CN117797181A - Application of Lactobacillus plantarum CCFM8661 in improving acne and facial redness - Google Patents
Application of Lactobacillus plantarum CCFM8661 in improving acne and facial redness Download PDFInfo
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- CN117797181A CN117797181A CN202311855765.9A CN202311855765A CN117797181A CN 117797181 A CN117797181 A CN 117797181A CN 202311855765 A CN202311855765 A CN 202311855765A CN 117797181 A CN117797181 A CN 117797181A
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- lactobacillus plantarum
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- acne
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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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
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
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Abstract
The invention discloses application of lactobacillus plantarum CCFM8661 in improving acne and facial redness, and belongs to the technical field of microorganisms. The invention applies the lactobacillus plantarum CCFM8661 to inhibit the growth of acne-related microorganisms, and respectively obtains the inhibition effects of 100%, 97%, 98%, 96%, 100% and 93% on escherichia coli, pseudomonas aeruginosa, candida albicans, propionibacterium acnes, staphylococcus aureus and staphylococcus epidermidis. In addition, the facial mask prepared by using the lactobacillus plantarum CCFM8661 can effectively improve acne and redness of the face.
Description
Technical Field
The invention relates to application of lactobacillus plantarum CCFM8661 in improving acne and facial redness, and belongs to the technical field of microorganisms.
Background
Acne, also known as acne, is a common skin disorder that occurs primarily during adolescence but may also occur in adults. The occurrence of acne is related to excessive secretion of sebaceous glands, blockage of follicular orifices, infection with propionibacterium acnes, and the like. Traditional acne treatment methods mainly comprise oral medicines and external medicines, but the methods can bring about side effects such as skin dryness, desquamation and the like.
In recent years, probiotics have been found to have a certain effect on improving acne. The probiotics can improve the micro-ecological environment of the skin by regulating the balance of intestinal flora, thereby reducing the occurrence of acne. In addition, probiotics can also reduce the occurrence of acne by inhibiting the growth of propionibacterium acnes. Based on these findings, related patents relating to the selection of probiotics, the preparation method of the probiotics, the acne treatment products containing the probiotics and the like are continuously emerging. For example, patent CN115181698A discloses a probiotic composition containing lactobacillus plantarum, which has a bacteriostasis rate on propionibacterium acnes of up to 90%. However, on the one hand, the occurrence of acne is not only related to the occurrence of propionibacterium acnes, but also has a close relationship with microbial infections such as pseudomonas aeruginosa, candida albicans, staphylococcus aureus, staphylococcus epidermidis, escherichia coli and the like; on the other hand, the inhibition effect on propionibacterium acnes and the improvement effect on acne in the prior art are still limited.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide application of lactobacillus plantarum (Lactobacillus plantarum) in reducing skin inflammation, improving facial health, and improving acne and facial redness.
The invention provides an application of lactobacillus plantarum (Lactobacillus plantarum) CCFM8661 or a progeny thereof in preparing a product for preventing and/or treating acne.
The lactobacillus plantarum (Lactobacillus plantarum) CCFM8661 is disclosed in the patent application publication No. CN 111869735A.
In one embodiment, the metazoan comprises a culture, a dried, an inactivated and/or a disrupted material of lactobacillus plantarum (Lactobacillus plantarum) CCFM 8661.
In one embodiment, the culture is a culture broth supernatant obtained by culturing lactobacillus plantarum (Lactobacillus plantarum) CCFM8661 with a fermentation medium.
In one embodiment, the dried product is powder prepared by drying lactobacillus plantarum (Lactobacillus plantarum) CCFM8661, and the drying method is not limited, and can be thermal drying, spray drying, vacuum freeze drying, fluidized bed drying or vacuum drying.
In one embodiment, the inactivated product is an inactivated cell obtained by inactivating lactobacillus plantarum (Lactobacillus plantarum) CCFM8661, and the inactivation method is not limited, and may be a heat treatment or a freeze-drying treatment.
In one embodiment, the disrupted material is a disrupted material obtained by disrupting lactobacillus plantarum (Lactobacillus plantarum) CCFM 8661.
In one embodiment, the product comprises a pharmaceutical, cosmetic or food product.
In one embodiment, the pharmaceutical product further comprises pharmaceutically acceptable excipients including, but not limited to, at least one of diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, co-solvents, solubilizing agents, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostats or buffers.
In one embodiment, the pharmaceutical product further comprises other drugs for preventing and/or treating acne, the other drugs being selected from at least one of retinoids (e.g., adapalene gel, tazarotene gel, isotretinoin, preferably isotretinoin), benzoyl peroxide, antibiotics (e.g., clindamycin, erythromycin, chloramphenicol, minocycline), azelaic acid, selenium disulfide, anti-androgens, or glucocorticoids.
In one embodiment, the medicine can be prepared into injection, oral liquid, tablet, capsule, dripping pill, spray, inhalant and other dosage forms by conventional methods.
In the present invention, the administration mode is not limited, and may be selected according to clinical actual needs. For example, administration may be by subcutaneous injection, intravenous injection, oral administration, application, respiratory inhalation administration, topical administration, sublingual administration, or the like.
In one embodiment, the formulation of the cosmetic is not limited, and may be formulated in the form of a solution, a topical ointment, a cream, a foam, a nutritional emollient, a soft emollient, a filler (pack), a soft water, a milky lotion, a cosmetic base, a fragrance, a soap, a liquid cleanser, a bathing agent, a sun block, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing cleanser, an oil, a foundation, an emulsion foundation, a wax foundation, a patch, and a spray.
In one embodiment, the cosmetic may further comprise at least one cosmetically acceptable carrier. The conventional components as cosmetically acceptable carriers are not limited thereto. For example, it may be oil, water, surfactant, humectant, lower alcohol, thickener, chelating agent, pigment, preservative, perfume.
In one embodiment, the food product includes, but is not limited to, a nutraceutical.
In one embodiment, the health food comprises a solid food, a liquid food, or a semi-solid food.
In one embodiment, the health food comprises a dairy product, a soy product, or a fruit and vegetable product.
In one embodiment, the dairy product comprises a fermented dairy product, a milk-containing beverage, or a milk powder.
In one embodiment, the soy product comprises soy milk, soy milk drink, or soy milk powder.
In one embodiment, the fruit and vegetable product comprises beet, cabbage, carrot, white radish, kelp, cucumber, yellow peach, litchi or waxberry product.
In one embodiment, the content of lactobacillus plantarum (Lactobacillus plantarum) CCFM8661 in the product is not less than 1×10 8 CFU/g。
The beneficial effects are that:
(1) Lactobacillus plantarum CCFM8661 is added at 1mL concentration of 1×10 9 When CFU/mL bacterial suspension is used, the clearance rate of DPPH free radical is 63.75% after 30min of reaction; lactobacillus plantarum CCFM8661 is added at a concentration of 200uL and 1×10 9 When CFU/mL bacterial suspension is adopted, the clearance rate of the hydroxyl radical reaches 66.34 percent after water bath at 37 ℃ for 15 min.
(2) The addition amount of the lactobacillus plantarum CCFM8661 is 1 multiplied by 10 of the final concentration of the thalli 9 The CFU/mL has 100%, 97%, 98%, 96%, 100% and 93% inhibitory effects on Escherichia coli, pseudomonas aeruginosa, candida albicans, propionibacterium acnes, staphylococcus aureus and Staphylococcus epidermidis, respectively.
(3) The facial mask prepared from Lactobacillus plantarum CCFM8661 as active ingredient has obvious improvement on acne and skin redness, and when Lactobacillus plantarum CCFM8661 bacterial suspension (bacterial concentration is 1×10) 9 When the adding amount of CFU/mL is more than 9% (wt%), the effective rate reaches 100%, and the obvious effective rate and the recovery rate reach 90%.
Detailed Description
The invention is not limited to the examples, which are given solely by way of illustration and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The preservation number of the lactobacillus plantarum (Lactobacillus plantarum) CCFM8661 is CGMCC No:5494, disclosed in the publication CN111869735 a.
Example 1: tolerance experiment of Lactobacillus plantarum CCFM8661 on artificial gastric juice and artificial intestinal juice
1. Tolerance test of gastric juice
Preparing artificial gastric juice: diluted hydrochloric acid with the concentration of 1mol/L is taken, water is added for dilution, the pH is respectively adjusted to 1.5,2.0,2.5,3.5, 1g of pepsin is added into each 100mL of liquid, the mixture is uniformly mixed, and a sterile filter head with the concentration of 0.2um is used for filtration.
The experimental method comprises the following steps: lactobacillus plantarum CCFM8661 is respectively inoculated into the artificial gastric juice with different pH values, and is cultivated in a constant-temperature water bath at 37 ℃ to simulate the temperature of a human body, and viable bacteria plate count is carried out at 0,0.5,1,1.5,2 and 3 hours. The experimental results are shown in table 1.
TABLE 1 Experimental results (CFU/mL) of the tolerance of Lactobacillus plantarum CCFM8661 to artificial gastric juice at different pH values
2. Tolerance test of artificial intestinal juice
Preparing artificial intestinal juice: 6.8g of dipotassium hydrogen phosphate is taken, 500mL of water is added for dissolution, the pH is adjusted to 6.8 by using sodium hydroxide solution with the mass fraction of 0.4%, and the mixture is diluted to 1000mL by adding water. Then 1g trypsin is added before use, the mixture is evenly dissolved by shaking, and the mixture is placed in a water bath shaker at 37 ℃ for warm water bath for 1h so as to simulate the temperature of a human body.
The experimental method comprises the following steps: lactobacillus plantarum CCFM8661 is inoculated into artificial intestinal juice, and is cultivated in a constant-temperature water bath at 37 ℃ to simulate the temperature of a human body, and viable bacteria plate counts are carried out at 0,0.5,1,1.5,2 and 3 hours. The experimental results are shown in table 2.
TABLE 2 Experimental results (CFU/mL) of Lactobacillus plantarum CCFM8661 tolerating artificial intestinal juice
Experimental results show that lactobacillus plantarum CCFM8661 cannot survive in an environment below ph 2.0; at pH above 2.0, the artificial gastric juice has little influence on the lactobacillus plantarum CCFM 8661; the artificial intestinal juice has almost no influence on lactobacillus plantarum CCFM 8661. The lactobacillus plantarum CCFM8661 is proved to have strong tolerance to artificial gastrointestinal fluids.
Example 2: measurement of antioxidant function of Lactobacillus plantarum CCFM8661
Enterococcus faecium CCFM1106 used in the present example is disclosed in patent CN111088184A, enterococcus faecium CCFM2002 is disclosed in literature (screening and identification of lactobacillus having excellent in vitro antioxidant ability, wang Gang, etc.), and lactobacillus plantarum ZH008 is disclosed in patent CN 1928071A.
1. Test of the tolerance of Lactobacillus plantarum CCFM8661 to different initial Hydrogen peroxide concentrations
Hydrogen peroxide is a relatively weak oxidant, has high diffusivity and long action time, and can directly cause oxidative damage to the body. Thus, the tolerance of lactobacillus plantarum CCFM8661 to different initial hydrogen peroxide concentrations is an in vitro evaluation index of its antioxidant capacity.
60mL of MRS culture medium is added into a 100mL sterilized triangular flask, hydrogen peroxide is added, the initial hydrogen peroxide concentration in the culture medium is respectively 0,0.5, 1.0, 1.5 and 2.0mmol/L, lactobacillus plantarum CCFM8661 and other control strains (CCFM 1106, CCFM2002 are enterococcus faecium and ZH008 are lactobacillus plantarum) are inoculated according to the inoculation amount of 1% by volume, and the culture medium is placed in a constant temperature incubator at 37 ℃ for culturing for 24 hours, and the growth conditions under different initial hydrogen peroxide concentrations are observed, and the results are shown in Table 3. It can be seen that Lactobacillus plantarum CCFM8661 is still able to grow when the hydrogen peroxide concentration reaches 1.5mmol/L, whereas Lactobacillus plantarum ZH008 is unable to grow at a hydrogen peroxide concentration of 1.0 mmol/L.
TABLE 3 viability of different lactic acid bacteria in hydrogen peroxide solutions of different concentrations (%)
2. Test of DPPH free radical scavenging ability of Lactobacillus plantarum CCFM8661
1mL of PBS bacterial suspension of Lactobacillus plantarum CCFM8661 (bacterial concentration 1X 10) 9 CFU/mL), 1mL of 0.4mM of the ready-prepared DPPH radical solution was added, and after mixing uniformly, the mixture was allowed to stand at room temperature for shading reaction for 30min, and then the absorbance of the sample at 517nm was measured. And taking an equal volume of PBS solution and DPPH-ethanol mixed solution as a control group, and performing blank zeroing by taking the equal volume of PBS bacterial suspension and ethanol mixed solution. DPPH radical scavenging was calculated according to the following formula:
clearance% = [1- (a) Sample of -A Blank space )/A Control ]*100％
The test results of the ability of Lactobacillus plantarum CCFM8661 to scavenge DPPH free radicals are shown in Table 4.
TABLE 4 DPPH radical scavenging Table of Lactobacillus plantarum CCFM8661
3. Test of the ability of Lactobacillus plantarum CCFM8661 to scavenge OH free radicals
100uL of sodium salicylate-ethanol solution with a concentration of 5mM, 100uL of ferrous sulfate with a concentration of 5mM, 500uL of deionized water and 200uL of Lactobacillus plantarum CCFM8661 bacterial suspension (bacterial cell concentration of 1X 10) 9 CFU/mL) was mixed, 100uL of a hydrogen peroxide solution having a concentration of 3mM was added thereto, and the absorbance of the sample at 510nm was measured after 15 minutes in a water bath at 37 ℃. The hydroxyl radical scavenging rate was calculated according to the following formula:
clearance% = (a Sample of -A Control of )/(A Blank space -A Control of )*100％
Wherein A is Control of For deionized water to replace the sample, A Blank space Substitution of deionized water for sample and H 2 O 2 Each set of experiments was set up in 3 replicates. The results of the test for the ability of Lactobacillus plantarum CCFM8661 to scavenge hydroxyl radicals are shown in Table 5.
TABLE 5 Lactobacillus plantarum CCFM8661 hydroxyl radical clearance Table
Example 3: cytotoxicity test of lactobacillus plantarum CCFM8661
Human sebaceous gland cell SZ95 was inoculated into 96 well plates with a cell density of 3 x 10 4 cell/ml. After culturing at 37℃under 5% carbon dioxide for 24 hours, the supernatant was removed. Grouping was according to the experimental protocol. Experimental group: 100uL of 1X 10 concentration was added to each well 9 CFU/mL of the Lactobacillus plantarum CCFM8661 bacterial suspension; control group: 100uL of DMEM (serum free) medium is added to each well. After culturing at 37℃for 24 hours in 5% CO, respectively, the supernatant was aspirated, 100uL of DMEM (serum-free) medium containing 5mg/mL MTT solution was added, after incubation at 37℃for 4 hours, MTT solution was removed, 100uL of DMSO was added, shaking at a low speed in a dark place for 10 minutes, absorbance of the culture was measured at 490nm using an enzyme-labeled instrument, and relative cell viability was calculated according to the following formula.
Cell relative survival% = a n /A 0 *100％
Wherein An is the absorbance of the experimental group, A 0 Absorbance was taken as control.
According to the toxicity evaluation standard described in International Standard ISO 10993-5:2009, the cell viability is defined as non-cytotoxicity when the cell viability is higher than 70%, and the result shows that the cell viability of the lactobacillus plantarum CCFM8661 bacterial suspension is 90.36%, which indicates that the lactobacillus plantarum CCFM8661 is non-cytotoxic and can be applied to skin coating preparations.
Example 4: antibacterial effect test of lactobacillus plantarum CCFM8661
1. Evaluation method
The most common 6 bacterial bacteriostatic efficacy evaluations that lead to acne growth are shown in table 6. Efficacy evaluation is divided into three stages: the bacterial inhibition rate of various strains shown in the table 6 reaches more than or equal to 90%, and the bacterial inhibition rate is judged to be capable of remarkably and effectively preventing and inhibiting the generation of acne; when the bacterial inhibition rate of various strains shown in the table 6 reaches more than or equal to 50% -90%, the bacterial inhibition rate is judged to be capable of effectively preventing and inhibiting the generation of acnes; when the bacterial inhibition rate of each strain shown in Table 6 is less than 50%, the bacterial inhibition rate is judged to be invalid.
Table 6 antibacterial efficacy evaluation chart
The most common 6 bacteria causing acne growth in Table 6 were used as pathogenic bacteria, and after the pathogenic bacteria were cultured in respective liquid media to the logarithmic growth phase (E.coli LB liquid medium, pseudomonas aeruginosa LB liquid medium, candida albicans YPD liquid medium, propionibacterium acnes BHI liquid medium, staphylococcus aureus LB liquid medium, staphylococcus epidermidis LB liquid medium), respectively, each of the pathogenic bacteria was inoculated into the corresponding culture medium to which the active material was added in an inoculum amount of 1% by volume. Wherein the final concentration of the active substance is 1×10 9 CFU/mL of Lactobacillus plantarum CCFM8661 bacterial suspension (experimental group) and clindamycin (positive control group) with final concentration of 100 mug/mL were used as negative control, the culture medium without adding active substances, the sample adding information is shown in Table 7,
TABLE 7 sample loading information table
| Group of | Sample addition (50 uL) | Final concentration |
| Negative control | Liquid culture medium | / |
| Positive control | Clindamycin | 100μg/ml |
| Experimental group | Lactobacillus plantarum CCFM8661 bacterial suspension | 1×10 9 CFU/mL |
After sample addition, the gun head is blown to resuspend after 16 hours of culture at 37 ℃, and the absorbance value is measured at 600nm of the enzyme label instrument (the absorbance value of the bacterial suspension at 600nm can reflect the number of bacteria to a certain extent, and the larger the absorbance value, the higher the concentration of bacteria). The bacteriostasis rate was calculated according to the following formula:
antibacterial ratio (%) = (OD A1 -OD A2 )/(OD A1 -OD A0 )×100。
OD A0 To add the OD value before the culture of the corresponding culture medium, the OD A1 To add OD value and OD after 16h of culture in the corresponding culture medium A2 OD values after 16h incubation after addition of Lactobacillus plantarum CCFM8661 bacterial suspension or positive control. The bacteriostasis rate of the lactobacillus plantarum CCFM8661 bacterial suspension on the 6 most common bacteria causing acne growth is experimentally measured as shown in table 8.
TABLE 8 results of antibacterial rate
The results show that the lactobacillus plantarum CCFM8661 bacterial suspension has obvious inhibition effects on escherichia coli (E.coli), pseudomonas aeruginosa (P.aeromonas), candida albicans (C.Albicans), propionibacterium acnes (P.acnes), staphylococcus aureus (S.aureus) and staphylococcus epidermidis (S.epideris), wherein the effects on pseudomonas aeruginosa (P.aeromonas) and propionibacterium acnes (P.acnes) are obviously better than those of positive control medicines clindamycin.
Example 5: human body test of lactobacillus plantarum CCFM8661 cosmetics
The mask was prepared according to the formulation of table 9 in a conventional manner. The concentration of thalli in the lactobacillus plantarum CCFM8661 bacterial suspension is 1 multiplied by 10 9 CFU/mL。
Table 9 mask formulation table
120 volunteers with obvious acne and skin redness, of which the ages of 18-25 years are respectively selected, 30 volunteers are respectively tested with the facial mask with the formula, 1 tablet is used every night at week 1,1 tablet is used every 3 days later, no other cosmetics for treating the acne and the skin redness are used in the period, and after 30 days of continuous test, the effect is observed. The face portion was divided into 5 regions using skin loss counting. I.e., forehead, left cheek, right cheek, nose and mandible, the number of each type of skin lesions (open and unopened acne, including comedo, papule, pustule, nodule, redness) in each region was recorded separately. Wherein: the skin damage is basically cured after 90 percent of the skin damage is resolved, 60 to 89 percent of the skin damage is resolved, 30 to 59 percent of the skin damage is resolved, and less than 30 percent of the skin damage is resolved, and the skin damage is resolved. The test results are shown in Table 10.
Table 10 mask test results table
| Group of | Healing of the wound | Has obvious effect | Effective and effective | Invalidation of |
| Formulation 1 | 10％ | 30％ | 30％ | 30％ |
| Formulation 2 | 20％ | 50％ | 20％ | 10％ |
| Formulation 3 | 20％ | 60％ | 20％ | 0％ |
| Formulation 4 | 30％ | 60％ | 10％ | 0％ |
As can be seen from Table 10, the Lactobacillus plantarum CCFM8661 bacterial suspension has good effects of treating acne and skin redness. At a content of 3% (wt%) of the lactobacillus plantarum CCFM8661 bacterial suspension, the acne and facial redness healed and a significant effective proportion was 40%. When the content of the lactobacillus plantarum CCFM8661 bacterial suspension reaches 5 percent (wt%), the significant and effective proportion of acne and facial redness is over 70 percent. When the content of the lactobacillus plantarum CCFM8661 bacterial suspension reaches 9 percent (wt%) or more, the significant and effective proportion of acne and facial redness is 90 percent or more.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. Use of lactobacillus plantarum (Lactobacillus plantarum) CCFM8661 or its progeny in the manufacture of a product for the prevention and/or treatment of acne.
2. Use according to claim 1, wherein the metazoan comprises a culture, a dried, an inactivated and/or a disrupted material of lactobacillus plantarum (Lactobacil lus plantarum) CCFM 8661.
3. The use according to claim 1, wherein the product comprises a pharmaceutical product, a cosmetic product or a health food product.
4. The use according to claim 3, wherein the pharmaceutical product further comprises pharmaceutically acceptable excipients, wherein the excipients comprise at least one of diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, co-solvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostats or buffers.
5. The use according to claim 3, wherein the pharmaceutical product comprises a dosage form selected from the group consisting of an injection, an oral liquid, a tablet, a capsule, a drop pill, a spray and an inhalant.
6. The use according to claim 3, wherein the pharmaceutical product further comprises other drugs for preventing and/or treating acne, said other drugs being selected from at least one of retinoids, benzoyl peroxide, antibiotics, azelaic acid, selenium sulphide, anti-androgens or glucocorticoids.
7. The use according to claim 3, wherein the cosmetic formulation comprises at least one of a solution, a topical ointment, a cream, a foam, a nutritional emollient, a soft emollient, a filler, a soft water, a milky lotion, a cosmetic base, a fragrance, a soap, a liquid cleanser, a body wash, a sunscreen, a suntan lotion, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a soap, a surfactant containing cleanser, an oil, a foundation, an emulsion foundation, a wax foundation, a patch, or a spray.
8. The use according to claim 3, wherein the cosmetic product further comprises at least one cosmetically acceptable carrier comprising oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives or fragrances.
9. Use according to claim 3, wherein the health food comprises dairy, soy or fruit and vegetable products.
10. The use according to any one of claims 1 to 9, wherein the content of lactobacillus plantarum (Lactob acillus plantarum) CCFM8661 in the product is not less than 1 x 10 8 CFU/g。
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