CN114891699A - Lactobacillus salivarius for regulating flora balance - Google Patents

Lactobacillus salivarius for regulating flora balance Download PDF

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CN114891699A
CN114891699A CN202210703954.3A CN202210703954A CN114891699A CN 114891699 A CN114891699 A CN 114891699A CN 202210703954 A CN202210703954 A CN 202210703954A CN 114891699 A CN114891699 A CN 114891699A
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lactobacillus salivarius
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廖梅香
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Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus salivarius for regulating flora balance. The lactobacillus salivarius with the preservation number of CCTCC NO: M20211389 can regulate the microbial flora of human skin, promote the proliferation and repair of epidermal cells, and has the effects of resisting inflammation and oxidation, and the application of the lactobacillus salivarius ProfMIC-201 and/or the fermentation liquid thereof in pharmaceuticals, foods, maintenance products or cosmetics.

Description

Lactobacillus salivarius for regulating flora balance
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus salivarius for regulating flora balance.
Background
The skin is a living area of a wide range of microorganisms as the largest organ of the human body, and the skin constitutes a habitat of various forms such as an entrapment part and a specialized space, and helps the survival of the widely distributed microorganisms. The microorganism forms a symbiotic relationship with the human host in which the skin microbiota performs an important and useful function, not only because of its ability to resist the adhesion and development of skin pathogens, but also because of its ability to interact and interact with the immune system. When dysbiosis occurs at the skin level, probiotics can act as regulators and restore the balance of the skin microbiota.
Lactic acid bacteria are one of the beneficial microorganisms in the human body, which colonize intestinal mucosal epithelial cells and are responsible for decomposing cellulose and complex proteins to become important nutritional components. In addition, the intestinal environment is kept acidic, and adhesion of other pathogenic bacteria is suppressed. In addition, the lactobacillus can also enhance the activity of macrophages and spleen, and show the effect of secreting and promoting substances related to immune response. Has immunoregulatory effect, and can be used for treating atopic diseases and allergy related diseases. Of the more than 400 anaerobic or aerobic microorganisms in the gastrointestinal tract, lactic acid bacteria account for about 10%.
With the development of biotechnology in recent years, various use values of lactic acid bacteria have been developed. In addition to being used in food and medicine, lactic acid bacteria are increasingly gaining attention for their development and application in the cosmetic industry. In the field of cosmetics, since commercialization in 1955, lactic acid bacteria culture solutions have been used, active studies have been conducted mainly using Streptococcus (Streptococcus) genus, Lactobacillus (Lactobacillus) genus, Lactococcus (Lactococcus) genus, Leuconostoc (Leuconostoc) genus, Bifidobacterium (Bifidobacterium) genus, and the like, and they include raw material forms such as fermentation, filtration, or extraction directly in culture solutions, and filtration or extraction after inoculation to active materials and fermentation. The effects of lactic acid bacteria on antibacterial, antioxidant and immunological activities have been reported. In recent years, research has been focused on whitening and moisturizing effects of lactic acid bacteria, and the possibility as a cosmetic material has been increasing.
Lactobacillus salivarius is a lactic acid bacterium widely distributed in animal and plant fermented products containing carbohydrates, and also found in oral cavity, vagina and intestinal tract of warm-blooded animals, and can be separated from plant body surface, dairy products, meat products, beer, wine, fruit juice, wort, fermented dough, sewage and human and animal feces. The strain has strong ability to decompose sugar and low ability to decompose protein. Some foods such as pickles, wines and yogurts are widely used in the production process. But the lactobacillus salivarius has not been reported to regulate the balance of flora on the surface of the skin.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide lactobacillus salivarius for regulating the balance of flora on the skin surface.
The invention provides lactobacillus salivarius with a preservation number of CCTCC NO: M20211389.
The invention also provides application of the lactobacillus salivarius with the preservation number of CCTCC NO: M20211389 in preparation of products for regulating flora.
In the present invention, the regulation of the bacterial population includes promotion of proliferation of beneficial bacteria and/or inhibition of harmful bacteria.
In the present invention, the inhibition of harmful bacteria includes inhibition of growth of harmful bacteria, inhibition of coagulation of harmful bacteria, and/or inhibition of formation of biofilm by harmful bacteria.
In some embodiments, the harmful bacteria include at least one of staphylococcus aureus, streptococcus mutans, staphylococcus hominis, staphylococcus haemolyticus, and/or pseudomonas aeruginosa. In some embodiments, the beneficial bacteria comprise staphylococcus epidermidis.
Specifically, the research of the invention shows that the lactobacillus salivarius with the preservation number of CCTCC NO: M20211389 can inhibit the activity, growth or proliferation of at least one of staphylococcus aureus, streptococcus mutans, staphylococcus hominis, staphylococcus haemolyticus and/or pseudomonas aeruginosa. The strain can also inhibit the agglutination and/or biofilm formation of Staphylococcus aureus.
The invention also provides application of the lactobacillus salivarius with the preservation number of CCTCC NO: M20211389 in preparing antioxidant products.
In the present invention, the anti-oxidation includes scavenging hydroxyl radicals and/or ABTS radicals. The antioxidant of the present invention also includes improving the total antioxidant capacity.
The invention also provides application of the lactobacillus salivarius with the preservation number of CCTCC NO: M20211389 in preparation of anti-inflammatory products.
In some embodiments, the anti-inflammatory is against inflammation induced by LPS. In particular, it is against inflammation of epidermal cells induced by LPS. The epidermal cells are keratinocytes. In the present invention, said anti-inflammatory comprises reducing the amount of NO released and/or down-regulating the level of inflammatory factors. In some embodiments, the inflammatory agent comprises at least one of IL-8, COX-2.
The invention also provides application of the lactobacillus salivarius with the preservation number of CCTCC NO: M20211389 in preparation of products for repairing skin.
Repairing skin as described herein includes repairing cell damage and/or repairing cell scarification.
In some embodiments, the repairing skin cell damage comprises protecting the viability of skin cells under SDS-induced conditions. In some embodiments, the skin cells are keratinocytes.
In some embodiments, repairing the cell scratch comprises repairing a scratch of a skin cell, the skin cell being a keratinocyte.
The invention also provides a medicine and/or a cosmetic, and the raw material of the medicine and/or the cosmetic comprises lactobacillus salivarius with the preservation number of CCTCC NO: M20211389.
In some embodiments, the product of the invention comprises at least one of the following i) to iv):
i) the preservation number is CCTCC NO: M20211389;
ii) inactivated lactobacillus salivarius with a preservation number of CCTCC NO: M20211389;
iii) a culture of Lactobacillus salivarius with a preservation number of CCTCC NO: M20211389;
iv) extract of Lactobacillus salivarius with preservation number CCTCC NO: M20211389.
In the present invention, the culture includes a culture solution obtained by culturing, or a supernatant or a cell obtained by separating the culture solution, or a suspension obtained by disrupting cells in the culture solution.
In the present invention, the extract comprises polysaccharides, proteins or other secondary metabolites obtained by extracting the culture.
The invention also provides a method of treating skin by administering the product of the invention.
The skin care according to the invention comprises flora regulation, antioxidant, anti-inflammatory and/or skin repair. The mode of administration includes spraying, painting, applying and/or introducing.
The lactobacillus salivarius with the preservation number of CCTCC NO: M20211389 can regulate the microbial flora of human skin, promote the proliferation and repair of epidermal cells, and has the effects of resisting inflammation and oxidation, and the application of the lactobacillus salivarius ProfMIC-201 and/or the fermentation liquid thereof in pharmaceuticals, foods, maintenance products or cosmetics.
Proof of biological preservation
Lactobacillus salivarius ProfMIC-201 is preserved in China center for type culture Collection at 11 month and 08 days 2021 at the preservation number of M20211389 CCTCC NO of university in Wuhan, China.
Drawings
FIG. 1 shows a ProfMIC-201 Staphylococcus aureus inhibition map;
FIG. 2 shows a ProfMIC-201 Staphylococcus aureus inhibition plot;
FIG. 3 is a graph showing that ProfMIC-201 inhibits biofilm formation by Staphylococcus aureus;
FIG. 4 is a graph showing the biofilm-OD change in ProfMIC-201 inhibition of Staphylococcus aureus formation;
FIG. 5 shows ProfMIC-201 coaggregation of Staphylococcus aureus;
FIG. 6 shows a plot of the inhibitory rate of ProfMIC-201 pathogenic bacteria;
FIG. 7 is a graph showing that ProfMIC-201 decreases NO release from Raw264.7 cells;
FIG. 8 shows a graph of ProfMIC-201 supernatant down-regulating expression of inflammation-associated factors;
FIG. 9 is a graph showing that the expression of inflammation-associated factors is down-regulated by inactivated ProfMIC-201 bacteria;
FIG. 10 is a graph showing that ProfMIC-201 promotes HaCaT cell repair;
FIG. 11 shows ProfMIC-201HaCaT cell scratch healing profiles;
FIG. 12 shows that ProfMIC-201 promotes HaCaT cell scratch healing-healing rate;
FIG. 13 shows a ProfMIC-201 radical scavenging potential diagram.
Detailed Description
The invention provides lactobacillus salivarius for regulating flora balance. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The Lactobacillus salivarius is separated from the nasal cavity of a healthy male infant with the age of 13 months and is marked as ProfMIC-201, and the strain is gram-positive and takes the shape of a short rod under a microscope, and has no spores and flagella; the micro-colonies grow on an MRS plate, pink needle-tip microcolonies with smooth and round surfaces can be formed, the edges are neat, and calcium dissolving rings are arranged; 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 optimum growth temperature is 35-38 ℃, and the pH is 3.0-7.0. It is preserved in China center for type culture Collection with a preservation number of CCTCC NO: M20211389.
Research shows that the lactobacillus salivarius can regulate human skin microbial flora, promote proliferation and repair of epidermal cells, has the effects of resisting inflammation and resisting oxidation, and can be applied to medicaments, foods, maintenance products or cosmetics.
Further, the invention provides the lactobacillus salivarius with the preservation number of CCTCC NO: M20211389, wherein the lactobacillus salivarius is used in a survival or inactivation mode, or in a lysate and/or extract mode, or in a bacterial product mode, or in a supernatant mode, or in a derivative mode. Such derivative forms include, but are not limited to: metabolites, metabolic biological products, prebiotics, cell walls or components related thereto, exopolysaccharides, or compounds containing immunogenic components. In the embodiment of the invention, the experimental verification is carried out by taking culture supernatant, thallus or inactivated thallus of lactobacillus salivarius with the preservation number of CCTCC NO: M20211389 as a test object.
In-vitro antibacterial tests show that the lactobacillus salivarius ProfMIC-201 has the effect of inhibiting staphylococcus aureus, the antibacterial zone can reach 14-25 mm, the antibacterial capacity is 25-75%, and the antibacterial rate is 45-80%.
In vitro agglutination experiments show that the lactobacillus salivarius PROFMIC-201 has the function of agglutinating staphylococcus aureus, and the agglutination capacity is 25-40%;
the lactobacillus salivarius ProfMIC-201 has the effect of inhibiting the formation of a film of staphylococcus aureus, and after the lactobacillus salivarius ProfMIC-201 is co-cultured with the staphylococcus aureus for 24 hours, the formation inhibition rate of a supernatant biological film of the ProfMIC-201 is 65-80%;
the lactobacillus salivarius ProfMIC-201 has the effect of inhibiting pathogenic bacteria such as streptococcus mutans, staphylococcus hominis, staphylococcus haemolyticus, pseudomonas aeruginosa and the like, and the bacteriostasis rate is 15.6-52.1%.
In vitro cell experiments show that the lactobacillus salivarius ProfMIC-201 has an anti-inflammatory effect, can reduce NO release amount of Raw264.7 cells induced by LPS, and can be reduced by 12-45% compared with a control group; the Lactobacillus salivarius ProfMIC-201 can down-regulate the expression of IL-8 and COX-2 genes, and can down-regulate 25-50.2%.
In vitro cell experiments show that the lactobacillus salivarius ProfMIC-201 has the effect of promoting epidermal cell repair, the scratch healing rate can reach 55-83%, and the scratch healing rate is improved by 5-42% compared with a control.
In vitro cell experiments show that the Lactobacillus salivarius ProfMIC-201 strain has the functions of eliminating free radicals and resisting oxidation, the hydroxyl radical clearance rate is 12.1-21.2%, the ABTS free radical clearance rate is 10.0-18.1%, and the total oxidation resistance is improved by 25.2-62.3%.
In the invention, the medicament is an external preparation, and the dosage form of the medicament comprises solution, lotion, emulsion, powder, ointment and paste. Wherein the lotion comprises an aqueous powder or suspension.
In the present invention, the cosmetics may be classified into: cleaning cosmetics, nursing cosmetics and beautifying/modifying cosmetics. The cleaning cosmetic is applied to the surface of human body (such as epidermis, hair, nail, lip and the like) by smearing, spraying or other similar methods, and has the effects of cleaning and sanitation or eliminating bad smell. The care cosmetic is applied to the surface of human body (such as epidermis, hair, nail, lip and the like) by smearing, spraying or other similar methods, and has the function of caring. The cosmetic/finishing cosmetic is applied to the surface of human body (such as epidermis, hair, nail, lip and the like) by smearing, spraying or other similar methods, and has the effects of beautifying, finishing and enhancing the charm of human body.
Cleansing cosmetic compositions suitable for application to the skin include: facial cleanser, makeup remover (milk), cleansing cream (honey), facial mask, toilet water, itching powder, toilet powder or bath lotion; skin-applicable care cosmetics include skin creams, lotions or lotions; cosmetic/finishing cosmetic compositions suitable for application to the skin include: pressed powder, rouge, eye shadow, eyeliner (liquid), eyebrow pencil, perfume or cologne; the hair cleansing cosmetic comprises shampoo, cream shampoo or shaving cream; suitable hair care cosmetic compositions include conditioners, creams, pomades/waxes or hair treatment creams; cosmetic/finishing-type cosmetics suitable for hair include: styling mousses/gels, hair dyes, perming agents, mascara, pilatory or depilatory agents; cleaning cosmetic for nail comprises nail lotion; nail-care cosmetic products include nail lotions (creams), nail hardeners; cosmetic/finishing type cosmetics suitable for nails include nail polish; cleansing cosmetics suitable for lips include lip cleansing liquids; care cosmetics suitable for the lips include lip balms; suitable and cosmetic/finishing cosmetic compositions for the lips include: lipstick, lip gloss, or lip liner.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
example 1 isolation of ProfMIC-201 Strain
MC agar medium: 5g of soybean peptone, 5g of beef extract, 5g of yeast extract, 20g of glucose, 20g of lactose, 10g of calcium carbonate, 15g of agar, 5ml of 1% neutral red solution, 1000ml of distilled water, pH value of 6 and sterilization at 121 ℃ for 15 min.
MRS liquid medium: 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of diammonium hydrogen citrate, 20g of glucose, 80lml of tween-80, 5g of sodium acetate, 2g of dipotassium phosphate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 1000ml of distilled water, 6.2-6.4 of pH value and 15min of sterilization at 121 ℃.
The sample is from the nasal cavity of a healthy male infant volunteer at 13 months of age. Aseptically sampling in a sterilized sampling tube containing 5ml of glycerol, and taking back and freezing at-20 deg.C. During separation, the sample is redissolved, 1g of the sample is taken to be uniformly shaken in 9ml of normal saline, the supernatant is taken and streaked on an MC plate, the MC plate is cultured at the constant temperature of 37 ℃ for 24-48 hours, then a colony with a red calcium-dissolving ring is selected, and the inoculation and screening are repeated until a uniform single colony is obtained, and the colony is named as ProfMIC-201.
Gram staining microscopy: the strain ProfFMIC-201 is G +, takes the shape of a short rod under a microscope, and has no spore and flagellum; the micro-colonies grow on the MC flat plate, pintip-shaped microcolonies which are pink and smooth in surface can be formed, the edges are neat, and calcium dissolving rings are arranged; 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.
Example 2 nucleic acid identification of ProfMIC-201 Strain
1. Primer:
according to the gene sequence of lactobacillus 16S rRNA registered in GenBank, the 368-charge 1049 site gene fragment is referred to, and a primer is designed:
F5-TCGGCTCGTAAAACTCTG-3’；
R5-GGACTTAACCCAACATCTCA-3’。
the primer is synthesized by Shanghai bioengineering GmbH, and the amplified fragment is V3-V7 and is 682bp long.
2. 16 srna gene sequence analysis:
and (3) picking a single colony, placing the single colony in an MRS centrifuge tube, culturing the single colony overnight at 37 ℃, centrifuging and collecting thalli, and operating according to the steps of the DNA extraction kit. The PCR amplification system is a 50 mu L system, and the pre-denaturation is carried out for 10min at 95 ℃; 35 cycles of 93 ℃ for 1min, 55 ℃ for 1min, 72 ℃ for 1 min; extension at 72 ℃ for 7 min.
3. Results
The PCR product was analyzed by 1.2% agarose gel electrophoresis, a specific target band appeared at 682bp, which was consistent with the expected size, and the sequence results were compared with the published standard sequence in GenBank to determine that the strain ProfMIC-201 was Lactobacillus salivarius.
Example 3 inhibition of Staphylococcus aureus by ProfMIC-201-zone of inhibition experiment
1. Preparing a lactobacillus salivarius ProfMIC-201 bacterial liquid:
culturing activated Lactobacillus salivarius ProfMIC-201 liquid with MRS in 37 deg.C incubator for 18 hr, measuring bacterial count with spectrophotometer OD600nm, and adjusting concentration of Lactobacillus salivarius liquid to 2 × 10 9 cells/mL, as viable bacteria solution. Centrifuging the viable bacteria solution, collecting supernatant to obtain viable bacteria solution supernatant, and boiling in water bath for 30min to obtain inactivated bacteria solution.
2. Preparation of staphylococcus aureus liquid:
culturing in broth culture at 140r/min and 37 deg.C overnight, measuring bacterial count with spectrophotometer OD600nm, and adjusting concentration of lactobacillus solution to 2 × 10 7 cells/mL。
3. Bacteriostatic disc test
Taking 100 μ l of bacterial liquid with concentration of 2 × 10 7 coating a plate (broth agar culture medium) with cells/mL staphylococcus aureus liquid; after 15min of coating, uniformly punching holes on the bacterium plate by using a 9mm puncher, removing culture medium in the holes, and adding 200 mul of bacterium liquid to be detected into each hole.
4. Bacteriostatic ability calculation method
Bacteriostatic ability (AU/mL) (the diameter of bacteriostatic circle is obtained by measuring the amount of lactic acid strain-the diameter of bacteriostatic circle is obtained by measuring the amount of control group)/volume of bacteria liquid in the adding disc
5. The results are shown in Table 1:
TABLE 1 inhibitory Activity of ProfMIC-201 against Staphylococcus aureus
Group of Antibacterial circle (mm) Bacteriostatic ability (AU/mL)
MRS 9 -
ProfMIC-201 live bacterial liquid 22.3 66.67
ProfMIC-201 inactivated bacterial liquid 15.3 31.67
Both the ProfMIC-201 live bacteria liquid and the inactivated bacteria liquid have an inhibiting effect on staphylococcus aureus, and the live bacteria liquid has stronger bacteriostatic ability. The results of the bacteriostatic ability of ProfMIC-201 Staphylococcus aureus are shown in FIG. 1.
Example 4ProfMIC-201 inhibition of Staphylococcus aureus bacterial liquid concentration Change experiment
1. Preparing a lactobacillus salivarius ProfMIC-201 bacterial liquid:
culturing activated Lactobacillus salivarius ProfMIC-201 liquid with MRS in 37 deg.C incubator for 18 hr, measuring bacterial count with spectrophotometer OD600nm, and adjusting concentration of Lactobacillus salivarius liquid to 2 × 10 9 cells/mL, centrifuging and taking the supernatant to obtain the supernatant of the viable bacteria liquid(ii) a And boiling in water bath for 30min to obtain inactivated supernatant.
2. Preparation of staphylococcus aureus liquid:
culturing in broth culture at 140r/min and 37 deg.C overnight, measuring bacterial count with spectrophotometer OD600nm, and adjusting concentration of lactobacillus solution to 1 × 10 8 cells/mL。
3. Staphylococcus aureus inhibition experiment
Respectively adding live lactobacillus salivarius liquid and inactivated bacterial liquid supernatant into staphylococcus aureus liquid in an amount of 5 vol%, standing at 37 ℃ for 4h, and evaluating the influence of ProfMIC-201 on the growth of staphylococcus aureus by the percentage reduction of bacterial liquid concentration (OD 600).
4. The results are shown in Table 2
TABLE 2ProfMIC-201 Staphylococcus aureus inhibition
Group of Bacteriostatic ratio (%)
Supernatant fluid 77.2
Inactivating the supernatant 65.9
After 24 hours of reaction, the lactobacillus salivarius ProfMIC-201 has the effect of inhibiting staphylococcus aureus, the viable bacteria supernatant inhibition rate is 77.2%, and the inactivated supernatant inhibition rate is 65.9%. The results of the inhibition rate of ProfMIC-201 Staphylococcus aureus are shown in FIG. 2.
EXAMPLE 5ProfMIC-201 film formation inhibition assay for Staphylococcus aureus
1. Preparing a lactobacillus salivarius ProfMIC-201 bacterial liquid:
activating saliva milkThe bacterial liquid of the bacillus ProfMIC-201 is cultured by MRS and is kept still for 18 hours in an incubator at 37 ℃, the bacterial number is measured by using a spectrophotometer OD600nm, and the concentration of the lactobacillus salivarius liquid is adjusted to be 2 multiplied by 10 9 cells/mL, centrifuging to obtain supernatant, and filtering with 0.22 μm filter membrane to obtain supernatant.
2. Preparation of staphylococcus aureus liquid:
staphylococcus aureus was grown overnight to log-extended periods, diluted with broth to an OD600 of 0.1, and added to 96-well plates at 200 μ l per well.
3. Experiment for inhibiting formation of biofilm of staphylococcus aureus
Adding the supernatant of the lactobacillus salivarius ProfMIC-201 into a 96-well plate inoculated with staphylococcus aureus in an amount of 5%, adding an MRS culture medium to serve as a blank control, repeating the steps for three times for each sample at 37 ℃, incubating for 24 hours in a 50rmp shaking table, removing the supernatant, sterilizing 1 x PBS, cleaning for 3 times, adding a fixing solution for fixing, performing crystal violet staining, and detecting OD 600. The effect of ProfMIC-201 on s.aureus biofilm formation was evaluated as the biofilm formation inhibition rate.
Calculating the formula:
biofilm formation inhibition (%) (Ax-Ay)/Ax × 100%
Note: ax is the value measured at 600nm of the control group; ay is a value measured by an experimental group at 600 nm;
4. the results are shown in Table 3:
TABLE 3ProfMIC-201 inhibition of Staphylococcus aureus biofilm formation
Group of OD600 value Inhibition ratio (%)
control 0.801 -
ProfMIC-201 supernatant 0.232 71.04
After 24h of culture, the biofilm formation inhibition rate of the PROFMIC-201 supernatant was 71.04%. The results of the ProfMIC-201 inhibiting the formation of the biofilm by Staphylococcus aureus are shown in FIGS. 3 and 4.
Example 6ProfMIC-201 promotion of Staphylococcus aureus agglutination Studies
1. Preparing a lactobacillus salivarius ProfMIC-201 bacterial liquid:
inoculating activated Lactobacillus salivarius ProfMIC-201 bacterial liquid into MRS culture medium, culturing in 37 deg.C incubator for 18 hr, inactivating at 121 deg.C under high pressure for 30min, measuring bacterial count with spectrophotometer OD600nm, centrifuging at 4000rpm for 15min, discarding supernatant, washing thallus with sterile 1 × PBS buffer solution twice, dissolving in 1 × PBS buffer solution, adjusting concentration of lactobacillus to 5 × 10 9 cells/mL。
2. Preparation of staphylococcus aureus liquid:
shake culturing at 37 deg.C for 18h in broth culture medium, measuring bacterial liquid concentration with spectrophotometer OD600nm, centrifuging at 4000rpm for 15min, discarding supernatant, washing thallus with sterilized 1 × PBS buffer solution twice, re-dissolving with 1 × PBS phosphate buffer solution, and adjusting golden concentration to 2.5 × 10 9 cells/mL。
3. Co-agglutination: and (3) mixing the staphylococcus aureus with the adjusted concentration of the bacterial liquid and the lactobacillus salivarius ProfMIC-201 bacterial liquid prepared in the step (1) according to the ratio of 2: mixing at a volume ratio of 1, taking staphylococcus aureus and PBS as a control, shaking for 15s, and observing flocculation.
4. Measurement indexes are as follows: the solution of the single lactic acid bacteria, the single pathogenic bacteria and the lactic acid bacteria and pathogenic bacteria mixed reaction tube is sampled at different time, 50 mu L of the solution is put into a 96-well plate from the top to the bottom of the solution, and the value is measured at 600 nm.
Calculating the formula:
percent flocculation ability (%) [ (Ax + Ay) -2Amix ]/(Ax + Ay) × 100%;
note: ax is the value of single lactobacillus measured in the reaction time of 600 nm; ay is the value of the staphylococcus aureus measured in the reaction time of 600 nm; amix is the value measured at the reaction time of 600nm after the mixing action of the lactobacillus and the pathogenic bacteria.
5. The results are shown in Table 4
TABLE 4ProfMIC-201 Co-agglutinated Staphylococcus aureus
Group of OD600 value Coagulation rate (%)
ProfMIC-201 0.366 -
Staphylococcus aureus 0.434 -
ProfMIC-201+ Staphylococcus aureus 0.266 33.5
After 2 hours of reaction, the agglutination rate of the ProfMIC-201 strain is 33.56%, so that the inactivated strain of ProfMIC-201 has the function of agglutinating staphylococcus aureus. The results of ProfMIC-201 coaggregation of Staphylococcus aureus are shown in FIG. 5.
Example 7ProfMIC-201 Strain inhibition of pathogenic bacteria experiment
1. Preparing a lactobacillus salivarius ProfMIC-201 bacterial liquid:
culturing activated Lactobacillus salivarius ProfMIC-201 bacterial liquid with MRS in 37 deg.C incubator for 18 hr, detecting and adjusting OD 600 Inactivating at 121 deg.C for 30min, centrifuging to obtain supernatant, and filtering with 0.22 μm filter membrane to obtain sample to be tested.
2. Preparing a pathogenic bacterium liquid:
the 4 pathogenic bacteria: human staphylococcus CGMCC No.1.493, lysostaphin CGMCC No.1.540, streptococcus mutans CGMCC No.1.2499, pseudomonas aeruginosa CGMCC No.1.1783 are cultured in BHI culture medium at 37 ℃ for 18h, and OD is detected 600 Measuring the number of bacteria, and adjusting the concentration of pathogenic bacteria liquid to 1 × 10 8 cells/mL。
3. Experiment for inhibiting pathogenic bacteria
Adding the lactobacillus salivarius supernatant into the pathogenic bacteria by volume fraction of 5%, standing at 37 ℃ for 4h, and evaluating the influence of ProfMIC-201 on the growth of the pathogenic bacteria by the percentage reduction of the bacterial liquid concentration (OD 600).
4. The results are shown in Table 5:
TABLE 5ProfMIC-201 inhibition of pathogen inhibition
Name of pathogenic bacterium Bacteriostatic ratio (%)
Streptococcus mutans 43.1
Human staphylococcus 20.3
Hemolytic staphylococcus 23.4
Pseudomonas aeruginosa 19.7
After 24 hours of reaction, the ProfMIC-201 has stronger inhibiting effect on pathogenic bacteria such as streptococcus mutans, staphylococcus hominis, staphylococcus haemolyticus, pseudomonas aeruginosa and the like. The results of the inhibition rate of ProfMIC-201 pathogenic bacteria are shown in FIG. 6.
Example 8ProfMIC-201 experiment for promoting the growth of Staphylococcus epidermidis
Culturing activated Lactobacillus salivarius ProfMIC-201 in MRS liquid culture medium in 37 deg.C incubator, standing for 16-2018h, and detecting OD 600 Measuring the number of bacteria and adjusting OD 600 Inactivating at 121 deg.C for 30min, centrifuging to obtain supernatant, and filtering with 0.22 μm filter membrane to obtain inactivated supernatant.
Inactivation supernatant was added to the starting OD at 10% volume fraction 600 The influence of lactobacillus salivarius ProfMIC c-201 on the growth of staphylococcus epidermidis was evaluated by the relative bacterial liquid concentration (sample OD600 to control ratio) of 0.2 in the bacterial liquid of staphylococcus epidermidis (model strain CGMCC No.1.4260) at 37 ℃ for 2 h.
The results are shown in Table 6:
TABLE 6ProfMIC-201 promotion of Staphylococcus epidermidis growth
Group of OD600 Acceleration Rate (%)
control 0.89 -
ProfMIC-201 1.0725 20.5
Compared with the control, the growth of staphylococcus epidermidis is promoted by adding the supernatant of the lactobacillus salivarius ProfMIC-201, and the promotion rate is 20.5%.
Example 9ProfMIC-201 Strain reduction of LPS-induced NO Release amount in Raw264.7 cells
1. Lactobacillus salivarius sample preparation
Culturing Lactobacillus salivarius ProfMIC-201 with MRS overnight, detecting OD600, and adjusting OD 600 Autoclaving at 121 deg.C for 30min, centrifuging, filtering the supernatant with 0.22 μm filter membrane, and dissolving the precipitate with 1ml PBS to obtain inactivated thallus.
2. Cell preparation
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-201 addition and LPS stimulation
Test group 1: adding 5% (volume fraction) of ProfMIC-201 supernatant into the tested cells;
test group 2: 10% (mass fraction) of ProfMIC-201 inactivated cells were added to the test cells.
Adding the two groups of test substances into Raw264.7 cells cultured overnight, inducing Raw264.7 cells to be inflamed by 200ng LPS after 2h, taking cell culture supernatant after 20h, detecting the NO content by using an NO content detection kit, and carrying out three experiments in total, wherein 3 holes are formed in each time. The experiment used the LPS-induced raw264.7 cell inflammation model without the addition of test substance as a control to calculate the inhibition rate of NO production in test group 1 and test group 2.
4. The results are shown in Table 7:
TABLE 7ProfMIC-201 reduction of NO release in Raw264.7 cells
Group of NO inhibition (%)
ProfMIC-201 supernatant 13.70
ProfMIC-201 inactivated thallus 19.06
Compared with an LPS control group, the NO inhibition rate of the supernatant of the ProfMIC-201 is 13.7%; the NO inhibition rate of the inactivated thallus is 19.1 percent, and the supernatant and the inactivated thallus of the ProfMIC-201 strain both have the inhibition effect on NO released by Raw264.7 cells induced by LPS. The results of the decrease of the NO release amount of Raw264.7 cells by ProfMIC-201 are shown in FIG. 7.
Example 10ProfMIC-201 Strain downregulation of Staphylococcus aureus-induced HaCaT inflammatory factor expression
1. Lactobacillus salivarius sample preparation
Culturing Lactobacillus salivarius ProfMIC-201 with MRS overnight, detecting OD600, and adjusting OD 600 The cells were autoclaved at 121 ℃ for 30min at 0.2, the centrifuged supernatant was filtered through a 0.22 μm filter, and the precipitate obtained by centrifugation was dissolved back in 1ml of sterile 1 × PBS to obtain inactivated cells.
2. Cell preparation
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 into broth culture medium, shaking culturing at 37 deg.C overnight, and adjusting the concentration of the culture medium to 3 × 10 with MEM serum-free medium 9 cell/ml, adding 100 μ l per well into HaCaT cell cultured overnight to stimulate cell to produce inflammatory factor, discarding cell culture after 3 hrThe medium was washed 5 times with PBS, and 1ml of MEM serum-free medium was added to each well.
4. Lactobacillus salivarius sample addition
And adding lactobacillus salivarius ProfMIC-201 supernatant with volume fraction of 5% and inactivated thallus with mass fraction of 10% into HaCaT cells stimulated by staphylococcus aureus, and culturing in 3 multiple wells for overnight.
5. Detecting the expression level of inflammatory factors
And (2) removing a culture medium from the cells, extracting RNA by using an RNA extraction kit, detecting the concentration and purity of the RNA, adjusting the total amount of the RNA of all samples to 1000ng for reverse transcription, performing qPCR (quantitative polymerase chain reaction) on inflammation-related genes, and calculating expression change multiples according to a formula.
The formula: f is 2 -ΔΔCT
The results are shown in tables 8 to 9:
TABLE 8ProfMIC-201 supernatant downregulating expression of inflammation-associated factors
Inflammatory factors Relative fold expression of mRNA Inhibition ratio (%)
IL-8 0.864 13.65
COX-2 0.747 25.26
TABLE 9 expression of inflammation-associated factors by inactivation of ProfMIC-201
Inflammatory factors Relative fold expression of mRNA Inhibition ratio (%)
IL-8 0.535 46.5
COX-2 0.688 31.23
The supernatant and the inactivated thallus of the Lactobacillus salivarius ProfMIC-201 can reduce the expression quantity of HaCaT inflammatory factors IL-8 and COX-2 which are induced by staphylococcus aureus to be down-regulated, so that the ProfMIC-201 has an anti-inflammatory effect. The results of ProfMIC-201 down-regulating the expression of inflammation-related factors are shown in FIGS. 8 and 9.
Example 11ProfMIC-201 Strain promotion of HaCaT cell Damage repair experiment
Inoculation of HaCaT cells (5X 10) 5 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,
the experiment was divided into three groups:
the control group contained no component containing SEUNEU-105;
test group 1 was added with 5 vol% of inactivated supernatant of SEUNEU-105;
the test group 2 was added with 10 wt% of inactivated cells of SEUNEU-105.
Then each group was incubated for 24h with 5% supernatant. Adding 10 μ l CCK-8 solution, incubating for 4h, and detecting the absorbance at 450 nm.
Cell viability ═ cell viability (experimental group-a blank)/(negative control group a-a blank).
Results table 10:
TABLE 10ProfMIC-201 promotes HaCat cell repair
Group of Cell survival rate (%)
SDS 100.00
SDS + ProfMIC-201 supernatant 114.65
SDS + ProfMIC-201 inactivated thallus 109.41
The survival rate of SDS (sodium dodecyl sulfate) damage repair cells of the ProfMIC-201 supernatant is 114.65%, which is increased by 14.65% compared with a control; the survival rate of the cells damaged and repaired by the inactivated thallus SDS is 109.41%, the survival rate is increased by 9.41% compared with a control, and both the ProfMIC-201 supernatant and the inactivated thallus have the repairing effect on HaCaT cells damaged by the SDS. The results of ProfMIC-201 in promoting HaCaT cell repair are shown in FIG. 10.
Example 12ProfMIC-201 promotion of HaCaT cell Scoring experiments
Inoculation of human immortalized keratinocytes HaCaT (5X 10) 5 cell/well) to a 6-well plate, culturing overnight until the cells adhere to the wall, and marking the bottom of the 6-well plate by using a 1ml gun head to be vertical to the plate bottom;
the experiment was divided into three groups:
the control group did not contain the ProfMIC-201 component;
test group 1 was added 5 vol% of inactivation supernatant of ProfMIC-201;
test group 2 was added with 10 wt% of inactivated bacteria of ProfMIC-201.
Photographs were taken, recorded as D1, and incubated. The picture was taken every other day as D2. Using image J to process data, according to the formula: the healing rate is (D1-D2)/D1. Data were plotted using GraphPad.
Results table 11:
TABLE 11ProfMIC-201 promotes HaCaT cell scratch healing
Figure BDA0003705497620000151
Compared with a control, the healing rate of the scratches of the supernatant of the Lactobacillus salivarius ProfMIC-201 strain is increased by 22.72 percent, the growth rate of the inactivated thallus is increased by 9.14 percent, and the Lactobacillus salivarius ProfMIC-201 has the effect of promoting the healing of the scratches of HaCaT cells. ProfMIC-201 results for promoting HaCaT cell scratch healing are shown in FIGS. 11 and 12.
Example 13 Total antioxidant Capacity assay of ProfMIC-201 Strain
1. Preparation of supernatant of Lactobacillus salivarius ProfMIC-201: culturing activated Lactobacillus salivarius ProfMIC-201 bacterial liquid with MRS in 37 deg.C incubator for 18 hr, measuring bacterial count with spectrophotometer OD600nm, and adjusting concentration of Lactobacillus salivarius to 1 × 10 9 cells/mL, centrifugation, and high pressure inactivation of the supernatant and passage through a 0.22 μm filter to obtain a sterilized supernatant.
2. Preparing a total antioxidant capacity (T-AOC) detection kit
3. Reagent and ProfMIC-201 supernatant were added using a 96-well 200. mu.L system according to the kit instructions, and after 10min of reaction, absorbance was measured at 593nm using a microplate reader. And carrying out calculation according to the light absorption value and the standard vertebral curve to obtain an X value.
4. Total antioxidant capacity (μmol/mL) ═ X × V anti-total ÷ V-like ═ X × 34
V, reverse total: the total volume of the reaction was 1.02 mL; and V sample: sample volume in reaction, 0.03 mL.
5. Results table 12:
TABLE 12ProfMIC-201 Total antioxidant Capacity
Group of Antioxidant capacity (mu mol/mL) Increase ratio (%)
control 1.1 0
ProfMIC-201 1.7 54.5
The total antioxidant capacity of the supernatant of the Lactobacillus salivarius ProfMIC-201 determined according to the total antioxidant capacity detection kit is 1.70 mu mol/mL, which is improved by 54.5 percent compared with a control.
Example 14ProfMIC-201 free radical scavenging Capacity test
1. Preparing a lactobacillus salivarius ProfMIC-201 bacterial liquid:
culturing the activated Lactobacillus salivarius ProfMIC-201 bacterial liquid in MRS liquid culture medium in a 37 ℃ incubator for standing culture for 18h, detecting and adjusting OD 600 Inactivating at 121 deg.C for 30min, centrifuging to obtain supernatant, and filtering with 0.22 μm filter membrane to obtain sample to be tested.
2. Measurement of hydroxyl radical scavenging ability
The reagent preparation and detection method are carried out according to the instruction of the Solebao hydroxyl radical scavenging capability detection kit. The absorbance at 536nm was measured for each sample, averaged and the clearance for each sample calculated as follows:
hydroxyl radical clearance D% ([ (a assay-a control) ÷ (a blank-a control) ] × 100%)
ABTS radical scavenging Capacity test
The reagent preparation and detection method are carried out according to the instruction of the Solebao hydroxyl radical scavenging capability detection kit. The absorbance at 405nm of each sample was measured, averaged and the clearance rate for each sample calculated as follows:
ABTS free radical clearance D% ([ a blank- (a assay-a control) ] ÷ a blank × 100%
The results are shown in Table 13:
TABLE 13ProfMIC-201 radical clearance
Group of Clearance (%)
Hydroxy radical 15.40
ABTS free radical 12.91
The results show that ProfMIC-201 has the effect of eliminating hydroxyl free radicals and ABTS free radicals, so that PROFMIC-201 has the effect of resisting oxidation. The results of ProfMIC-201 scavenging free radicals are shown in FIG. 13.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims (10)

1. Lactobacillus salivarius with a preservation number of CCTCC NO: M20211389.
2. Application of Lactobacillus salivarius with a preservation number of CCTCC NO: M20211389 in preparing products for regulating flora.
3. The use according to claim 2, wherein said modulating the population comprises promoting the proliferation of beneficial bacteria and/or inhibiting harmful bacteria; the inhibiting harmful bacteria comprises inhibiting the growth of the harmful bacteria, inhibiting the coagulation of the harmful bacteria and/or inhibiting the harmful bacteria from forming a biofilm;
the harmful bacteria comprise at least one of staphylococcus aureus, streptococcus mutans, staphylococcus hominis, staphylococcus haemolyticus and/or pseudomonas aeruginosa;
the beneficial bacteria comprise staphylococcus epidermidis.
4. The application of the lactobacillus salivarius with the preservation number of CCTCC NO: M20211389 in preparing antioxidant products.
5. Use according to claim 4, wherein the antioxidant properties comprise scavenging hydroxyl radicals and/or ABTS radicals, increasing the total antioxidant capacity.
6. Application of Lactobacillus salivarius with a preservation number of CCTCC NO: M20211389 in preparing anti-inflammatory products.
7. The use according to claim 6, wherein the anti-inflammatory agent comprises a reduced NO release and/or down-regulated level of an inflammatory factor comprising at least one of IL-8, COX-2.
8. Application of Lactobacillus salivarius with a preservation number of CCTCC NO: M20211389 in preparation of products for repairing skin.
9. Use according to claim 8, wherein the repairing of the skin comprises repairing cell damage and/or repairing cell scarification; the cell is a keratinocyte.
10. A medicine and/or cosmetic is characterized in that the raw material comprises Lactobacillus salivarius with the preservation number of CCTCC NO: M20211389.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116103208A (en) * 2023-04-07 2023-05-12 西南科技大学 Application of lactobacillus salivarius in antioxidation

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116103208A (en) * 2023-04-07 2023-05-12 西南科技大学 Application of lactobacillus salivarius in antioxidation
CN116103208B (en) * 2023-04-07 2024-01-16 西南科技大学 Application of lactobacillus salivarius in antioxidation

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