CN118222461B - Saliva combined lactobacillus Lumine-102 with whitening and antioxidation effects and application thereof - Google Patents
Saliva combined lactobacillus Lumine-102 with whitening and antioxidation effects and application thereof Download PDFInfo
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
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- 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
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- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
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- Gerontology & Geriatric Medicine (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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Abstract
The invention provides saliva combined lactobacillus Lumine-102 with whitening and antioxidation effects and application thereof, and relates to the technical field of microorganisms and application thereof. The saliva combined lactobacillus Lumine-102 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.29489. The saliva combined lactobacillus Lumine-102 provided by the invention can effectively inhibit tyrosinase activity under in vitro and in vivo conditions, and the Lumin-102 strain also has excellent hydroxyl radical scavenging capacity and higher reducing power, and shows strong antioxidation. Therefore, the saliva combined lactobacillus Lumine-102 strain has wide market application prospect in the aspect of preparing skin care products such as whitening, antioxidation, anti-aging and the like.
Description
Technical Field
The invention relates to the technical field of microorganisms and application thereof, in particular to saliva combined lactobacillus (Ligilactobacillus salivarius) Lumine-102 with melanin formation inhibiting and antioxidant activities and application thereof.
Background
Melanin is a pigment produced by melanocytes in the skin, which determines the color of the skin. Skin melanin formation is a complex biochemical process, tyrosine is a precursor of melanin formation, and when skin is stimulated by hormones, neurotransmitters, cytokines, and external stimuli (such as ultraviolet radiation), tyrosinase converts tyrosine to dopa, which in turn produces dopaquinone. As a key intermediate in the melanogenesis process, dopaquinone is oxidized by dopaquinase to form melanin precursors, ultimately leading to the formation of melanin. Excessive melanin production can lead to problems of skin pigmentation, spots, etc., such as freckles, sunburn, chloasma, etc. Thus, inhibiting tyrosinase activity can effectively reduce melanin production. In addition, oxidative stress is also considered as a factor that stimulates melanogenesis, and free radicals having strong oxidizing properties are generated during metabolism of the body, resulting in skin aging and darkness. The antioxidant activity of the whitening product is expressed as the capability of scavenging free radicals in the body, and the whitening product can play a role in repairing oxidative damage, thereby achieving the effects of whitening and resisting aging. At present, the method for evaluating the efficacy of the whitening product mainly carries out analysis and evaluation on the tyrosinase activity inhibition and antioxidation capability of the whitening product.
Probiotics are a group of microorganisms that are beneficial to human health and are commonly used as modulators of the digestive system to help maintain the balance of intestinal flora. Recent studies have shown that probiotics may also have some inhibitory effect on melanogenesis.
Several studies have shown that probiotics and their metabolites have melanogenesis inhibiting and antioxidant activity. They can neutralize free radicals and reduce damage to the skin from oxidative stress. Probiotics can also affect melanogenesis by affecting the activities of the neuroendocrine system, regulating melanogenesis-related hormone levels, such as melanogenesis-promoting hormone (MSH) and cortisol, and the like. Compared with the existing whitening products, the probiotic bacteria have the defects of unstable effect, stimulation to skin, side effect and the like, are taken as natural antioxidant substances, are mild in effect, safe and nontoxic, and have better antioxidant and whitening capabilities. Therefore, searching and identifying probiotics with melanin generation inhibition and high antioxidant activity provides a brand new thought for developing related products of whitening and skin care, and has huge market and application prospects.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a salivary lactobacillus (Ligilactobacillus salivarius) Lumine-102 with melanin formation inhibiting and high oxidation resistance effects and application thereof.
In one aspect, the invention provides a saliva combined lactobacillus (Ligilactobacillus salivarius) strain Lumine-102 which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 29489.
In one aspect, the invention provides the use of the saliva in combination with lactobacillus Lumine-102 or a culture supernatant or dead cells thereof to inhibit melanin production.
In one aspect, the invention provides the use of said Lactobacillus salivarius Lumine-102, or a culture supernatant or dead cells thereof, to inhibit tyrosinase activity.
In one aspect, the invention provides the use of the saliva in combination with lactobacillus Lumine-102 dead cells in the preparation of a skin care product.
In another aspect, the invention provides a microbial inoculum comprising said Lactobacillus salivarius Lumine-102 or a culture supernatant or dead cells thereof. Preferably, the composition is a solid preparation or a liquid preparation. More preferably, the solid microbial inoculum is powder prepared by a freeze drying method.
In another aspect, the invention provides a cosmetic comprising said saliva in combination with lactobacillus Lumine-102 dead cells. Preferably, the cosmetic is a skin care product.
In another aspect, the present invention provides a method of preparing a product composition, the method comprising: adding the bacterial substance and/or metabolite of lactobacillus salivarius Lumine-102 in combination with claim 1 to a product substrate to obtain the product composition.
Biological sample preservation information: the Lactobacillus salivarius (Ligilactobacillus salivarius) strain Lumine-102, which has been deposited with the China general microbiological culture Collection center, with the accession number: CGMCC No. 29489; preservation address: the institute of microbiology, academy of sciences, china, no. 3, north Star West way 1, the Korean area of Beijing, china, postal code 100101.
The Lumine-102 strain can obviously inhibit tyrosinase activity, and in addition, the Lumine-102 strain also has higher free radical scavenging capacity and total reducing power, and shows antioxidant activity. Therefore, the strain has the potential of inhibiting melanin generation and being applied to whitening products.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention.
FIG. 1 is a gram of the saliva of the present invention in combination with Lactobacillus Lumine-102.
FIG. 2 is a genomic circle map of Lactobacillus salivarius Lumine-102 in the present invention.
FIG. 3 shows the effect of culture supernatant of Lactobacillus salivarius Lumine-102 on tyrosinase activity in accordance with the present invention. (A) In vitro tyrosinase activity inhibition rate, kojic acid as positive control, n=3; (B) Inhibition of tyrosinase activity in vivo xanthine oxidase in vitro inhibition, arbutin as positive control, n=3. * p <0.05, p <0.01, p <0.001.
The graph shows the clearance of hydroxyl radicals from the supernatant of the culture of Lactobacillus salivarius Lumine-102 in combination with the present invention. Vc as positive control, n=3. * p <0.05, p <0.01, p <0.001.
FIG. 5 shows the total reducing power test of Lactobacillus salivarius Lumine-102 in the present invention. Vc as positive control, n=3. * p <0.05, p <0.01, p <0.001.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. In the following examples and comparative examples, the experimental methods used were conventional methods unless otherwise specified, and the materials, reagents and the like used, unless otherwise specified, were all commercially available.
According to the invention, saliva combined lactobacillus strain Lumine-102 is separated from the feces of the people living in the centella and middle-aged in Hainan province for a long time, and experiments prove that the Lumine-102 strain not only has the capability of inhibiting tyrosinase activity in vitro and in vivo, but also has higher antioxidant activity, and the strain has the potential of inhibiting melanin generation.
Example 1 cultivation and preparation of Lactobacillus salivarius Lumine-102 Strain
(1) Identification of strains
The saliva combined lactobacillus Lumine-102 strain is separated from fecal samples of Hainan-Cheng-Bainian old people, and the target people do not take antibiotics before collection, so that the oral history of probiotics and the gastrointestinal history are avoided. The collected fecal samples are diluted and coated on MRS solid culture medium (peptone 10g, beef extract 10g, yeast extract 5g, diammonium citrate 2g, glucose 20 g, tween 80 mL, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, agar 18g and distilled water 1000 mL), anaerobic culture is carried out at 37 ℃ for 24-48 h, different single colonies on a plate are obtained after separation, each single colony is respectively streaked on a new MRS solid culture medium plate, purified colonies are obtained after anaerobic culture at 37 ℃ for 48 h, and each purified single colony is stored in a refrigerator at-80 ℃ after subsequent identification for later use.
Among them, gram staining results (FIG. 1) of Lumine-102 strain according to the present invention show that Lactobacillus salivarius Lumine-102 according to the present invention is a gram positive bacterium.
According to sequencing identification, the analysis result shows that the FastANI value of the Lumine-102 strain and the reference genome of the Lactobacillus salivarius is 99.15, and the similarity is extremely high, so that the Lumine-102 strain is named as Lactobacillus salivarius Lumine-102. Further, lumine-102 was subjected to genome assembly and Binning (Binning), and the resulting genomic sequence. Gene annotation of the genome was performed using Prokka (https:// gitsub.com/tseemann/prokka), and finally a genomic circle map of Lumine-102 was drawn using cgview (https:// cgview.ca /). The results showed Lumine-102 genome size 1.96Mb, a circular chromosome containing 1,957,224 base pairs with 1898 CDSs, 27 tRNAs, 4 rRNAs, 1 tmRNAs, 48 misc RNAs.
(2) Strain culture
When the strain is activated and cultured, saliva stored in a refrigerator at the temperature of minus 80 ℃ is inoculated into fresh MRS liquid culture medium (agar is not added in the formula of the solid culture medium) under aseptic operation condition, the strain is cultured overnight in a 37 ℃ hypoxia workstation, and bacterial liquid after 3 generations of activation is used as seed liquid for culture, and the final concentration is 5 multiplied by 10 9 CFU/mL. The seed liquid is inoculated into fresh MRS liquid culture medium with the inoculation amount of 4 percent of volume fraction, and the seed liquid can be used as strain working liquid after being cultured in a constant temperature incubator at 37 ℃ for 18 h.
(3) Strain sample preparation
The resulting working strain mixture was centrifuged at 12000 r/min for 10: 10min at 4℃and the supernatant and strain pellet were collected, respectively. The supernatant was taken and filtered off with a 0.22 μm filter membrane to obtain a culture supernatant of Lactobacillus salivarius Lumine-102 for subsequent experiments, and the samples were kept at 4℃for further use.
Example 2 Effect of saliva on tyrosinase activity by Lactobacillus bifidus Lumine-102
This example is a graph showing the inhibitory effect of Lactobacillus salivarius Lumine-102 of the present invention on tyrosinase activity.
Since tyrosinase is a key enzyme for melanin production, the present practice evaluates the inhibition of tyrosinase activity by saliva in combination with lactobacillus Lumine-102 culture supernatants through in vitro and in vivo enzyme activity inhibition experiments.
(1) In vitro tyrosinase activity inhibition assay
The reaction system was prepared as in Table 1 below, specifically, each set of samples was placed in an EP tube of 1.5 mL (the reaction system was prepared as 350. Mu.L) using a micropipette, and the remaining volume was made up with PBS, 100. Mu.g/mL kojic acid was used as a positive control. After the reaction system was uniformly mixed, incubation was performed at 37℃in the absence of light for 10 min. Mu.L, and then 100. Mu.L of the mixed solution was transferred to a 96-well plate, 3 parallel plates were set, and absorbance at 490, 490 nm was read with an ELISA reader. Wherein the method comprises the steps of
Sample group tyrosinase inhibition rate (%) = [ 1- (OD Sample of -OD sample blank )/(OD Negative control - OD Negative blank ) ] ×100 (%);
Positive group tyrosinase inhibition rate (%) = [ 1- (OD Kojic acid positive control - OD Kojic acid blank control )/(OD Kojic acid negative control - OD Kojic acid negative blank ) ]x100 (%).
TABLE 1 in vitro tyrosinase activity inhibition experiment system
The experimental result is shown in fig. 3A, under the in vitro reaction condition, the inhibition rate of positive control kojic acid on tyrosinase activity is 42.89%, the average value of the inhibition rate of Lumine-102 strain culture supernatant in the invention is 52.84%, and the inhibition rate is obviously higher than that of positive control kojic acid, which indicates that the saliva combined lactobacillus Lumine-102 can effectively inhibit tyrosinase activity.
(2) In vivo tyrosinase activity inhibition assay
Mouse melanoma cells B16F10 in the logarithmic growth phase were inoculated into 96-well plates, 100. Mu.L of cell suspension was added to each well, and the cells were incubated at 4X 10 4 cells/well, 37℃and 5% CO 2 in a constant temperature incubator for 24. 24 h. And when the cell growth is converged to 70% -80%, discarding the supernatant. Each set of samples was removed using a micropipette and placed in 96-well plates (100 μl of reaction system was prepared per well) and mixed well. After incubation of the supernatant with cells for 24 h hours, the medium was discarded, washed twice with PBS, 50. Mu.L of 1% Triton X-100 solution was added, and the cells were disrupted by shaking 5min on a shaker. After shaking, 50 mu L of 10mM L-dopa is added into each hole, the reaction is carried out at 37 ℃ in a dark place for 30min, and the absorbance is measured at a position of a microplate reader 490 nm. Each set was set up in 3 replicates and the experiment was repeated 3 times. The positive control group was 1mM arbutin solution.
Tyrosinase inhibition rate (%) = [ (OD Negative control - OD Sample of )/OD Negative control ] ×100 (%) of the sample group to be tested;
positive control tyrosinase inhibition ratio (%) = [ (OD Blank control - OD Positive control )/OD Blank control ] ×100 (%)).
The experimental results of this example show that the inhibition rate of the culture supernatant of the saliva combined lactobacillus Lumine-102 on tyrosinase activity in B16F10 cells is 85.98%, the inhibition rate of the positive control arbutin is 72.24%, and the effect of the Lumine-102 strain is obviously higher than that of arbutin (figure 3B).
According to in-vivo and in-vitro tyrosinase activity inhibition experiments, the saliva combined lactobacillus Lumine-102 disclosed by the invention can effectively inhibit the activity of tyrosinase, is beneficial to reducing melanin generation, and has the potential of being developed into a whitening related product.
Example 3 determination of the hydroxy radical scavenging ability of Lactobacillus salivarius Lumine-102 in combination
This example illustrates the scavenging efficacy of Lactobacillus salivarius Lumine-102 on free radicals.
The sample solutions of each group were transferred to an EP tube (190. Mu.L of the prepared reaction system) of 1.5 mL using a micropipette according to Table 2, and after mixing uniformly, reacted in a water bath at 37℃for 30 min. 50 μl of the mixed solution was added to each well of the 96-well plate, 3 replicates were set for each group, and absorbance values were measured at 510 nm. The positive control group used vitamin C (Vc) and the concentration was set at 2 mg/mL.
TABLE 2 hydroxyl radical scavenging ability detection experiment System
Sample hydroxyl radical clearance= [ 1- (OD Experimental group - OD Sample blank ）/（OD Negative control group -OD Negative blank ) ] ×100 (%);
vc hydroxyl radical clearance= [ 1- (OD Vc-ODVc Blank control group ）/（ODVc Negative control group -ODVc Negative blank ) ] ×100 (%);
According to the graph shown in FIG. 4, the supernatant sample of the saliva combined lactobacillus Lumine-102 can efficiently remove free radicals, the removal rate can reach 74.72 percent, and the removal rate is higher than the removal rate (63.02 percent) of positive group vitamin C (Vc). Since oxidative stress is also a factor that stimulates melanogenesis, free radicals having strong oxidizing properties are generated during the metabolism of the body, resulting in skin aging and darkness. The excellent hydroxyl free radical scavenging ability helps to relieve oxidative stress, helps to resist skin aging and reduces melanin generation, so that the saliva combined lactobacillus Lumine-102 has the effect of inhibiting melanin generation, can also help to scavenge free radicals and resists oxidative stress.
Example four saliva Total reducing force determination of Lactobacillus Lumine-102
This example is presented to demonstrate the antioxidant capacity of Lactobacillus salivarius Lumine-102.
Mu.L of bacterial liquid samples with different concentrations are added into a 1.5 mL centrifuge tube, and simultaneously 60 mu.L of PBS buffer (pH 6.6) and 60 mu.L of 1% potassium ferricyanide solution are added into the centrifuge tube, and water bath is performed at 4 ℃ for 20: 20 min. After rapidly cooling to room temperature with ice water, 60. Mu.L of 10% trichloroacetic acid was added, and the mixture was stirred well, and centrifuged at 3000 r/min in a centrifuge for 10: 10 min (210. Mu.L of reaction system was prepared). 90. Mu.L of the supernatant was removed, and 90. Mu.L of deionized water and 18. Mu.L of a 0.1% ferric chloride solution were added thereto to react at room temperature for 10 min. After the completion of the reaction, 100. Mu.L of pure water was added to the centrifuge tube to dilute the system. Then 70 μl was taken in 96-well plates and three-well replicates were performed, and after shaking 1 min, the absorbance at 700 nm was determined, the higher this value indicated the stronger the reducibility of the samples. The blank control group is pure water, and the positive control group is vitamin C (Vc) solution of 0.2 mg/mL.
According to the analysis result (FIG. 5), the absorbance reading of the blank at 700nm is 0.102, the average value of absorbance of the Lactobacillus salivarius Lumin-102 in the present invention is 0.164, vc is used as a strong reducing agent for the positive control group, and the absorbance thereof is 0.215; the Lumine-102 strain had a significantly higher number than the blank, indicating that it was strongly reducing, but slightly lower than the Vc solution.
According to the results of the embodiment, it can be determined that the saliva combined lactobacillus Lumine-102 provided by the invention can effectively inhibit the activity of melanin generation key enzyme tyrosinase under two conditions of in vitro and in vivo, and can directly influence the generation of melanin; the Lumine-102 strain also has excellent capability of scavenging hydroxyl free radicals and strong reducing power, and the characteristics are that the saliva combined lactobacillus Lumine-102 has strong antioxidation capability, is favorable for resisting oxidative stress and reducing oxidative damage, so that the Lumine-102 strain has great development potential and wide application prospect in the aspect of preparing novel whitening and anti-aging probiotic products.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. Saliva with the preservation number of CGMCC No.29489 is combined with lactobacillus Lumine-102 (Ligilactobacillus salivarius) or culture supernatant or dead bacteria thereof.
2. Use of lactobacillus salivarius Lumine-102 in combination with a culture supernatant or dead cells thereof as claimed in claim 1 to inhibit melanogenesis.
3. Use of lactobacillus salivarius Lumine-102 in combination with a culture supernatant or dead cells thereof as claimed in claim 1 to inhibit tyrosinase activity.
4. Use of the saliva of claim 1 in combination with lactobacillus Lumine-102 dead cells for the preparation of a skin care product.
5. A microbial inoculum comprising the Lactobacillus salivarius Lumine-102 or a culture supernatant or dead cells thereof according to claim 1.
6. The microbial agent of claim 5, wherein the microbial agent is a solid formulation or a liquid formulation.
7. The microbial inoculum of claim 6, wherein the solid preparation is a powder prepared by a freeze-drying method.
8. A cosmetic comprising the saliva of claim 1 in combination with lactobacillus Lumine-102 dead cells.
9. The cosmetic product of claim 8, wherein the cosmetic product is a skin care product.
10. A method of preparing a product composition, the method comprising: adding the bacterial substance and/or metabolite of lactobacillus salivarius Lumine-102 in combination with claim 1 to a product substrate to obtain the product composition.
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