CN116515661A - Antioxidant antibacterial staphylococcus caprae and application thereof - Google Patents
Antioxidant antibacterial staphylococcus caprae and application thereof Download PDFInfo
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- CN116515661A CN116515661A CN202210937198.0A CN202210937198A CN116515661A CN 116515661 A CN116515661 A CN 116515661A CN 202210937198 A CN202210937198 A CN 202210937198A CN 116515661 A CN116515661 A CN 116515661A
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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
- 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
- A61P31/02—Local antiseptics
-
- 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
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- 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
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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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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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
-
- 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
- C12R2001/44—Staphylococcus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an antioxidant antibacterial staphylococcus caprae and application thereof. The strain of the invention is staphylococcus caprae (Staphylococcus caprae) CCSM0335, is isolated from healthy human sources, and is preserved in China Center for Type Culture Collection (CCTCC) at the 1 st month of 2022, and the preservation number is CCTCC No: m2022776. Experiments prove that the fermentation broth supernatant of the strain has good scavenging activity on DPPH free radicals, can reduce the ROS content of human keratinocytes caused by vitamin K3, has a strong antibacterial effect on staphylococcus aureus, and has good application prospect in the aspects of development of antioxidant and antibacterial microecological skin care products.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to an antioxidant and antibacterial staphylococcus caprae seu ovis and application thereof.
Background
The skin is the largest organ of the human body and serves as a barrier for the body to protect it from foreign microorganisms, antigens or toxic substances. A large number of microorganisms inhabit the surface layers or follicles of human skin, constituting the human skin flora, which plays an important role in maintaining skin health, known as the microbial barrier of the skin. The skin microbiota, the host skin and the environment form a skin micro-ecological system, and the interaction and the mutual restriction between the skin microbiota, the host skin and the environment keep the coordinated, physiological and dynamic balance. Balance skin microecology and is beneficial to skin health. In daily life, frequent use of cosmetics, excessive cleaning, excessive care, chemicals, medicines, excessive uv exposure, bad life style, environmental pollution, etc. may cause skin dysbiosis. The skin biological barrier is weakened or destroyed to influence the normal physiological functions of the skin and even the skin is infected, so that the aging of the skin is promoted.
Free radicals are continuously generated in the human body in the metabolic process of the body and in the presence of external pollution, solar irradiation and the like, and excessive free radicals can cause various diseases and skin aging of the body. Studies have shown that in photoaged skin, UV irradiation increases ROS in skin cells, inducing increased levels of MMPs, resulting in degradation of collagen and elastin of the skin, and roughening, sagging, and wrinkling of the skin. The staphylococcus epidermidis, propionibacterium acnes and the like on the skin have the protection effect on skin injury caused by UV, can obviously reduce the ROS level induced by ultraviolet radiation, can directly regulate the expression level of MMPs of skin cells by influencing a plurality of signal paths, reduce the degradation of collagen and elastin after UV irradiation, and maintain the health of the skin.
With the development of analytical techniques such as high throughput sequencing, metagenomics, metabonomics and bioinformatics, we have a more thorough understanding of the microbial composition, structure and function of human skin. The skin flora, which is an important role in skin microecology, plays an important role in maintaining skin health. There are a large number of potential beneficial bacteria on healthy skin and a large number of studies have shown that: the antibacterial peptide is secreted by coagulase negative staphylococci represented by Staphylococcus epidermidis, and decomposes sebum to generate Short Chain Fatty Acids (SCFAs), nucleosides and other metabolites, has various physiological functions such as antibacterial, anti-inflammatory, immunoregulatory, skin barrier function enhancing and anticancer, and is a potential skin beneficial bacterium (Stacy A, belkaid Y.Microbial guardians of skin health [ J ]. Science,2019,363 (6424):227-228.).
Based on the important role of dermatophytes in maintaining skin health, it has been studied in 2015 that microorganisms isolated from autologous skin and mixed with gel to prepare a novel cosmetic have been remarkably improved in skin moisture retention (yuich Nodakea, saki Matsumotoa, b.pilot study on novel skin care method by augmentation with Staphylococcus epidermidis, an autologous skin microbe-A blinded randomized clinical trial [ J ]. Journal of Dermatological Science 79,2015,119-126). Therefore, the application of searching beneficial bacteria in the skin to cosmetics has wide application prospect.
At present, screening of skin bacteria and functional strains from healthy people is lacking in China, and reports about screening of staphylococcus caprae are not found. The strain is separated from healthy skin, and the screening and verification of the efficacy strain can lay a foundation for the development of skin beneficial bacteria and the development of microecological skin care products.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a coagulase negative staphylococcus-staphylococcus caprae (Staphylococcus caprae) CCSM0335 and application, and experiments prove that the supernatant of the fermentation broth of the strain has good scavenging activity on DPPH free radicals, can reduce the ROS content of human keratinocytes caused by vitamin K3, has a strong antibacterial effect on staphylococcus aureus, and has good application prospects in the aspects of development of antioxidant and antibacterial microecological skin care products.
The first object of the present invention is to provide a staphylococcus caprae (Staphylococcus caprae) CCSM0335 isolated from healthy human source (on skin of healthy human) and preserved in the China center for type culture Collection (preservation Address: university of martial arts, china) at 1/6 of 2022, with a preservation number of cctccc No: m2022776.
The colony characteristics of the strain are as follows: as shown in FIG. 1A, the bacterial strain CCSM0335 has white and small colony on a TSA blood plate, forms circular bulges, has regular edges, has the colony diameter of about 0.5-1 mm, has smooth surface, is moist and opaque, and can be picked up to be drawn into a wire without a hemolysis ring.
The thallus characteristics of the strain are as follows: as can be seen from fig. 1B, gram staining was positive. The thallus is spherical and has a diameter of about 0.9-1.0 μm, is arranged into grape shape, and has single arrangement without flagellum, can not move and can not produce spores.
A second object of the present invention is to provide a fermentation method of Staphylococcus caprae (Staphylococcus caprae) CCSM0335, wherein Staphylococcus caprae CCSM0335 is inoculated into TSB liquid medium, shake-cultured at 30-40deg.C for 16-20 h, and the OD of the cells is adjusted 600 0.9 to 1.1 percent as seed liquid; then inoculating the seed liquid into TSB liquid culture medium according to the volume ratio of 1-3%, and culturing for 10-15 h at 30-40 ℃ to obtain fermentation liquor of staphylococcus caprae; and centrifuging the fermentation liquor to obtain fermentation supernatant. TSB broth (trypticase soy broth): 17.0g/L tryptone, 3.0g/L soybean papain digest, 5.0g/L sodium chloride, 2.5g/L potassium dihydrogen phosphate and 2.5g/L glucose, and autoclaving at 121 ℃ for 15min for later use.
A third object of the invention is to provide the use of Staphylococcus caprae (Staphylococcus caprae) CCSM0335 and the above-described broth supernatants for antioxidant and bacteriostatic aspects of the skin.
The fourth purpose of the invention is to provide application of staphylococcus caprae (Staphylococcus caprae) CCSM0335 and the supernatant of the fermentation broth in preparing antioxidant and antibacterial microecological skin care products for resisting damage of free radicals to skin.
The antioxidant includes scavenging DPPH free radical and reducing ROS content of human keratinocyte caused by vitamin K3. The above antibacterial includes inhibiting Staphylococcus aureus.
At present, no report on screening of staphylococcus caprae, namely skin bacteria, is seen, and the invention provides a staphylococcus caprae CCSM0335 which is a healthy human source (separated from skin of a healthy person). Experiments prove that the staphylococcus caprae CCSM0335 has good antioxidant effect and antibacterial effect, the clearance of DPPH free radical of the fermentation supernatant is 95.60%, the clearance of ROS is 17.7%, and the inhibition rate of staphylococcus aureus is 15.7%. Therefore, the method has good application prospect in resisting skin injury and skin infection caused by free radicals and developing antioxidant and antibacterial microecological skin care products, and fills the blank of screening the antioxidant and antibacterial effects of the staphylococcus caprae.
Drawings
FIG. 1 shows colony morphology and photomicrographs of Staphylococcus caprae (Staphylococcus caprae) CCSM0335, wherein Panel A is a colony morphology graph and Panel B is a photomicrograph;
FIG. 2 growth curve of Staphylococcus caprae (Staphylococcus caprae) CCSM 0335;
FIG. 3 cytotoxicity of Staphylococcus caprae (Staphylococcus caprae) CCSM0335 fermentation supernatant on human skin keratinocytes;
FIG. 4 effect of Staphylococcus caprae (Staphylococcus caprae) CCSM0335 fermentation supernatant on scavenging reactive oxygen species ROS in human keratinocytes.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention.
Example 1: isolation, screening and purification of the Strain CCSM0335
(1) Recruiting perfect skin volunteers
The skin is required to be healthy, the skin is fine and smooth, pores are small, acne, pustule, inflammation, desquamation and the like are avoided, and a person with spots can participate without wiping ointment for 3 months; age 18-30 years, and the sex is unlimited.
(2) Sample collection
The volunteers were required to sample the skin (water, milk, etc.) on a basis after the previous evening of the face wash, and the next morning could not wash the face, typically at noon or afternoon. An area of about 4cm x 4cm was selected at the sampling skin site, a sterile cotton swab of polymeric fiber material was wetted in a wetting solution (containing 0.9% sodium chloride and 0.1% tween-20), rubbed back and forth 50 times in the selected area (the swab was rubbed with a certain force), placed in a sampling tube with sterile forceps, and sealed with a sealing film. And (3) putting the collected sample into an ice box for refrigeration, taking the sample back to a laboratory, and placing the sample into a refrigerator at the temperature of 4 ℃ for separating the bacterial strain as soon as possible.
(3) Sample pretreatment
The aseptic cotton swab head with the sample is sheared off by aseptic scissors in an ultra-clean workbench, placed in a 5mL centrifuge tube (EP tube), and 5mL of aseptic water is sucked into the centrifuge tube and fully and uniformly mixed.
(4) Plate screening
Taking 0.5mL of the sample liquid obtained in the step (3), adding 4.5mL of sterile water for gradient dilution, selecting proper dilution, absorbing 0.1mL of the dilution liquid, coating the dilution liquid on a TSA blood plate, coating 2 plates at each dilution, and respectively placing the plates in a 37 ℃ incubator for aerobic culture for 24 hours.
Trypticase soy agar blood medium (TSA): 15.0g/L tryptone, 5.0g/L soyase peptone, 5.0g/L sodium chloride, 15.0g/L agar, sterilizing at 121deg.C for 15min, cooling to about 50deg.C, adding 5% sterilized defibrinated sheep blood, mixing, and plating.
(5) Score line purification and preservation
According to the colony characteristics of coagulase negative staphylococcus strains on blood plates, the differences of hemolysis circle, colony size, color, wetting, gloss and the like are not produced, single colonies are selected to respectively score TSA blood plates, the blood plates are placed in a constant temperature incubator at 37 ℃ for culture for 16 hours, after multiple score separation, a preservation culture medium which adopts skim milk powder as a protective agent is preserved in a preservation tube, and the blood plates are preserved in a cryogenic refrigerator at-85 ℃ after freeze drying.
Preservation medium: 120g of skim milk powder, 880mL of distilled water, and autoclaving at 118 ℃ for 15min for later use.
The strain CCSM0335 was obtained by the above method, and was subjected to microbiological identification by the following method.
Example 2: microbiological identification of the Strain CCSM0335
(1) Colony characteristics:
strain CCSM0335 was streaked onto TSA platelets and after 16h incubation at 37 ℃ the colony morphology of the strain on the plates was observed. As shown in FIG. 1A, it can be seen from FIG. 1A that the colony of the strain CCSM0335 on the TSA blood plate is white and small, forms circular bulges with regular edges, has a colony diameter of about 0.5-1 mm, has a smooth surface, is moist and opaque, and can be drawn without hemolysis rings.
(2) Characteristics of the cells:
and (3) selecting a small amount of bacterial strain CCSM0335 obtained in the step (1) on a glass slide, smearing, gram staining and observing the morphological characteristics of the bacterial strain under a microscope. The results are shown in FIG. 1B, and it can be seen from FIG. 1B that gram staining is positive. The thallus is spherical and has a diameter of about 0.9-1.0 μm, is arranged into grape shape, and has single arrangement without flagellum, can not move and can not produce spores.
(3) Culture characteristics:
inoculating a strain CCSM0335 into a TSB liquid culture medium, and culturing at different temperatures, wherein researches show that the minimum growth temperature of the CCSM0335 is 15 ℃, the maximum growth temperature is 45 ℃, and the growth temperature is optimal at 30-40 ℃; inoculating into TSB liquid culture medium with different pH, culturing at 37deg.C, and culturing CCSM0335 strain with highest growth pH of 9.0, lowest growth pH of 4.0, and optimal growth pH of 6.0.
Trypticase soytone broth (TSB broth): 17.0g/L tryptone, 3.0g/L soybean papain digest, 5.0g/L sodium chloride, 2.5g/L potassium dihydrogen phosphate and 2.5g/L glucose, and autoclaving at 121 ℃ for 15min for later use.
(4) Genetic characterization (16S rDNA sequence analysis):
CCSM0335 genomic DNA extraction method: the purified CCSM0335 single colony is selected and inoculated into 10mL TSB liquid culture medium, and after culturing for 14-16h at 37 ℃, bacterial liquid is centrifuged (8000 r/min,15 min) to collect bacterial cells.
The DNA was extracted using a genomic DNA extraction kit (Shanghai Co., ltd.). The PCR amplification uses two synthesized universal primers (16s 27F:GAGAGTTTGATCCTGGCTCAG;16s 1492R:CGGCTACCTTGTTACGACTT), the PCR product is recovered by a column type PCR product purification kit (Shanghai) and sent to the Meiji bioengineering (Shanghai) for sequencing after purification. The nucleotide sequence of the 16S rDNA of the CCSM0335 is shown in SEQ NO.1, and the sequence length is as follows: 1383bp, the specific sequence is as follows:
CAAAGGTTACTCCACCGGCTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGTAGCATGCTGATCTACGATTACTAGCGATTCCAGCTTCATGTAGTCGAGTTGCAGACTACAATCCGAACTGAGAACAACTTTATGGGATTTGCTTGACCTCGCGGTTTCGCTACCCTTTGTATTGTCCATTGTAGCACGTGTGTAGCCCAAATCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCAACTTAGAGTGCCCAACTTAATGATGGCAACTAAGCTTAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGTCCCCCGAAGGGGAAAACTCTATCTCTAGAGGGGTCAGAGGATGTCAAGATTTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGATCCCCACGCTTTCGCACATCAGCGTCAGTTACAGACCAGAAAGTCGCCTTCGCCACTGGTGTTCCTCCATATCTCTGCGCATTTCACCGCTACACATGGAATTCCACTTTCCTCTTCTGCACTCAAGTTTTCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAAAAACCGCCTACGCGCGCTTTACGCCCAATAATTCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGATTAGGTACCGTCAAGACGTGCATAGTTACTTACACATTTGTTCTTCCCTAATAACAGAGTTTTACGATCCGAAGACCTTCTTCACTCACGCGGCGTTGCTCCGTCAGGCTTTCGCCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCGGCGCGGATCCATCTATAAGTGACAGCAAGACCGTCTTTCACTGTTGAACCATGCGGTTCAACATGTTATCCGGTATTAGCTCCGGTTTCCCGAAGTTATCCCAGTCTTATAGGTAGGTTATCCACGTGTTACTCACCCGTCCGCCGCTAACGTCAGAGGAGCAAGCT。
send GenBank to Blast analysis. The highest homology of the strain CCSM0335 is MT023404.1, and the homology is 100%. Species having G+C (mol%) 10% or less and 12% or more and 16S rRNA having a sequence homology of 95% or more according to the DNA described by Goodfeldow and O' Donnell can be classified as a genus, and Embley and Stackebrangdt consider that 16S rRNA has a sequence homology of 97% or more can be considered as a species. From this it can be deduced that: the strains CCSM0335 and Staphylococcus caprae belong to the same species.
According to the microbiological characteristics such as colony, fungus body shape, culture, physiological biochemical identification and the like, the 16srDNA identification strain CCSM0335 is staphylococcus caprae (Staphylococcus caprae), and the strain is preserved in China Center for Type Culture Collection (CCTCC) in 6-month-1 of 2022, and the preservation number is CCTCC NO: m2022776.
Example 3: drawing of a growth curve of Staphylococcus caprae CCSM0335
Inoculating activated staphylococcus caprae (Staphylococcus caprae) CCSM0335 into TSB liquid culture medium according to 2% (v/v) inoculum size, culturing at 37deg.C under shaking for 24 hr, measuring OD value of culture solution at 600nm every 2 hr, and regulating OD 600 The results of the growth curve of staphylococcus caprae (Staphylococcus caprae) CCSM0335 in TSB broth were plotted against time, and as shown in fig. 2, staphylococcus caprae (Staphylococcus caprae) CCSM0335 grew rapidly in TSB broth, entering the log phase at about 2h and entering the stationary phase at about 12 h.
Example 4: DPPH radical clearance determination of goat staphylococcus CCSM0335 fermentation supernatant
(1) Preparation of goat staphylococcus (Staphylococcus caprae) CCSM0335 fermentation supernatant
The staphylococcus caprae (Staphylococcus caprae) CCSM0335 strain is streaked on a TSA plate, placed at 37 ℃ and cultured for 16-20 hours at constant temperature, and activated for 2 times. Inoculating activated staphylococcus caprae (Staphylococcus caprae) CCSM0335 into TSB liquid culture medium, shake culturing at 37deg.C for 16-20 hr, and regulating thallus OD 600 In the range of 0.9 to 1.1 as seed liquid. Inoculating the seed solution into a TSB liquid culture medium of a 250mL triangular flask according to the volume ratio of 2%, and culturing at 37 ℃ at the speed of 160r/min for 12 hours to obtain a fermentation broth of staphylococcus caprae; centrifuging the fermentation broth at 10000r/min for 15min to obtain a centrifugal supernatant, namely fermentation supernatant.
TSB broth (trypticase soy broth): 17.0g/L tryptone, 3.0g/L soybean papain digest, 5.0g/L sodium chloride, 2.5g/L potassium dihydrogen phosphate and 2.5g/L glucose, and autoclaving at 121 ℃ for 15min for later use.
(2) The DPPH radical scavenging rate in the metabolite of Staphylococcus caprae (Staphylococcus caprae) CCSM0335 was measured by the DPPH scavenging radical method as follows:
(1) preparing mother solution (DPPH solution) with DPPH concentration of 0.1mmol/L with absolute ethyl alcohol, and preserving in dark at low temperature.
100. Mu.L of the sample solution and 100. Mu.L of the DPPH solution were added to a 96-well plate and designated as solution s;
100 mu L of sample solution and 100 mu L of absolute ethanol solution are placed in a 96-well plate and marked as solution b;
100. Mu.L of 50% ethanol solution and 100. Mu.L of DPPH solution were placed in a 96-well plate and designated as solution c;
uniformly mixing, and reacting for 30min in a dark environment at a constant temperature of 37 ℃;
(2) and respectively measuring OD values of the reaction solutions obtained in the As, ab and Ac holes at 517nm by using an enzyme-labeled instrument, wherein each reaction solution obtained in the As, ab and Ac holes is at least parallel for 3 times, and taking an average value according to a DPPH free radical clearance formula:
clearance = 100% × [ a ] c -(A s -A b )]/Ac
DPPH radical clearance of the goat staphylococcus (Staphylococcus caprae) CCSM0335 fermentation supernatant was calculated to be 95.60 + -4.8%.
Example 5: ROS assay for scavenging human keratinocytes from goat staphylococcal CCSM0335 fermentation supernatant
(1) Preparation of goat staphylococcus (Staphylococcus caprae) CCSM0335 fermentation supernatant
The preparation method is the same as in the step (1) of the example 4, the fermentation supernatant of the staphylococcus caprae (Staphylococcus caprae) CCSM0335 is obtained, and the fermentation supernatant is concentrated to 1/4 of the original volume, namely the concentrated fermentation supernatant of the staphylococcus caprae.
(2) Cell culture
Human primary skin keratinocytes (NEKs) were isolated from normal skin tissue using Promocell-specific complete medium (Keratinocyte Growth Medium, C-20011) at 37℃with 5% CO 2 Conventional culture under conditions, after cells grew to near confluence, passaging was done by trypsinization, 1 passage per 5 d.
(3) Cell viability assay
Get atNEKs cells in optimal growth state, conventional treatment, cell suspension liquid density was adjusted to 8X 10 4 -1×10 5 mu.L of cell suspension per well was inoculated into 96-well plates at 37℃and 5% CO 2 Culturing in an incubator. 0.1%, 0.5%, 1%, 5% and 10% of the concentrate of the ccsm0335 fermentation supernatant was added to the culture for 24 hours, and a control group was set, and 3 parallel wells were set for each experimental group. After 24h of incubation, 10. Mu.L of CCK-8 reagent (Japanese homozygote CK-04) was added to each well and incubated for 2h in a conventional manner. The absorbance was measured at a wavelength of 450nm using an enzyme-labeled instrument, and the reference wavelength was 600nm or more.
(4) ROS detection
NEKs cells in optimal growth state are cultured in Promocell special culture medium, and the density of cell suspension is regulated to 8×10 4 -1×10 5 mu.L of cell suspension per well was inoculated into 96-well plates at 37℃and 5% CO 2 Culturing in an incubator. 10. Mu. Mol/L vitamin K3 was added to each well, 5% CCSM0335 fermentation supernatant concentrate was added to the experimental group, 200. Mu. Mol/L vitamin E was used as positive control, and after incubation for 24h, 1. Mu.L ROS fluorescent chromogenic reagent (CellRox, thermo) was added to each well, and incubation was performed for 4h. The fluorescence value was measured using an enzyme-labeled instrument, the excitation light wavelength was 485nm, and the emission light was 520nm.
As can be seen from fig. 3, when the amount of the concentrate of CCSM0335 fermentation supernatant added was less than 5% as compared with the control NT, cytotoxicity was not shown and the growth of human primary skin keratinocytes was significantly promoted. A concentrate of 5% ccsm0335 fermentation supernatant was chosen here for ROS experiments. Experimental results show (figure 4) that after the induction group treatment of vitamin K3, keratinocytes secrete a large amount of ROS, and the addition of CCSM00335 to ferment supernatant concentrate can remarkably reduce the amount of ROS, and the ROS clearance rate is 17.7%. Therefore, the goat staphylococcus CCSM0335 fermentation supernatant can obviously reduce the expression of ROS caused by vitamin K3, and shows good antioxidant effect.
Example 6: antibacterial measurement of staphylococcus aureus by goat staphylococcus CCSM0335 fermentation supernatant
(1) Preparation of goat staphylococcus (Staphylococcus caprae) CCSM0335 fermentation supernatant
A method for preparing a fermentation supernatant of Staphylococcus caprae seu ovis (Staphylococcus caprae) CCSM0335 in step (1) of example 4 was carried out.
(2) Indicator staphylococcus aureus activation:
streaking staphylococcus aureus on TSA blood plate, culturing at 37 ℃ for 18-24 h, activating for 2 times, diluting with TSB liquid culture medium after culturing, adjusting OD 660 Between 0.08 and 0.1.
(3) Bacteriostasis experiment
200 mu L of sterile water is added to the outer ring of the 96-well plate; column 2 was filled with 100 μl of TSB broth and 100 μl of sterile water as blank; column 3 was charged with 100. Mu.L of TSB broth, 50. Mu.L of sterile water and 50. Mu.L of Staphylococcus aureus broth as positive controls. 100. Mu.L of TSB broth and 50. Mu.L of Staphylococcus aureus broth and 50. Mu.L of CCSM0335 fermentation supernatant were added to column 4 and mixed well. 4 multiple wells are made on each sample, the culture is carried out for 16 hours at the constant temperature of 37 ℃, and OD is measured after the culture is finished 660 . According to the bacteriostasis rate = ((OD) Yang (Yang) -OD Blank space )-(OD Sample -OD Blank space ))/(OD Yang (Yang) -OD Blank space ) To calculate the bacteriostasis rate of the sample.
The CCSM0335 fermentation supernatant and staphylococcus aureus are cultured simultaneously, the inhibition rate of the CCSM0335 fermentation supernatant and the staphylococcus aureus is 15.7%, and the CCSM0335 fermentation supernatant shows a strong antibacterial effect.
Claims (8)
1. A staphylococcus caprae (Staphylococcus caprae) CCSM0335 with a preservation number of CCTCC No: m2022776.
2. The fermentation process of staphylococcus caprae (Staphylococcus caprae) CCSM0335 of claim 1, wherein fermentation is performed using TSB broth; and centrifuging the fermentation liquor to obtain fermentation supernatant.
3. The fermentation method according to claim 2, wherein staphylococcus caprae CCSM0335 is inoculated into TSB liquid culture medium, shake-cultured at 30-40 ℃ for 16-20 h, and the OD of the thallus is adjusted 600 0.9 to 1.1 as seedSub-liquid; then inoculating the seed liquid into TSB liquid culture medium according to the volume ratio of 1-3%, and culturing for 10-15 h at 30-40 ℃ to obtain fermentation liquor of staphylococcus caprae; and centrifuging the fermentation liquor to obtain fermentation supernatant.
4. A fermentation broth supernatant produced by the fermentation process of claim 2 or 3.
5. Use of staphylococcus caprae (Staphylococcus caprae) CCSM0335 as claimed in claim 1 or the supernatant of a fermentation broth as claimed in claim 4 for antioxidant and bacteriostatic aspects of the skin.
6. Use of staphylococcus caprae (Staphylococcus caprae) CCSM0335 as defined in claim 1 or the supernatant of fermentation broth as defined in claim 4 for preparing an antioxidant and bacteriostatic microecological skin care product against damage of free radicals to skin.
7. The use according to claim 5 or 6, characterized in that said antioxidant comprises scavenging DPPH free radicals and reducing the ROS content of human keratinocytes caused by vitamin K3.
8. The use according to claim 5 or 6, wherein said inhibiting bacteria comprises inhibiting staphylococcus aureus.
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