CN116286475A - Post-natal cells with improved skin cell HA content prepared from Lactobacillus paracasei CCFM1294 - Google Patents
Post-natal cells with improved skin cell HA content prepared from Lactobacillus paracasei CCFM1294 Download PDFInfo
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Abstract
The invention discloses a metagen with the function of improving the HA content of skin cells, which is prepared by cheese bacillus paracasei CCFM1294, and belongs to the technical field of microorganisms. The metagen prepared by the cheese bacillus paracasei CCFM1294 HAs the effect of promoting the synthesis of a host HA: (1) significantly up-regulating UGDH mRNA expression by skin cells; (2) significantly up-regulating HAS1mRNA expression by skin cells; (3) significantly up-regulating HAS2mRNA expression by skin cells; (4) significantly up-regulating HAS3mRNA expression by skin cells; (5) increasing skin cell HA content; therefore, the metagen prepared by the cheese-making bacillus paracasei CCFM1294 HAs the effect of promoting the synthesis of host HA and preventing and/or repairing relevant symptoms such as skin dryness, aging and the like, and HAs great application prospect.
Description
Technical Field
The invention relates to a metagen with the function of improving the HA content of skin cells, which is prepared by cheese bacillus paracasei CCFM1294, and belongs to the technical field of microorganisms.
Background
Hyaluronic Acid (HA) is the most important moisturizing substance in skin tissue, and HAs the highest HA content in skin during teenagers, and gradually decreases with age after adulthood. The accompanying aging behavior of dry skin, roughness, wrinkles, loss of elasticity and moisturization is associated with a decrease in HA content.
It HAs been reported that the use of epidermal growth factor, transforming growth factor beta, retinoic acid, N-methylserine, cytokines, estradiol derivatives increases cellular HA synthesis (Journal of Investigative Dermatology.120 (6), 1038-1044,Journal of Biological Chemistry,276 (23), 20428-20435,Biochemical Journal,404,327-336,Circulation Research,94 (5), 592-600,Journal of Biological Chemistry,285 (32), 24639-24645.).
CN111902149a discloses a method for promoting HA synthesis by contacting a hyaluronic acid-producing cell with a polysaccharide derivative or a salt thereof, thereby promoting the synthesis of hyaluronic acid in the hyaluronic acid-producing cell.
CN 113081878A discloses a composition for promoting skin HA synthesis, said composition comprising 0.01-5.0 parts by weight of L lactic acid.
The safety of the substances for improving the HA content also needs to be further evaluated through clinical experiments, and the metazoan substances prepared by probiotics can improve the HA content of skin cells in a targeted way, and innovatively relate the microorganism field to the HA synthesis.
HA is a linear glycosaminoglycan formed by alternately connecting two precursors UDP-GlcA and UDP-GlcNAc under the action of HAS, and HAs remarkable improvement effect on skin problems caused by skin exposure to UV environment, such as skin inflammation, lipid peroxidation, apoptosis and the like. The synthesis of the HA precursors is derived from the glycolytic product G6P, UDP-GlcA is synthesized from G1P under the mediation of UDP-glucose dehydrogenase (UGDH), UDP-GlcNAc is synthesized from F6P according to the mediation of hexosamine biosynthesis pathway. Two major factors affecting HA synthesis are the activity of the enzyme hyaluronate synthase (HAS) and the supply of UDP-GlcA and UDP-GlcNAc, the precursors for HA synthesis. In mammals, three isoenzymes, HAS1, HAS2, HAS3, respectively, can all be involved in HA synthesis. In the HA synthesis pathway, UGDH is the only rate-limiting enzyme mediating UDP-GlcA synthesis, and expression changes thereof can influence the HA level by affecting UDP-GlcA accumulation, and research shows that during the HA synthesis process, the expression of UGDH mRNA, HAS1mRNA, HAS2mRNA and HAS3mRNA can be up-regulated to promote the synthesis of HA, thereby increasing the intracellular HA content. Therefore, the metagen prepared by utilizing the probiotics can increase the HA level of skin cells by regulating the expression of UGDH mRNA, HAS1mRNA, HAS2mRNA and HAS3mRNA so as to improve the skin health condition, and HAS practical application significance.
Disclosure of Invention
The present invention provides a metagen which can be prepared from Lactobacillus paracasei (Lacticaseibacillus paracasei) and can increase the HA content of skin cells.
The invention provides a strain of cheese bacillus paracasei (Lacticaseibacillus paracasei) CCFM1294, wherein the cheese bacillus paracasei (Lacticaseibacillus paracasei) is stored in the microorganism strain collection in Guangdong province at the date of 2022, 11 months and 13 days, and the storage number is GDMCC No:62972, the preservation address is Guangzhou Mr. first 100 college No. 59 building.
The Lactobacillus paracasei (Lacticaseibacillus paracasei) CCFM1294 is separated from the feces of a healthy human body, the strain is subjected to sequencing analysis, the sequence obtained by sequencing is subjected to nucleic acid sequence comparison in NCBI, and after the comparison result is obtained, the identification result is the Lactobacillus paracasei.
The colony of the Lactobacillus paracasei (Lacticaseibacillus paracasei) CCFM1294 on the MRS solid medium is round, white and smooth.
The Lactobacillus paracasei (Lacticaseibacillus paracasei) CCFM1294 is a gram positive bacterium, is facultative anaerobic, has a temperature preference, has an optimal growth temperature of 35-40 ℃ and an optimal growth pH of 6.0-7.0.
The invention provides a composition comprising the above-described Lactobacillus paracasei CCFM1294 and/or a metagen prepared from the above-described Lactobacillus paracasei CCFM1294.
In one embodiment of the invention, the metazoan comprises dead cells, fermentation supernatant, bacterial lysate and/or fermentation broth.
In one embodiment of the invention, the preparation method of the fermentation broth comprises inoculating the above Lactobacillus paracasei CCFM1294 into a fermentation medium to obtain a bacterial liquid, and performing heat treatment to obtain the fermentation broth.
In one embodiment of the present invention, the heat treatment is performed at 60 to 70℃for 25 to 35 minutes.
In one embodiment of the present invention, the preparation method of the bacterial lysate comprises high-pressure homogenizing the fermentation broth obtained by the preparation method, and centrifuging to obtain the bacterial lysate.
In one embodiment of the present invention, the dead cells are inactivated bacterial cells obtained by heat treatment or freeze-drying a bacterial sludge precipitate obtained by centrifuging the fermentation liquid.
In one embodiment of the present invention, the metazoan may be dried into powder or directly used by various drying methods such as vacuum drying, spray drying, vacuum freeze drying, fluidized bed drying, etc.
In one embodiment of the invention, the product comprises a cosmetic, a skin care product, a pharmaceutical product.
In one embodiment of the invention, the cosmetic comprises the above composition, a base material and/or conventional adjuvants.
In one embodiment of the present invention, the matrix material includes a lipid material, a wax material, a synthetic lipid material, a powdered material, a gum material, a coagulant, and a surfactant.
In one embodiment of the present invention, the conventional adjuvants include one or more of moisturizers, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film agents, antioxidants, emulsifiers, and cosmetic nutritional additives.
In one embodiment of the invention, the skin care product comprises the above composition, a base stock and/or conventional adjuvants.
In one embodiment of the present invention, the base stock comprises hydrocarbon, wax, grease stock.
In one embodiment of the present invention, the hydrocarbon comprises liquid paraffin, solid paraffin, microcrystalline paraffin, ceresin, or vaseline.
In one embodiment of the invention, the waxes include palm wax, candelilla wax, jojoba wax, wood wax, lanolin, beeswax.
In one embodiment of the present invention, the oil and fat raw material includes olive oil, coconut oil, castor oil, cotton seed oil, rice bran oil, tea seed oil.
In one embodiment of the present invention, the conventional auxiliary materials include solvents, emollients, penetrants, stabilizers, chelating agents, thickeners, dispersants.
In one embodiment of the invention, the pharmaceutical product comprises a composition, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.
In one embodiment of the invention, the pharmaceutical excipients comprise excipients and additives.
In one embodiment of the invention, the pharmaceutical excipients comprise solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integration agents, permeation enhancers, pH modifiers, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, and release retarders.
In one embodiment of the invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.
In one embodiment of the invention, the dosage form of the pharmaceutical product comprises a capsule, granule, mixture, tablet or tincture.
The invention also provides a product, which contains the lactobacillus paracasei CCFM1294 or the composition; the product has effects of preventing and/or repairing skin dryness, aging, etc.
In one embodiment of the invention, the symptoms associated with dry skin, including skin becoming less elastic, rough, fine lines, etc.
In one embodiment of the invention, symptoms associated with skin aging include increased sensitivity of the skin to temperature, sagging, accompanied by irregular pigmentation, a marked increase in wrinkles, decreased skin resistance, and the like.
In one embodiment of the invention, the product comprises at least one of the following effects:
(1) Significantly up-regulating the expression level of UGDH mRNA of skin cells;
(2) Significantly up-regulating the expression level of the skin cell HAS1 mRNA;
(3) Significantly up-regulating the expression level of the skin cell HAS2 mRNA;
(4) Significantly up-regulating expression level of skin cell HAS3 mRNA;
(5) Significantly increasing the HA content of skin cells.
In one embodiment of the invention, the product comprises a cosmetic, a skin care product, a pharmaceutical product.
In one embodiment of the invention, the cosmetic comprises the above composition, a base material and/or conventional adjuvants.
In one embodiment of the present invention, the matrix material includes a lipid material, a wax material, a synthetic lipid material, a powdered material, a gum material, a coagulant, and a surfactant.
In one embodiment of the present invention, the conventional adjuvants include one or more of moisturizers, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film agents, antioxidants, emulsifiers, and cosmetic nutritional additives.
In one embodiment of the invention, the skin care product comprises the above composition, a base stock and/or conventional adjuvants.
In one embodiment of the present invention, the base stock comprises hydrocarbon, wax, grease stock.
In one embodiment of the present invention, the hydrocarbon comprises liquid paraffin, solid paraffin, microcrystalline paraffin, ceresin, or vaseline.
In one embodiment of the invention, the waxes include palm wax, candelilla wax, jojoba wax, wood wax, lanolin, beeswax.
In one embodiment of the present invention, the fat and oil raw material includes animal fat and vegetable fat.
In one embodiment of the invention, the animal fat comprises mink oil, egg yolk oil, lanolin, lecithin.
In one embodiment of the present invention, the vegetable oil comprises olive oil, coconut oil, castor oil, cotton seed oil, rice bran oil, tea seed oil.
In one embodiment of the present invention, the conventional auxiliary materials include solvents, emollients, penetrants, stabilizers, chelating agents, thickeners, dispersants.
In one embodiment of the invention, the pharmaceutical product comprises a composition, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.
In one embodiment of the invention, the pharmaceutical excipients comprise excipients and additives.
In one embodiment of the invention, the pharmaceutical excipients comprise solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integration agents, permeation enhancers, pH modifiers, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, and release retarders.
In one embodiment of the invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.
In one embodiment of the invention, the dosage form of the pharmaceutical product comprises a capsule, granule, mixture, tablet or tincture.
The invention also provides application of the lactobacillus paracasei CCFM1294 or the composition in preparing a product for increasing the content of hyaluronic acid in cells.
Advantageous effects
The invention screens and obtains a strain of cheese bacillus (Lacticaseibacillus paracasei) CCFM1294, and the metagen prepared by the cheese bacillus (Lacticaseibacillus paracasei) CCFM1294 has the effect of repairing related symptoms such as skin dryness, aging and the like, and is specifically expressed in the following steps:
(1) Significantly up-regulating expression of the skin cell UGDH mRNA;
(2) Significantly up-regulating expression of skin cell HAS1 mRNA;
(3) Significantly up-regulating expression of skin cell HAS2 mRNA;
(4) Significantly up-regulating expression of skin cell HAS3 mRNA;
(5) Significantly increasing the HA content of skin cells;
therefore, the metagen prepared by the cheese bacillus paracasei (Lacticaseibacillus paracasei) CCFM1294 has great application prospect in preparing products for preventing and/or repairing skin dryness and aging and even preventing and/or relieving skin problems related to aging and metabolic disorder.
Preservation of biological materials
A strain of Lactobacillus paracasei (Lacticaseibacillus paracasei) CCFM1294, taxonomically designated Lacticaseibacillus paracasei, was deposited on the microorganism strain collection, accession number GDMCC No:62972, the preservation address is Guangzhou Mr. first 100 college No. 59 building.
Drawings
Fig. 1: the effect of different metants on the expression of UGDH mRNA in skin cells.
Fig. 2: effect of different metants on expression of HAS1mRNA in skin cells.
Fig. 3: effect of different metants on expression of HAS2mRNA in skin cells.
Fig. 4: effect of different metants on expression of HAS3mRNA in skin cells.
Fig. 5: effect of different metants on HA content of skin cells.
The "signs" indicated a statistical difference from the Model group (P < 0.05), the "signs" indicated a significant statistical difference from the Model group (P < 0.01), and the "signs" indicated a very significant statistical difference from the Model group (P < 0.001).
Detailed Description
Human immortalized keratinocytes (HaCaT) referred to in the following examples were purchased from: shanghai cell bank.
The post-production of Lactobacillus paracasei FXJWS3M2 and Lactobacillus plantarum 23 are from the university of south China food biotechnology center laboratory.
The invention is further illustrated below in conjunction with specific examples.
The following examples relate to the following media:
MRS liquid medium: 5.0g/L of yeast powder, 10.0g/L of beef extract, 10.0g/L of peptone, 20.0g/L of glucose, 2.0g/L of anhydrous sodium acetate, 2.0g/L of diamine hydrogen citrate, 2.6g/L of dipotassium hydrogen phosphate, 0.25g/L of manganese sulfate monohydrate, 0.5g/L of magnesium sulfate heptahydrate and 1mL/L of tween-80, and the pH value is 6.2-6.4.
MRS solid medium: 5.0g/L of yeast powder, 10.0g/L of beef extract, 10.0g/L of peptone, 20.0g/L of glucose, 2.0g/L of anhydrous sodium acetate, 2.0g/L of diamine hydrogen citrate, 2.6g/L of dipotassium hydrogen phosphate, 0.25g/L of manganese sulfate monohydrate, 0.5g/L of magnesium sulfate heptahydrate, 20.0g/L of tween-80 and agar, and pH value of 6.2-6.4.
Cell culture medium: 89% (v/v) DMEM medium+10% (v/v) fetal bovine serum+1% (v/v) 100 Xpenicillin and streptomycin mixed solution (penicillin content 10000U/mL, streptomycin concentration 10mg/mL in mixed solution).
Example 1: screening and identification of Lactobacillus paracasei
The method comprises the following specific steps:
1. screening
The method comprises the steps of (1) pretreating a sample from healthy human body excrement, storing the sample in 30% glycerol in a refrigerator with the temperature of-80 ℃, taking out the sample for thawing, uniformly mixing and absorbing 0.5mL of the sample, adding the sample into 4.5mL of physiological saline, carrying out gradient dilution by using the physiological saline with the concentration of 9g/L, selecting proper gradient dilution to be coated on an MRS solid culture medium, culturing at the temperature of 37 ℃ for 48 hours, picking a typical colony of the cheese bacillus paracasei, carrying out streak purification on the MRS solid culture medium, picking a single colony, transferring the single colony to the MRS liquid culture medium for enrichment, and preserving 30% glycerol to obtain the cheese bacillus CCFM1294; of these, the typical colony of Lactobacillus paracasei is round, milky white, smooth raised.
2. Authentication
The genome of the strain CCFM1294 was extracted, and the 16S rDNA of the strain CCFM1294 was amplified and sequenced (the nucleotide sequence of the 16S rDNA amplification of CCFM1294 was aligned in NCBI by Jin Weizhi Biotechnology Co., st., which showed that the strain was Lactobacillus paracasei, designated as Lactobacillus paracasei (Lacticaseibacillus paracasei) CCFM 1294).
Example 2: cell resuscitation and culture
Firstly taking out frozen human immortalized keratinocytes (HaCaT), rapidly thawing in a water bath at 37deg.C, centrifuging at 1000r/min for 3min, discarding supernatant, adding appropriate volume of cell culture medium to resuspend cells, placing in a culture dish, and adding a culture medium containing 5% CO 2 Culturing in a 37 ℃ incubator, and carrying out cell passage when the cells grow for 1-2 days to 70% -80% fusion.
Example 3: preparation of metazoan by cheese bacillus paracasei CCFM1294
(1) Culturing in a 37 ℃ water-proof constant temperature incubator for 24-48 hours by using an MRS solid culture medium to obtain single colonies; single colony is selected and inoculated into MRS liquid culture medium, and cultured for 12-18 hours at 37 ℃ to obtain culture solution 1;
inoculating the culture solution 1 into MRS liquid culture medium with an inoculum size of 2% (v/v), and culturing at 37 ℃ for 12h to obtain seed solution;
inoculating 3-5% (v/v) of seed solution into MRS liquid culture medium for expansion culture, and culturing at 37 ℃ for 18-24 hours to obtain bacterial solution, wherein the bacterial concentration is as follows: 4.2X10 10 CFU/ml。
And (3) carrying out heat treatment on the bacterial liquid (65 ℃ for 30 min), carrying out high-pressure homogenization (800-1200 MPa,3 times) in a high-pressure homogenizer to obtain bacterial lysate, and carrying out freeze-drying to obtain post-raw cell freeze-dried powder for later use to prepare the post-raw cell of the cheese bacillus paracasei CCFM1294.
(2) And (3) preparing the post-metazoan of the Lactobacillus paracasei FXJWS3M2 and the Lactobacillus plantarum 23 according to the method of the step (1).
Example 4: effect of post-production of Lactobacillus paracasei CCFM1294 on UGDH mRNA in HaCaT cells
The method comprises the following specific steps:
(1) HaCaT cells were grown at 2.5X10 6 The cells were cultured overnight with cells attached to the wall after seeding/ml on 12-well plates. Old medium was discarded, washed 3 times with PBS, and Control and treatment groups were set;
the control group is a group without adding metagen;
the treatment group comprises re-suspending metazoan with cell culture medium (the amount of metazoan after re-suspending and fermentation to a concentration of 1.0X10) 7 The amount of metazoan prepared by CFU/ml bacterial liquid is equivalent):
2ml of the post-cell of Lactobacillus paracasei CCFM1294 and post-cell of Lactobacillus paracasei FXJWS3M2 prepared in example 3 and post-cell of Lactobacillus plantarum 23 were added to the well plate containing HaCaT cells, respectively;
each of the above groups was incubated in an incubator at 37℃for 4 hours, three for each sample.
(2) After co-culturing metazoan and cells, discarding culture supernatant, rapidly washing 3 times with PBS (phosphate buffer solution) in each well, adding 1mL of cell lysate in each well, repeatedly blowing, sucking cell lysate to extract RNA (ribonucleic acid), performing reverse transcription into cDNA (complementary deoxyribonucleic acid) by using an RT-PCR (reverse transcription kit), detecting the expression condition of genes in HaCaT cells by using a real-time fluorescence quantitative method, and using 2 -△△Ct The expression level of UGDH mRNA was calculated by the formula, wherein the internal reference is GAPDH, and the primers are described in the following Table 1, and the results are shown in FIG. 1.
Table 1: primer sequences
As shown in FIG. 1, the relative expression amounts of UGDH mRNA of the Control group (Control) were 1, the relative expression amounts of UGDH mRNA of the post-cell of Lactobacillus paracasei CCFM1294, the post-cell of Lactobacillus paracasei FXJWS3M2 and the post-cell of Lactobacillus plantarum 23 were 1.94, 1.21 and 1.35, respectively, and the post-cell of Lactobacillus paracasei CCFM1294 significantly promoted the expression of UGDH mRNA (94% higher than that of the Control group), whereas the post-cell of Lactobacillus paracasei FXJWS3M2 was only 21% higher than that of the Control group.
As can be seen from the results of the cell experiments, the post-tuple CCFM1294 from Lactobacillus paracasei has the ability to up-regulate UGDH mRNA expression.
Example 5: effect of the metazoan produced by Lactobacillus paracasei CCFM1294 on HAS1mRNA, HAS2mRNA and HAS3mRNA in HaCaT cells
The method comprises the following specific steps:
(1) HaCaT cells were grown at 2.5X10 6 The cells were cultured overnight with cells attached to the wall after seeding/ml on 12-well plates. Old medium was discarded, washed 3 times with PBS, and control and treatment groups were set;
the control group is a group without adding metagen;
the treatment group comprises re-suspending metazoan with cell culture medium (the amount of metazoan after re-suspending and fermentation to a concentration of 1.0X10) 7 The amount of metazoan prepared by CFU/ml bacterial liquid is equivalent):
2ml of the post-cell of Lactobacillus paracasei CCFM1294 and post-cell of Lactobacillus paracasei FXJWS3M2 prepared in example 3 and post-cell of Lactobacillus plantarum 23 were added to the well plate containing HaCaT cells, respectively;
each of the above groups was incubated in an incubator at 37℃for 4 hours, three for each sample.
(2) After co-culturing metazoan and cells, discarding culture supernatant, rapidly washing 3 times with PBS (phosphate buffer solution) in each well, adding 1mL of cell lysate in each well, repeatedly blowing, sucking cell lysate to extract RNA (ribonucleic acid), performing reverse transcription into cDNA (complementary deoxyribonucleic acid) by using an RT-PCR (reverse transcription kit), detecting the expression condition of genes in HaCaT cells by using a real-time fluorescence quantitative method, and using 2 -△△Ct The expression levels of HAS1mRNA, HAS2mRNA and HAS3mRNA were calculated by the formula, wherein the internal reference was GAPDH, and the primers are described in the following Table 2, and the results are shown in FIGS. 2 to 4.
Table 2: primer sequences
The results showed that the expression levels of HAS1mRNA, HAS2mRNA, and HAS3mRNA in the Control group (Control) were 1;
the relative expression amounts of HAS1mRNA of the metagenome prepared by Lactobacillus paracasei CCFM1294, lactobacillus paracasei FXJWS3M2 and Lactobacillus plantarum 23 were 1.68, 0.33 and 0.91, respectively, and the relative expression amounts of HAS2mRNA were 1.44, 0.68 and 1.01, respectively; the relative expression levels of HAS3mRNA were 1.49, 0.74 and 1.24, respectively.
From this it can be seen that the lactobacillus paracasei CCFM1294 significantly promoted expression of HAS1mRNA (68% higher than control), HAS2mRNA (44% higher than control), HAS3mRNA (49% higher than control).
As can be seen from the results of the cell experiments, the metagenome prepared from Lactobacillus paracasei CCFM1294 HAs the ability to up-regulate the expression of HA synthase.
Example 6: the effect of the metazoan produced by Lactobacillus paracasei CCFM1294 on the HA content in HaCaT cells is specifically as follows:
(1) HaCaT cells were grown at 2.5X10 6 The cells were cultured overnight with cells attached to the wall after seeding/ml on 12-well plates. Old medium was discarded, washed 3 times with PBS, and control and treatment groups were set;
the control group is a group without adding metagen;
the treatment group uses cell culture medium to re-suspend metazoan (the amount of metazoan after re-suspension and fermentation to a concentration of 1.0X10) 7 The amount of metazoan prepared by CFU/ml bacterial liquid is equivalent):
2ml of the metazoans prepared by Lactobacillus paracasei CCFM1294, lactobacillus paracasei FXJWS3M2 and Lactobacillus plantarum 23 were added to the well plate containing HaCaT cells, respectively, and cultured for 4 hours, three in parallel.
(2) After the co-culture of metazoan and cells is completed, the culture supernatant is discarded, each well is rapidly washed 3 times by PBS, 1mL of cell lysate is added into each well, the mixture is repeatedly blown, the cell lysate is sucked, and the HA content of skin cells is detected by an ELISA method, and the result is shown in figure 5.
As a result, it was found from FIG. 5 that the HA content of the Control group (Control) was 29.61. Mu.g/l, the HA content of the post-tuple produced by Lactobacillus paracasei CCFM1294 was 46.79. Mu.g/l, the HA content of the post-tuple produced by Lactobacillus paracasei FXJWS3M2 was 31.39. Mu.g/l, and the HA content of the post-tuple produced by Lactobacillus plantarum 23 was 33.60. Mu.g/l.
The metants prepared from lactobacillus paracasei CCFM1294 significantly increased the HA content of the skin cells (up-regulated by 58% compared to the control group) compared to the control group and other metants, which were up-regulated by 13.48% compared to the control group.
As can be seen from the results of the cell experiments, the metagen prepared from Lactobacillus paracasei CCFM1294 promotes the synthesis of HA by up-regulating the expression of the precursor enzymes UGDH and HA synthase during the synthesis of HA, thereby increasing the HA content of the skin cells.
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. A strain of Lactobacillus paracasei (Lacticaseibacillus paracasei) CCFM1294, deposited with the microorganism strain collection, canon province, at 11/13 2022, under the accession number GDMCC No:62972, the preservation address is Guangzhou Mr. first 100 college No. 59 building.
2. A composition comprising the lactobacillus paracasei CCFM1294 of claim 1 and/or containing the metazoan produced by the lactobacillus paracasei CCFM1294 of claim 1.
3. The composition of claim 2, wherein the metazoan comprises dead cells, fermentation supernatant, bacterial lysate and/or fermentation broth, or a powder thereof prepared by any drying means.
4. A method for preparing a post-metazoan of the Lactobacillus paracasei CCFM1294, which is characterized in that the method comprises inoculating the Lactobacillus paracasei CCFM1294 of claim 1 into a fermentation medium for culture to obtain a bacterial liquid, and then carrying out heat treatment and homogenization pyrolysis on the bacterial liquid to obtain the post-metazoan.
5. Use of a lactobacillus paracasei CCFM1294 according to claim 1 or a composition according to any of claims 2 to 3 for the preparation of a product for preventing and/or repairing dry skin and delaying ageing.
6. The use according to claim 5, wherein the product is a food, a cosmetic, a skin care product or a pharmaceutical product.
7. A product comprising the lactobacillus paracasei CCFM1294 of claim 1 or the composition of any of claims 2 to 3.
8. The product according to claim 7, wherein the product is a food, a cosmetic, a skin care product or a pharmaceutical for external use.
9. The product of claim 8, wherein the product comprises at least one of the following effects:
(1) Significantly up-regulating the expression level of UGDH mRNA of skin cells;
(2) Significantly up-regulating the expression level of the skin cell HAS1 mRNA;
(3) Significantly up-regulating the expression level of the skin cell HAS2 mRNA;
(4) Significantly up-regulating expression level of skin cell HAS3 mRNA;
(5) Significantly increasing the hyaluronic acid HA content of skin cells.
10. Use of the lactobacillus paracasei CCFM1294 of claim 1 or the composition of any of claims 2 to 3 for the preparation of a product for increasing the hyaluronic acid HA content in a cell.
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