same volume) is added into an experimental group 2, each group is arranged in 3 parallel, after the groups are respectively cultured for 24 hours, lysate is added, total RNA of cells is extracted, the concentration and the purity of the RNA are detected, all samples are adjusted to be 1 mu g and are reversely transcribed into cDNA, GAPDH is used as an internal reference gene, and qPCR is carried out to detect the expression of FLG and LOR genes. And calculating expression change multiples according to a formula.
The formula: f =2 -ΔΔCT
As a result:
experimental group 1 (ProfMIC-203 supernatant) Up-regulated barrier repair-related Gene expression
Experimental group 2 (ProfMIC-203 inactivated bacteria) Up-regulated barrier repair-related gene expression
The results show that the addition of ProfMIC-203 has the effect of promoting skin barrier repair.
Example five: profMIC-203 reduces NO production of Raw264.7 cells
1. ProfMIC-203 bacterial liquid preparation
ProfMIC-203 was cultured overnight in MRS medium and OD was detected 600 And adjusting the concentration of the bacterial liquid to OD with PBS buffer 600 =0.2, after centrifugation, the supernatant was filtered through 0.22 μm filter, the cells were washed twice with PBS and resuspended to OD with PBS 600 =0.2, and the supernatant and the bacterial sample are finally inactivated by autoclaving at 121 ℃ for 30 min.
2. Preparation of Raw264.7 cells
Raw264.7 cells were digested and then digested at 2X 10 5 cells/well were inoculated into 24-well plates and incubated overnight at 37 ℃ in a 5% carbon dioxide incubator.
3. ProfMIC-203 addition and LPS stimulation
Culturing overnight Raw264.7 cells, adding 5% (V/V, corresponding control group is PBS with equal volume) of supernatant into the experimental group 1, adding 10% (V/V, corresponding control group is PBS with equal volume) of inactivated thallus into the experimental group 2, adding 0.5ml of LPS solution with the concentration of 0.2 mu g/ml after 2 hours, inducing Raw264.7 cells to be inflamed, taking cell culture supernatant after 20 hours, drawing a standard curve according to the method of the Biyunnan NO detection kit, and calculating the concentration and inhibition rate of NO in the sample.
ProfMIC-203 reduces NO production of Raw264.7 cells
The results show that ProfMIC-203 has anti-inflammatory effect, can reduce the NO production of Raw264.7 cells induced by LPS, and compared with an LPS control group, the supernatant is reduced by 24.70%, and the inactivated thallus is reduced by 28.11%.
Example six: profMIC-203 downregulates the expression of HaCaT cell inflammatory factors
1. Preparing ProfMIC-203 bacterial liquid (same as the fifth example);
2. preparation of HaCaT cells:
HaCaT cells were digested and then treated at 2X 10 5 cells/well were inoculated into 24-well plates and incubated overnight at 37 ℃ in a 5% carbon dioxide incubator.
3. Preparation and addition of staphylococcus aureus
Inoculating Staphylococcus aureus to nutrient broth culture medium, shake culturing at 37 deg.C overnight, and adjusting bacterial liquid concentration to OD with MEM serum-free culture medium 600 =6.0, 100 μ l per well was added to overnight cultured HaCaT cells to stimulate the production of inflammatory factors, the cell culture medium was discarded after 3h, washed 5 times with PBS, and 1ml of MEM serum-free medium was added to each well.
4. ProfMIC-203 sample addition
The ProfMIC-203 inactivated supernatant was added at 5% (V/V, equal volume of PBS for the control group) to staphyloccal stimulated HaCaT cells, 3 replicates per group, and incubated overnight.
5. qPCR method for detecting relative expression multiple of cell inflammatory factor mRNA
After the culture medium of the cells is removed, RNA is extracted by using an RNA extraction kit, the concentration and the purity of the RNA are detected, all samples are adjusted to 1 mu g, RT-PCR and qPCR are carried out by using a reverse transcription kit and a SYBRGreen qPCR kit, and the relative expression multiple F of inflammatory factor genes IL-8, IL-22, COX-2 and TRPV1 is calculated.
The formula: f =2 -ΔΔCT
As a result:
ProfMIC-203 inactivated supernatant down-regulating expression of inflammatory factor genes
The result shows that the ProfMIC-203 can reduce the expression of HaCaT cell inflammatory factor related genes induced by staphylococcus aureus, and the expression level is reduced by 30-66%. Thus, profMIC-203 has an anti-inflammatory effect.
In addition, all the fermentation supernatants or inactivated bacteria, which are verified to have the corresponding effects, are the derivatives (lysate or extract) of ProfMIC-203 obtained by further processing, extraction, etc., or the primary metabolite or secondary metabolite of ProfMIC-203 in the fermentation supernatants.
Claims (7)
1. A strain of Lactobacillus oralis (Lactobacillus oris) is characterized in that the strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M20211438.
2. The application of the lactobacillus oralis is characterized in that the lactobacillus oralis is used for repairing skin barriers and resisting inflammation, and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M20211438.
3. The application of the lactobacillus buchneri is characterized in that the lactobacillus buchneri is used for preparing food, medicines and cosmetics which have the functions of promoting skin barrier repair and resisting inflammation, and is preserved in China center for type culture collection with the preservation number of CCTCC NO: M20211438.
4. Use of a Lactobacillus buchneri according to claim 3, wherein the Lactobacillus buchneri can be its bacterial bodies or/and its derivatives or/and its metabolites.
5. The use of Lactobacillus bucinum according to claim 4, wherein the bacterial strain is live or/and inactivated.
6. Use of a lactobacillus buchneri according to claim 4, wherein the derivative is a lysate or/and extract of lactobacillus buchneri.
7. Use of a lactobacillus buchneri according to claim 4, wherein the metabolite is a primary metabolite or a secondary metabolite of lactobacillus buchneri.
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