CN117625456A - Lactobacillus plantarum CCFM1283 with anti-saccharification and anti-aging functions and progeny thereof - Google Patents
Lactobacillus plantarum CCFM1283 with anti-saccharification and anti-aging functions and progeny thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a lactobacillus plantarum CCFM1283 with anti-saccharification and anti-aging functions and a progeny thereof, belonging to the technical field of microorganisms and medicines. The lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1283 and the metazoan prepared by the lactobacillus plantarum (Lactiplantibacillus plantarum) can be used for relieving saccharification injury and/or resisting aging of an individual in an external or oral mode. The lactobacillus plantarum CCFM1283 provided by the invention is taken as a food safety strain, and the strain and/or the metazoan prepared by the strain have great application prospects in the fields of foods, health-care products, medicaments or cosmetics.
Description
Technical Field
The invention relates to a lactobacillus plantarum CCFM1283 with anti-saccharification and anti-aging functions and a progeny thereof, belonging to the technical field of microorganisms and medicines.
Background
Advanced glycation end products (AGEs) are caused by non-enzymatic glycosylation of protein amino and glucose reducing sugars. AGEs can form both intra-and extracellular, their presence in biological molecules altering their biomechanical and functional properties. Proteins, lipids and nucleic acids can be targets for advanced glycosylation, interfering with many physiological functions of organisms by modifying enzyme-substrate interactions, protein-DNA interactions, protein-protein interactions, DNA regulation and epigenetic regulation. One prominent feature of aging at the molecular level is the gradual accumulation of non-enzymatically modified proteins, the most common of which is glycosylation. The reaction of the reducing sugar with free amino groups on the protein (and other molecules) results in the reversible production of reactive intermediates and ultimately in the irreversible advanced glycation end products (AGEs).
At the skin level, glycosylation is involved in the aging process and affects cells (endothelial cells, fibroblasts) and structural proteins, such as collagen, elastin, glycoproteins. The glycosylation-modified dermal extracellular matrix (ECM) further affects fibroblast growth, differentiation, motility, cytokine responses, enzymatic activity (metalloproteases), and vascular hemostasis. One common consequence of AGE accumulation is covalent cross-linking of AGEs with proteins, which results in increased stiffness of the protein matrix, impedes function and increases resistance to removal of cross-linked proteins by proteolysis in various tissues and organs, resulting in impaired organ function. CN116602977a discloses an active component with antioxidant, anti-saccharification and anti-inflammatory effects, and a preparation method and application thereof, wherein the component comprises terligustrin, diosmin, neodimine, rosmarinic acid, oleuropein, salvianolic acid B, linarin, salangid phylloside, ligustrin G13, 6' -O-trans-cinnamoyl-8-epi Jin Jigan acid, and has good in vitro antioxidant, anti-saccharification and anti-inflammatory activity.
In order to develop a more efficient and comprehensive anti-glycation product, a 4-path anti-glycation strategy can be proposed according to the occurrence and development of glycation injury, and potential effective biological raw materials can be screened based on the paths: (1) reducing AGE formation; (2) blocking AGE-RAGE binding; (3) disrupting the cross-linking of AGE with protein; (4) inhibiting post-AGE-RAGE binding signal activation. The most studied at present is to compete for saccharification reaction intermediates or limit conditions under which the intermediates react to form AGEs, thereby reducing the production and accumulation of AGEs; however, it is not enough to limit AGE production, and other anti-glycation pathways have been studied intensively.
Disclosure of Invention
The invention provides application of lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1283 and progeny thereof in preparing anti-saccharification and anti-aging products.
The invention provides a lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1283, which is deposited in the Guangdong province microorganism strain collection center with the deposit number of: GDMCC No:62885, the preservation date is 2022, 10 month and 14 days.
The lactobacillus plantarum CCFM1283 is derived from feces of healthy people, and the 16S rDNA sequence of the lactobacillus plantarum CCFM1283 is shown as SEQ ID NO. 1.
The lactobacillus plantarum CCFM1283 has raised colony on the MRS solid culture medium, smooth and fine surface, white round shape and diameter of about 3mm.
The invention also provides a metagen prepared by using the lactobacillus plantarum CCFM 1283.
In one embodiment, the metazoan comprises inactivated or inactivated cells, cell cultures, and/or cell lysates.
In one embodiment, the metazoan is a powder prepared by drying one or more of an inactivated or inactivated cell, cell culture, cell lysate.
In one embodiment, the inactivated or inactivated cells are prepared as follows: culturing the lactobacillus plantarum CCFM1283 in a culture medium for a period of time, collecting the somatic cells in a cell culture solution, and carrying out heat treatment or freeze-drying to obtain the inactivated somatic cells.
In one embodiment, the conditions of the heat treatment are: 60-70 ℃ for 25-35 min.
In one embodiment, the method for preparing the bacterial lysate comprises the following steps: culturing the lactobacillus plantarum CCFM1283 in a culture medium for a period of time, collecting the bacterial cells, homogenizing at 8000r/min and 30min under high pressure, and centrifugally collecting the supernatant to be used as a bacterial lysate.
In one embodiment, drying includes, but is not limited to, preparation via spray drying, vacuum freeze drying, fluid bed drying, vacuum drying.
The invention also provides a composition containing the lactobacillus plantarum CCFM1283 and/or a metathereof.
In one embodiment, the composition includes, but is not limited to, a food, a pharmaceutical, a nutraceutical, or a cosmetic.
In one embodiment, the composition is the product of the lactobacillus plantarum CCFM1283 after fermentation in a peanut coat-containing medium.
The invention provides application of the lactobacillus plantarum CCFM1283, the metaplasia or the composition in preparation of anti-saccharification and anti-aging products.
In one embodiment, the product comprises a food product, a nutraceutical product, a pharmaceutical product, or a cosmetic product.
In one embodiment, the product comprises at least one of the following effects:
(1) Inhibiting the generation of fluorescent AGE;
(2) Preventing damage and reduced function of skin fibroblasts (HSF) caused by high glucose and AGE formation intermediates (methylglyoxal);
(3) Reducing the aging characteristics (blood biochemical index, skin appearance) of the individual;
(4) Reducing AGE content in blood and skin tissue of an individual;
(5) Reducing AGE-RAGE binding in skin tissue of an individual;
(6) Reducing inflammatory responses and oxidative stress in skin tissue of an individual;
(7) Relieving skin elasticity and collagen decrease of aging individuals.
In one embodiment, the symptoms associated with aging are directed to the accumulation of the glycation senescence marker AGE in skin aging, blood and other tissue organs.
In one embodiment, skin aging comprises dry skin, reduced elasticity, sagging, wrinkling, oxidative damage, collagen loss.
In one embodiment, the product is applied by topical and oral use.
In one embodiment, the content of lactobacillus plantarum CCFM1283 in the product is not less than 1×10 6 CFU/mL or 1X 10 6 CFU/g。
In one embodiment, the post-natal prepared by lactobacillus plantarum CCFM1283 is used at a dose of not less than 10mg/kg body weight.
The invention provides an application of lactobacillus plantarum CCFM1283 and/or a metagen thereof in preparing a medicament for preventing and/or relieving skin aging caused by saccharification.
In one embodiment, the symptoms associated with skin aging include dry skin, reduced elasticity, sagging, wrinkling, oxidative damage, collagen loss.
In one embodiment, the food product contains the lactobacillus plantarum CCFM1283 and/or its metazoan, and conventional excipients.
In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional supplements.
In one embodiment, the health product contains the lactobacillus plantarum CCFM1283 and/or its metazoan, and conventional adjuvants.
In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional supplements.
In one embodiment, the medicament contains the lactobacillus plantarum CCFM1283 and/or its metazoan, and a pharmaceutical carrier and/or pharmaceutical adjuvant.
In one embodiment, the pharmaceutical excipients comprise excipients and additives.
In one embodiment, 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, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, and release retarders.
In one embodiment, the cosmetic comprises the lactobacillus plantarum CCFM1283 and/or its progeny, a base material and/or conventional adjuvants.
In one embodiment, the matrix material comprises a lipid material, a wax material, a synthetic lipid material, a powdered material, a gum material, a coagulant, a surfactant.
In one embodiment, the conventional adjuvants include one or more of moisturizers, whitening agents, flavoring agents, binders, lubricants, preservatives, film agents, antioxidants, emulsifiers, and cosmetic nutritional additives.
The invention also provides application of the lactobacillus plantarum CCFM1283 or the metazoan in preparing food.
Advantageous effects
The lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1283 and the metazoan prepared by the same have the capability of relieving saccharification damage to a host and reducing corresponding aging problems, and are specifically expressed in the following steps:
(1) Preventing a decrease in cell viability in a methylglyoxal to skin fibroblast (HSF) injury model;
(2) Preventing abnormal expression of DDOST mRNA and MMP-9mRNA after skin fibroblast (HSF) is damaged by sugar under high glucose culture;
(3) Reducing AGE content in serum and skin of aging mice;
(4) Reducing the content of an inflammation marker TNF-alpha in serum and skin of a aged mouse;
(5) Relieving the condition of the reduced back skin elasticity of aged mice caused by saccharification injury;
(6) Reducing the content of III type collagen in the back skin of the aging mice caused by saccharification injury;
(7) Alleviating exacerbation of back skin glycation lesions in aging mice mediated by AGE-RAGE binding;
(8) Alleviating collagen synthesis, degradation and inflammatory reaction caused by saccharification injury of aging mice.
Therefore, the lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1283 and the metaplasia prepared by the same have great application prospect in products for relieving saccharification damage to hosts and reducing corresponding aging.
Preservation of biological materials
Lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1283, taxonomically designated Lactiplantibacillus plantarum, was deposited at the collection of microorganisms of the Guangdong province on day 14 of 2022 under the accession number GDMCC No:62885, the preservation address is Guangzhou Mr. first 100 college No. 59 building.
Drawings
FIG. 1 shows the effect of different metants on HSF cell proliferation;
FIG. 2 shows the effect of different metants on HSF cell viability under the action of methylglyoxal, an intermediate in saccharification reactions;
FIG. 3 is a graph showing the effects of different metants on the prevention of anti-glycation related gene expression in HSF cells under high glucose culture (DDOST mRNA, MMP-9 mRNA);
FIG. 4 is a flow chart of a mouse experiment;
FIG. 5 shows the effect of Lactobacillus plantarum CCFM1283 and its prepared metazoan on AGE content in blood and skin tissue of mice;
FIG. 6 shows the effect of Lactobacillus plantarum CCFM1283 and its prepared metazoan on the TNF- α content of inflammatory markers in serum and skin of aging mice;
FIG. 7 shows the effect of Lactobacillus plantarum CCFM1283 and its prepared metazoan on skin elasticity properties;
FIG. 8 shows the effect of Lactobacillus plantarum CCFM1283 and its prepared metazoan on skin type III collagen synthesis, degradation and content;
FIG. 9 is a graph showing the effect of Lactobacillus plantarum CCFM1283 and its prepared metants on inhibition of AGE-RAGE binding and oxidative stress relief of skin glycation lesions;
FIG. 10 is a graph showing the effect of Lactobacillus plantarum CCFM1283 fermented peanut coat on enhancing the in vitro anti-saccharification ability of peanut coat;
FIG. 11 is a graph showing the effect of Lactobacillus plantarum CCFM1283 fermented peanut coat on enhancing the oral inhibition of AGE accumulation in serum and subsequent receptor binding of peanut coat;
FIG. 12 is a graph showing the effect of Lactobacillus plantarum CCFM1283 fermented peanut coat on improving peanut coat oral relief of skin elasticity decline caused by saccharification;
FIG. 13 is a graph showing the effect of Lactobacillus plantarum CCFM1283 fermented peanut coat on the target of oral release of skin glycation lesions from peanut coat;
FIG. 14 is a graph showing the effect of Lactobacillus plantarum CCFM1283 fermented peanut coat on improving peanut coat oral administration to alleviate skin inflammation and oxidative stress.
"" indicates a statistical difference from the Model group (P < 0.05), "" indicates a significant statistical difference from the Model group (P < 0.01), "" indicates a very significant statistical difference from the Model group (P < 0.001); "" indicates that there was a very significant statistical difference from the Model group (P < 0.0001).
Detailed Description
The invention is further illustrated below in conjunction with specific examples.
Human Skin Fibroblasts (HSFs) referred to in the following examples were purchased from: kunming cell bank.
BALB/c mice referred to in the examples below were purchased from Vetong Liwa.
The Lactobacillus plantarum CCFM1283, lactobacillus plantarum FXJCJ22M3, lactobacillus plantarum FSCDJY93L1 and Lactobacillus plantarum FXJCJ26M6 referred to in the examples below are from the food biotechnology center self-screening strains of university in Jiangnan.
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.
MRS (simplified) liquid medium: glucose 8g/L, yeast powder 5g/L, calcium carbonate 6g/L, anhydrous sodium acetate 2g/L, citric acid diamine acid 2g/L, dipotassium hydrogen phosphate 2.6g/L, manganese sulfate monohydrate 0.25g/L, magnesium sulfate heptahydrate 0.5g/L, tween-80 1mL and pH 6.2-6.4.
Peanut coat fermentation medium: adding 5g/L of commercial peanut coat extract, 4g/L of glucose, 5g/L of yeast powder and 4g/L of calcium carbonate, regulating the pH to 6.8-7.2, and sterilizing at 115 ℃ for 20min to prepare culture solution (abbreviated as hsp) for peanut coat fermentation; among them, peanut coat extract was purchased from Shaanxi Cheng Heng biotechnology Co., ltd, lot number SH20220328.
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: cell resuscitation and culture
Firstly taking out frozen human skin fibroblast cell strain (HSF), rapidly thawing in a water bath kettle at 37 ℃, centrifuging at 1000r/min for 3min, discarding the supernatant, adding a proper volume of cell culture medium to resuspend cells, placing the cells in a culture dish, placing the culture dish in a 37 ℃ incubator containing 5% CO2 for culture, and carrying out cell passage when the cell growth is recovered and the viability is grown for 1-2 d and reaches 70% -80% fusion.
Example 2: screening of lactobacillus plantarum CCFM1283 and metazoan preparation
The sample is derived from healthy human body excrement, the sample is preserved in a refrigerator at the temperature of minus 80 ℃ in 20% glycerol after being pretreated, the sample is taken out and thawed, 0.5mL of the sample is added into 4.5mL of physiological saline, the physiological saline is used for gradient dilution, proper gradient diluent is selected to be coated on an MRS solid culture medium, the culture is carried out for 48 hours at the temperature of 37 ℃, typical bacterial colonies of lactobacillus plantarum are selected to be streaked and purified on the MRS solid culture medium, single bacterial colonies are selected to be transferred to the MRS liquid culture medium for enrichment, 30% glycerol is preserved, and the bacterial strain is obtained and named as lactobacillus plantarum CCFM1283. The genome of the extracted strain was amplified and sequenced (by Jin Weizhi Biotechnology Co., st. Job) with 16S rDNA, and the result was determined to be Lactobacillus plantarum by NCBI sequence alignment, designated Lactobacillus plantarum CCFM1283.
(1) Marking and resuscitating the lactobacillus plantarum CCFM1283 from a bacteria-retaining tube, and culturing in a 37 ℃ water-proof constant-temperature incubator for 24-48 hours by utilizing an MRS solid culture medium to obtain single colonies; selecting single colony, inoculating into MRS liquid culture medium, and culturing at 37deg.C for 12-18 hr 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 2% (v/v) of seed solution into MRS liquid culture medium and MRS (simplified) liquid culture medium, culturing at 37deg.C for 18-24 hr, and regulating the concentration of two bacterial solutions to a corresponding level (bacterial concentration of 1.5X10) 9 CFU/mL) to obtain bacterial liquid a and bacterial liquid b.
Centrifuging the bacterial liquid a at 8000r/min for 30min to obtain supernatant, performing heat treatment (65 ℃ for 30 min) on the supernatant, and freeze-drying to obtain powder for later use, thereby preparing the bacterial liquid a: lactobacillus plantarum CCFM1283 fermentation supernatant (designated CCFM 1283_M).
Centrifuging the bacterial liquid b at 8000r/min for 30min to obtain bacterial mud, re-suspending the bacterial mud with double distilled water accounting for 75% of the volume of the original bacterial liquid, performing heat treatment (65 ℃ for 30 min), homogenizing (1000-1200 MPa for 10 times) in a high-pressure homogenizer at high pressure, centrifuging to collect supernatant, obtaining bacterial lysate (marked as CCFM 1283_Z), and freeze-drying to obtain the metaplastic freeze-dried powder for standby.
The preparation method of the lactobacillus plantarum CCFM1283 viable bacteria is the same as that of the metaplasia, except that bacterial liquid b is centrifuged at 8000r/min for 30min to obtain bacterial mud, and the bacterial mud is resuspended in a ratio of 1g to 2mL of freeze-drying protective agent and then is directly freeze-dried to obtain lactobacillus plantarum CCFM1283 viable bacteria powder, which is marked as CCFM1283.
The preparation method comprises the following steps: lactobacillus plantarum CCFM1283 metazoan (thallus lysate CCFM1283_Z and fermentation supernatant CCFM 1283_M).
(2) And (3) preparing the metazoan of the lactobacillus plantarum FXJCJ22M3, the lactobacillus plantarum FSCDJY93L1 and the lactobacillus plantarum FXJCJ26M6 according to the method of the step (1).
Example 3: peanut coat fermentation liquid prepared by fermenting peanut coats with lactobacillus plantarum CCFM1283
Dipping a inoculating loop in a bacterial solution of lactobacillus plantarum CCFM1283, streaking on an MRS solid culture medium, and inversely culturing for 48 hours at 37 ℃; taking single bacterial colony to MRS liquid culture medium, aerobically culturing at 37 ℃ for 18h, uniformly mixing, inoculating bacterial liquid to new MRS liquid culture medium according to inoculum size of 2% (v/v), and continuously activating for 3 times to finally obtain activated bacterial liquid.
The obtained activated bacterial liquid was inoculated into peanut coat fermentation medium at an inoculum size of 2% (v/v), and shake-cultured at 200rpm under 37℃for 72 hours. Taking fermentation liquor at a culturing place for 72 hours, centrifuging the bacterial liquor at 8000r/min for 30min to obtain supernatant, then carrying out heat treatment (65 ℃ for 30 min) on the supernatant, and freeze-drying to obtain powder for later use, thus obtaining the microbial inoculum: lactobacillus plantarum CCFM1283 fermented peanut coat supernatant (noted ccfm1283_h).
Example 4: determination of fluorescent AGE production in vitro fructose-bovine serum albumin system
10mg/mL bovine serum albumin and 0.5. 0.5M d- (+) -fructose were prepared in 0.1M phosphate buffer (pH 7.4), and a sterile condition of fructose-bovine serum albumin saccharification system was obtained by passing through a 0.22 μm aqueous filter. And respectively adding different metaseduction samples to be tested and a fructose-bovine serum albumin saccharification system, and incubating for 7 days at 37 ℃. The incubated samples were used to detect the formation of fluorescent AGEs 7 days after incubation.
After incubation, fluorescence AGE in glucose-modified BSA was detected with excitation wavelength of 370nm and emission wavelength of 440nm, and the percent inhibition of fluorescence AGE generation was calculated as 1 minus the difference in fluorescence intensity of the sample compared to the control (bsa+/glucose+).
Example 5: effect of the metazoan produced by Lactobacillus plantarum CCFM1283 on HSF cell proliferation
The method comprises the following specific steps:
(1) Taking 100. Mu.L of human skin fibroblast (HSF cell) in logarithmic growth phase at 3×10 4 Inoculating the concentration of individual cells/holes into a 96-well plate, wherein the outermost ring is filled with PBS solution, edge effect is prevented, culturing for 24 hours, and setting a blank group, a control group and a metazoan treatment group after the cells/holes are attached to the wall;
blank group: contains only cell culture medium and no HSF cells;
Control group: contains cell culture medium and HSF cells, but does not contain metazoan;
treatment group: collectingResuspension of metants with cell culture medium (amount of metants after resuspension and fermentation to a concentration of 5.0X10) 7 The amount of the metazoan prepared from the bacterial liquid of CFU/mL was equivalent, and 100. Mu.L of metazoan prepared from Lactobacillus plantarum CCFM1283, lactobacillus plantarum FXJCJ22M3, lactobacillus plantarum FSCDJY93L1, or Lactobacillus plantarum FXJCJ26M6 was added, respectively.
(2) The above-mentioned orifice plates were each set at a temperature of: incubation was performed in an incubator at 37℃for 24h, and after incubation, 10. Mu.LCCK 8 solution was added to each well and incubated for 2h to determine absorbance (OD) at 450 nm.
Cell viability was calculated according to the following formula: cell viability (%) = (treatment OD value-blank OD value)/(control OD value-blank OD value) ×100%.
As shown in FIG. 1, the effect on cell proliferation was compared with the control group (cell proliferation rate 100%), and the concentration of the cell inactivated was 5.0X10 by adding the metagen (CCFM 1283_M or CCFM 1283_Z) derived from Lactobacillus plantarum CCFM1283, the metagen (FXJCJ22M3_M or FXJCJ22M3_Z) derived from Lactobacillus plantarum FJCJY 22M3, the metagen (FSCDJY 93L1 (FSCDJY 93L1_M or FSCDJY93 L1_Z)) derived from Lactobacillus plantarum FXJCJ26M6 (FXJCJ26M6_M or FXJCJ26M6_Z) derived from Lactobacillus plantarum CCJCJ26M 6 (FXJCJ26M6_M or FXJCJ26M6_Z) 7 The cell proliferation rates at CFU/mL were 102.90%, 133.10%, 108.48%, 105.30%, 98.50%, 97.90%, 99.99%, 94.76%, 107.73%, respectively.
According to the toxicity grading evaluation method in ISO 10993-5:2009, if the cell activity is more than 70%, the cell is considered to be nontoxic. The above results indicate that the concentration of the inactivated bacteria is 5.0X10 7 The cell viability of HSF at the metagen concentration of CFU/mL is higher than 90%, and the optional inactivated cell concentration is 5.0X10 in consideration of no cytotoxicity 7 CFU/mL is the appropriate metagen concentration for subsequent cell experiments.
Example 6: the metagen prepared from lactobacillus plantarum CCFM1283 prevents the influence of methylglyoxal on saccharification damage caused by HSF cells
The method comprises the following specific steps:
(1) 100. Mu.L of HSF cells in logarithmic growth phase was taken at 3X 10 4 The individual cell/well concentrations were seeded in 96-well plates, with the outermost ring filled with PBS solution, preventing edge effects,culturing for 24h, and setting a blank group, a control group 1 and a treatment group 1 after the culture is attached to the surface of the culture medium;
blank group: contains only cell culture medium and no HSF cells;
control group 1: contains cell culture medium and HSF cells, and does not contain metagen;
treatment group 1: comprises a cell culture medium and HSF cells, and also comprises metazoan,
The metazoan comprises Lactobacillus plantarum CCFM1283, lactobacillus plantarum FXJCJ22M3, lactobacillus plantarum FSCDJY93L1 and metazoan prepared from Lactobacillus plantarum FXJCJ26M 6. Resuspension of metazoan with cell culture medium (amount of metazoan after resuspension and fermentation to a concentration of 5.0X10) 7 The amount of metagen prepared by CFU/mL bacterial liquid is equivalent).
(2) The above-mentioned orifice plates were each set at a temperature of: incubators at 37 ℃ were incubated for 24h, old media of control and modeling groups were discarded after incubation was completed, and PBS was used for 3 times to set up control, model and treatment groups:
control group: after the liquid is changed in the control group 1, the liquid contains a cell culture medium and HSF cells, and is not treated by metagen and does not contain a methylglyoxal molding agent;
model group: changing the liquid of the control group 1 into a cell culture medium containing a methylglyoxal molding agent, containing original HSF cells without post-metaplasia treatment,
the cell culture medium containing the methylglyoxal molding agent is that methylglyoxal is uniformly mixed in a common cell culture medium and sterilized by a 0.22 mu m water-based filter membrane, and the final concentration of methylglyoxal in the cell culture medium is 400 mu mol/L;
treatment group: the treatment group 1 is changed into a cell culture medium containing a methylglyoxal molding agent, contains original HSF cells and is subjected to metaplasia treatment.
(3) The above-mentioned orifice plates were each set at a temperature of: incubation was performed in an incubator at 37℃for 24h, and after incubation, 10. Mu.LCCK 8 solution was added to each well and incubated for 2h to determine absorbance (OD) at 450 nm.
Cell viability was calculated according to the following formula: model group cell viability (%) = (model group OD value-blank group OD value)/(control group OD value-blank group OD value) ×100%; treatment cell viability (%) = (treatment OD value-blank OD value)/(control OD value-blank OD value) ×100%.
The result of preventing the damage of methylglyoxal to HSF cells is shown in fig. 2, and compared with the control group (cell viability 100%), the cell viability of the model group is 54.93%, and the methylglyoxal model causes significant damage to HSF cells.
The cell viability of the treated group after CCFM1283_M and CCFM1283_Z are 70.70% and 69.97% respectively, wherein compared with 54.93% of the treated group, the CCFM1283_Z and the CCFM1283_M remarkably improve the cell viability of HSF compared with the treated group, which proves that the metagen of the CCFM1283 of the lactobacillus plantarum can effectively prevent saccharification damage caused by methylglyoxal to the HSF cells;
the post-metants of other treatment groups lactobacillus plantarum fxjcj22M3 (fxjcj22m3_m and fxjcj22m3_z), lactobacillus plantarum fscdjj93l1_m and fscdjj93l1_z), lactobacillus plantarum fxjcjcj26m6 (fxjcj26m6_m and fxjcj26m6_z) showed HSF cell viability of 37.99%, 43.54%, 42.92%, 35.04%, 49.72%, 36.02%, respectively, i.e. the post-metants of other lactobacillus plantarum did not possess the ability of lactobacillus plantarum CCFM1283 to significantly prevent methylglyoxal from damaging HSF cells.
Example 7: the metagen prepared by the lactobacillus plantarum CCFM1283 prevents the influence of the related gene expression in HSF cells under high glucose culture.
(1) HSF cells were grown at 1X 10 5 The cells were cultured overnight with cells attached to the wall after inoculating the cells/mL onto a 6-well plate. Old medium was discarded, washed 3 times with PBS, and control and treatment groups were set;
the control group 1 is a group without adding metazoan;
the treatment components are lactobacillus plantarum CCFM1283 lysate (CCFM 1283_Z), lactobacillus plantarum FXJCJ22M3 lysate (FXJCJ 22M 3_Z), lactobacillus plantarum FSCDJY93L1 lysate (FSCDJY 93 L1_Z) and lactobacillus plantarum FXJCJ26M6 lysate (FXJCJ 26M 6_Z), and the cells are adopted to re-suspend metazoan after treatment (the amount and the fermentation concentration of metazoan after re-suspension are 5.0x10) 7 The amount of metagen prepared by CFU/mL bacterial liquid is equivalent).
2mL of a Lactobacillus plantarum CCFM1283 lysate (CCFM 1283_Z), a Lactobacillus plantarum FXJCJ22M3 lysate (FXJCJ 22M 3_Z), a Lactobacillus plantarum FSCDJY93L1 lysate (FSCDJY 93 L1_Z) and a Lactobacillus plantarum FXJCJ26M6 lysate (FXJCJ 26M 6_Z) were respectively aspirated, and the mixture was added to a 6-well plate and cultured for 24 hours, wherein three samples were obtained in parallel.
(2) The above-mentioned well plate was incubated in an incubator at 37℃for 24 hours, after the incubation was completed, the old medium of the control group and the modeling agent group was discarded, washed 3 times with PBS, and the control group, model group and treatment group were set up:
The control group is that after the liquid change of the control group 1, the original HSF cells are not treated by metagen and added with 2mL of common cell culture medium;
the model group is that the liquid of the control group 1 is changed into a cell culture medium containing 35mmol/L glucose, and the cell culture medium contains original HSF cells and is not treated by metagen;
the treatment group is that the liquid of the control group 1 is changed into a cell culture medium containing 35mmol/L glucose, and the cell culture medium contains original HSF cells and is subjected to metaplasia treatment.
(2) Incubating the above-mentioned pore plate in an incubator at 37deg.C for 24h, discarding culture supernatant, rapidly washing 3 times with PBS per well, adding 1mL of cell lysate per well, repeatedly blowing, sucking cell lysate to extract RNA, reverse transcribing into cDNA using RT-PCR reverse transcription kit, detecting gene expression in HSF cells by real-time fluorescence quantification method, and using 2 -△△Ct The expression levels of DDOST mRNA and MMP-9mRNA were calculated by the formula, wherein the internal reference is beta-actin, and the primers are described in the following Table 1, and the results are shown in FIG. 3.
TABLE 1 primer sequences
| Oligomeric (Oligo) designation | Sequence(s) | Description of the invention |
| F-qPCR-DDOST | GAGACTCATTCGCTTTTCTTCCG | Competitive binding sites with RAGE (AGER 1) |
| R-qPCR-DDOST | CTCCAAAATCTTCTACCGAAGGG | Competitive binding sites with RAGE (AGER 1) |
| F-qPCR-MMP9 | AGACCTGGGCAGATTCCAAAC | Matrix metalloproteinase 9 (MMP-9) |
| R-qPCR-MMP9 | CGGCAAGTCTTCCGAGTAGT | Matrix metalloproteinase 9 (MMP-9) |
AGER1 (also known as DDOST) is a protein with strong AGE-specific binding capacity that has been demonstrated to directly accelerate the absorption and clearance of AGE, prevent cellular AGE-RAGE-mediated increases in reactive oxygen species and pro-inflammatory cytokines, and inhibit RAGE signaling by competing with RAGE for AGE; saccharification damage causes a decrease in DDOST mRNA expression level, exacerbating AGE-RAGE binding, thus targeting alleviating AGE-RAGE damage and enhancing the anti-saccharification function of DDOST by increasing DDOST mRNA expression level. As a result, as shown in FIG. 3, the expression level of DDOST mRNA in the control group was about 1, and the expression level in the high sugar medium dry prognosis model group was reduced to 0.58; the metagen (CCFM 1283_Z) prepared by the lactobacillus plantarum CCFM1283 remarkably increases the expression level of DDOST mRNA in HSF cells to 1.15 (110.7 percent higher than that of a model group); however, the expression levels of DDOST mRNA after the treatment of the metazoan FXJCJ22M3_ Z, FSCDJY93L1_ Z, FXJCJ M6_Z) of other lactobacillus plantarum are only 0.71, 0.45 and 0.77 respectively, and the metazoan has no obvious up-regulation effect on the expression of the DDOST mRNA compared with the model group.
Matrix metalloproteinase 9 (MMP-9) is a class of enzymes belonging to the zinc-metalloproteinase family, an enzyme that mainly degrades type IV collagen and elastin, and extracellular matrix degradation involved in normal physiological and pathological processes; under high sugar conditions, MMP-9 expression increases, resulting in reduced proliferation of dermal fibroblasts, reduced viability, migration and reduced collagen secretion. FIG. 3 shows that the expression level of MMP-9mRNA in the control group was about 1, and the expression level in the high sugar medium dry prognosis model group was increased to 2.16; the metagen (CCFM 1283_Z) prepared by the lactobacillus plantarum CCFM1283 obviously reduces the MMP-9mRNA expression level in HSF cells to 0.75 (70.5 percent of the reduction compared with a model group); in contrast, the expression levels of MMP-9mRNA after the treatment of the metazoan FXJCJ22M3_ Z, FSCDJY93L1_ Z, FXJCJ M6_Z) of other Lactobacillus plantarum were about 6.11, 2.49 and 1.07, and none of them showed down-regulation effect on MMP-9mRNA expression compared with the model group.
According to experimental results, the metagen (thallus lysate) prepared by the lactobacillus plantarum CCFM1283 can down regulate the expression of MMP-9mRNA in high glucose culture to relieve protein dysfunction caused by saccharification, and up regulate the expression of DDOST mRNA to prevent AGE-RAGE from combining, so that the sugar damage to HSF cells caused by high glucose culture conditions is prevented.
Example 8: effect of Lactobacillus plantarum CCFM1283 and metazoan prepared therefrom on AGE levels in blood and skin of aging mice
The preparation methods of the progenitors (CCFM 1283_M and CCFM 1283_Z) of the Lactobacillus plantarum CCFM1283 in this example are the same as those of example 2, except that the CCFM1283_M is centrifuged after obtaining the bacterial liquid and then the bacterial sludge is collected without high-pressure homogenization.
The method comprises the following specific steps:
(1) 45 healthy male BALB/c mice of 8 weeks old are randomly divided into 9 cages, each cage is 5, and the 9 cages are respectively: the Model group (Model) was divided into 2 cages, and the blank group and the remaining group were each 1 cage, respectively:
blank (Control): physiological saline was used as a control;
model set (Model): physiological saline was used as a control;
CCFM1283 group:the lactobacillus plantarum CCFM1283 viable bacteria are used, and the dosage is as follows: 5X 10 9 CFU/kg mouse body weight;
CCFM1283_Z group: the lactobacillus plantarum CCFM1283 metazoan (thallus lysate) is used, and the dosage is as follows: 500mg/kg mouse body weight;
CCFM1283_M group: lactobacillus plantarum CCFM1283 metabolite (fermentation supernatant) was used at the dose: 500mg/kg mouse body weight;
wherein, the cleavage liquid or metabolite in each group above: dead bacteria or metabolites obtained by preparing bacterial liquid after fermenting corresponding bacterial amounts of living bacteria and the like.
Experiments were performed for 7 weeks: after mice were acclimatized for one week, D-galactose (1000 mg/kg) was subcutaneously injected into the remaining groups at 0.1 mL/day except for the blank group, and from the second week, each intervention group was gastric lavaged with the corresponding strain of lyophilized powder or strain-prepared metagen lyophilized powder (lysate and broth) in the corresponding dose, and the blank group and model group were lavaged with the same amount of physiological saline as the control until the end of the experiment. All groups were free-drinking and ingestion, and the experimental flow is shown in fig. 4.
After the experiment is finished, killing the mice to obtain eyeball blood, standing for 40min, centrifuging at a speed of 3000r/min for 20min, and obtaining blood supernatant for ELISA detection; the back skin tissue is cut and ground to be homogenate according to the weight-volume ratio of PBS1:10, the homogenate is centrifuged at 3000r/min for 20min, and the skin supernatant is taken and checked by ELISA kit.
Detection of AGE content in mouse serum and skin by ELISA kit is shown in figure 5:
serum AGE content: compared with a control group (262.70 ng/L), the AGE content in serum of a model group is obviously increased to 406.22ng/L, the oral lactobacillus plantarum CCFM1283 and a metaplastic comparison model group prepared by the oral lactobacillus plantarum CCFM1283 obviously reduce the AGE content of a saccharification aging marker in serum of mice, and the CCFM1283_Z group, the CCFM1283_M group and the CCFM1283 viable bacteria group respectively reduce the AGE content in serum to 329.72ng/L,267.61ng/L and 184.27ng/L, and 18.8 percent, 34.1 percent and 54.6 percent of the oral lactobacillus plantarum CCFM1283 and metaplastic (lysate and supernatant) prepared by the oral lactobacillus plantarum CCFM1283 can reduce the AGE content in serum of aging mice and reduce the accumulation of saccharification loss.
Skin AGE content: compared with a control group (336.76 ng/L), the AGE content in serum of the model group is obviously increased to 463.21ng/L, the content of the saccharification aging marker AGE in the skin of a mouse is obviously reduced by oral administration of a metaplasia comparison model group prepared from lactobacillus plantarum CCFM1283, the content of the AGE in the skin of the mouse is respectively reduced to 403.80ng/L and 413.09ng/L by the CCFM 1283-Z group and the CCFM 1283-M group, the content of the AGE in the skin is respectively reduced by 12.8 percent and 10.8 percent compared with the model group, and the content of the AGE in the skin of a aged mouse can be reduced by oral administration of metaplasia (lysate and supernatant) prepared from lactobacillus plantarum CCFM 1283.
The results show that the metazoan prepared by the lactobacillus plantarum CCFM1283 has the capability of relieving the accumulation of AGE content in serum and skin of the aging mice, and the improvement is obvious compared with a model group.
Example 9: effect of Lactobacillus plantarum CCFM1283 and metazoan prepared from same on content of inflammatory markers in serum and skin of aging mice
The design of the animal experiments and the lavage group involved in this example are the same as in example 8, and the TNF- α content in serum and skin of the aged mice is shown in fig. 6.
(1) Serum TNF- α content: compared with the control group (297.24 ng/L), the TNF-alpha content in the serum of the model group is obviously increased to 382.15ng/L, the content of TNF-alpha in the serum of mice is obviously reduced by taking the metazoan prepared from lactobacillus plantarum CCFM1283 orally, the TNF-alpha content in the serum of the mice is respectively reached to 278.78ng/L, 468.95ng/L, 346.62ng/L by the CCFM1283_Z group, the inflammatory factor TNF-alpha content in the serum of the model group corresponding to the CCFM1283 active bacterial group is respectively reduced by 27.0 percent and 9.3 percent by the CCFM1283_M group, and the TNF-alpha content in the serum is conversely increased by the CCFM1283_M group.
(2) Skin TNF-alpha content: the TNF- α content in the skin of the model group increased significantly to 486.73ng/L compared to the control group (351.25 ng/L), and the oral administration of the metazoans prepared from lactobacillus plantarum CCFM1283 significantly reduced the TNF- α content in the skin of mice, with the CCFM1283_z group, the CCFM1283_m group and the CCFM1283 viable bacteria group achieving TNF- α content in the skin of 383.09ng/L, 426.98ng/L, 434.11ng/L (21.3%, 12.3% and 10.8% reduction, respectively, compared to the model group).
The result of the related biochemical indexes in animal serum is comprehensive, and the metagen prepared by the lactobacillus plantarum CCFM1283 can reduce the content of inflammatory factor TNF-alpha in the serum and skin of the aging mice, and relieve inflammation to resist the overall aging condition of the host. Example 10: effects of Lactobacillus plantarum CCFM1283 and its prepared metazoan use on skin cuticle moisture content and skin elasticity of aging mice
The animal experiment design and the stomach lavage group in this example were the same as in example 8, and the skin elasticity performance of the back of each mouse was measured at the end of the experiment by using the skin elasticity tester MPA580 (Germany CK company), and the results are shown in FIG. 7.
(1) As can be seen from fig. 7, the skin elasticity R2 significantly reduced to 53.7% in the model group compared to the blank group (82.42%), 37.9% in the model group compared to the skin elasticity (74.03%) in the lactobacillus plantarum ccfm1283_z group, and 28.4% in the model group compared to the skin elasticity (68.95%) in the ccfm1283_m group. The lactobacillus plantarum CCFM1283 viable bacteria group also has obvious elastic recovery effect in this respect, and the skin elasticity performance is recovered to 82.12 percent.
According to experimental results, the metagen prepared from the lactobacillus plantarum CCFM1283 can obviously increase the skin elasticity performance of the back of the aged mice, the concentration of AGE is gradually increased in the aging process, collagen synthesis is reduced after the AGE and the receptor thereof interact, the original structural protein function is damaged, the cytoskeleton and cell space supporting capacity is reduced, and the exogenous complementary anti-saccharification functional product can relieve the change of the skin texture in the aging process, so that the metagen prepared from the lactobacillus plantarum CCFM1283 can obviously increase the back skin elasticity performance of the aged mice.
Example 11: influence of Lactobacillus plantarum CCFM1283 and use of metazoan prepared from same on synthesis and content of skin type 3 collagen of mice with glucose-impaired aging
The animal experiment design and the lavage group related to the present example are the same as those of example 8, and the type 3 collagen synthesis, degradation related gene expression and specific type 3 collagen content in the skin of the aging mice are shown in fig. 8.
The Trizol method is used for extracting skin RNA, and cDNA is used for detecting gene expression of key targets in the synthesis and degradation process of the skin type 3 collagen, and primers of COL3A1 mRNA and MMP-2mRNA are described in the following table 2.
Table 2: primer sequences
The type III collagen content in the back skin of the mice is shown in FIG. 8, the type 3 collagen content of the model group is significantly reduced to 6.09 μg/L compared with the blank group (7.17 μg/L), the type 3 collagen content of the CCFM1283_M group is increased by 31.2% compared with the model group (7.99 μg/L), but the type 3 collagen content of the CCFM1283_Z group is only 5.34 μg/L. The lactobacillus plantarum CCFM1283 viable bacteria also have similar function of maintaining the type 3 collagen content (6.66 mug/L, 9.4% higher than the model group), but no CCFM1283 self-fermentation supernatant is good (CCFM 1283_M).
As can be seen from the detection of the expression of COL3A1 mRNA, lactobacillus plantarum CCFM1283 and the metagen CCFM1283_Z prepared by the lactobacillus plantarum CCFM1283 can obviously up-regulate the expression of the type III collagen synthase COL3A1 mRNA to enable the relative expression quantity to be 1.23 and 1.86 (which are respectively increased by 180.1 percent and 323.2 percent compared with the model group 0.44), the abnormal reduction of the type III collagen synthesis caused by saccharification loss is relieved, and the normal function of the collagen is maintained.
By detecting the expression of MMP-2mRNA, lactobacillus plantarum CCFM1283 and the metagen CCFM1283_Z prepared by the lactobacillus plantarum CCFM1283 can obviously reduce the expression of matrix metalloproteinase MMP-2mRNA to make the relative expression quantity of the matrix metalloproteinase MMP-2mRNA be 0.08 and 0.31 (which are respectively reduced by 97.1 percent and 88.7 percent compared with a model group 2.77), thus relieving abnormal elevation of matrix metalloproteinase 2 caused by saccharification loss and maintaining normal function of protein.
Example 12: lactobacillus plantarum CCFM1283 and effects of use of metazoan prepared from same on relieving skin multi-angle saccharification damage
The design of animal experiments, lavage group and RNA extraction and detection in this example are the same as in example 11, and the primers of the key genes DDOST, cu/Zn-SOD in the skin of aged mice are described in Table 3 below. The effect of inhibiting AGE-RAGE binding on alleviating skin glycation lesions the results of gene expression and the effect of inhibiting downstream sugar damage inflammatory pathways and oxidative stress pathways are shown in figure 9.
Table 3: primer sequences
(1) Effect of the metagen prepared from Lactobacillus plantarum CCFM1283 on inhibiting AGE-RAGE binding to alleviate skin glycation injury
The detection of the expression of the DDOST mRNA shows that the expression level of the DDOST mRNA is still only 0.99 after the CCFM1283 viable bacteria is increased to 1.35 (139.8 percent higher than the model group 0.56) by the metagen CCFM1283_Z prepared by the lactobacillus plantarum CCFM1283, and no obvious improvement effect is achieved. Namely, oral administration of the post-natal (lysate) of Lactobacillus plantarum CCFM1283 can inhibit the binding of AGE-RAGE by increasing the expression of DDOST to compete for the binding site of RAGE and AGE, thereby alleviating the effect of skin glycation injury.
(2) Effect of the metazoan produced by Lactobacillus plantarum CCFM1283 on inhibition of downstream sugar injury oxidative stress pathways
By detecting the expression of Cu/Zn-SOD mRNA, the metagen CCFM1283_Z prepared from lactobacillus plantarum CCFM1283 can improve the expression of Cu/Zn-SOD mRNA to 1.71 (204.4 percent higher than that of a model group 0.49); the CCFM1238 viable bacterial group has no therapeutic effect of the target. Namely, the metaplasia (lysate) prepared by orally taking the lactobacillus plantarum CCFM1283 can relieve the subsequent saccharification damage of the skin by inhibiting the influence of downstream sugar damage oxidative stress channels.
Example 13: influence of Lactobacillus plantarum CCFM1283 fermented peanut coat on improvement of external anti-saccharification capability of peanut coat
Preparation of peanut coat fermentation broth (hsp) and lactobacillus plantarum CCFM1283 fermented peanut coat supernatant (CCFM 1283_H) was as previously described. Construction of in vitro fructose-bovine serum albumin System and detection of fluorescent AGE production As shown in example 4
Detection of prevention of glycation lesions caused by methylglyoxal on HSF cells is shown in example 6.
(1) The lactobacillus plantarum CCFM1283 fermented peanut coat supernatant improves the capacity of inhibiting fluorescent AGE generation of peanut coats in vitro:
the peanut coat extract mainly contains flavonoid compounds such as proanthocyanidin, resveratrol, quercetin and the like, and the oligomeric proanthocyanidin has the biological activities of scavenging free radicals, resisting oxidation, protecting heart and cerebral vessels, resisting inflammation, inhibiting tumors and the like, and the capacity of resisting saccharification damage is verified in a cell experiment. However, the peanut coat has low content of anti-glycation active substances and low bioavailability, and in order to better exert the anti-sugar function characteristic of the peanut coat, lactobacillus plantarum CCFM1283 is used for fermenting the peanut coat to prepare lactobacillus plantarum CCFM1283 fermented peanut coat supernatant (CCFM 1283_H), and the influence of lactobacillus plantarum CCFM1283 on the sugar resistance capability of the peanut coat is verified by using 100 mug/mL of culture solution for peanut coat fermentation and the concentration of the fermented peanut coat supernatant.
As shown in fig. 10, the inhibition rate of the original peanut coat on fluorescent AGE production of fructose-bovine serum albumin system is 61.70%, while the inhibition rate of the supernatant after fermentation of lactobacillus plantarum CCFM1283 reaches 75.05%, which is 21.6% higher than that of the unfermented group, and the ability of inhibiting AGE production is significantly improved.
(2) The capacity of the peanut coat supernatant obtained by fermenting the lactobacillus plantarum CCFM1283 for improving the in-vitro reduction of the HSF cell viability caused by methylglyoxal is improved:
as can be seen from fig. 10, the original HSF cell viability after peanut coat action was 52.23% (5.5% higher than model 54.93%), but after fermentation with lactobacillus plantarum CCFM1283, the HSF cell viability after CCFM1283_h action was 78.43% (47.3% higher than model 54.93%). The anti-saccharification effect of peanut coats is significantly improved by lactobacillus plantarum CCFM 1283.
Example 14: effect of Lactobacillus plantarum CCFM1283 fermented peanut coat on improving peanut coat oral inhibition of AGE accumulation in serum and skin
Experimental construction for aged mice as shown in example 8, preparation of peanut coat fermentation broth (hsp) and lactobacillus plantarum CCFM1283 fermented peanut coat supernatant (ccfm1283_h) as shown in example 3; the peanut coat synthesis preparation is added into the mixture for gastric lavage (viable bacteria lactobacillus plantarum CCFM1283 and peanut coat fermentation culture solution (hsp) which is marked as CCFM 1283+hsp).
(1) Accumulation of AGE in serum: as can be seen from FIG. 11, although peanut coat itself reduced AGE content in serum of aged mice to 246.95ng/L (262.70 ng/L in control group, 406.22ng/L in model group), by 39.2% compared to model group; however, the peanut coat fermentation liquor obtained by the lactobacillus plantarum CCFM1283 has better effect, and the AGE content is reduced to 219.98ng/L which is reduced by 45.85 percent compared with a model group; the peanut coat synthesis formulation group was not as effective as the other two groups, but only reduced 41.04% of the model group (AGE content 239.52 ng/L).
(2) Accumulation of AGE in skin: as can be seen from FIG. 11, although peanut coat itself reduced AGE content in the skin of the aged mice to 376.40ng/L (control 336.76ng/L, model 456.72 ng/L), 17.59% lower than model; however, the peanut coat fermentation liquor obtained by the lactobacillus plantarum CCFM1283 has better effect, and the AGE content is reduced to 352.38ng/L which is reduced by 17.59 percent compared with a model group; the peanut coat synthesis formulation group was not as effective as the other two groups, but only reduced 14.63% of the model group (AGE content 389.92 ng/L).
Example 15: effects of Lactobacillus plantarum CCFM1283 fermented peanut coat on improving peanut coat oral administration and relieving skin inflammation and oxidative stress caused by saccharification
Experimental construction for the aging mice is shown in example 8; the experimental set of the culture broth (hsp) for peanut coat fermentation, the culture supernatant (CCFM 1283_H) for peanut coat fermentation by Lactobacillus plantarum CCFM1283, and the gastric lavage group of the peanut coat synthesis preparation (live Lactobacillus plantarum CCFM1283 and the culture broth (hsp, referred to as CCFM 1283+hsp) for peanut coat fermentation) was set as in example 14, and the detection method was set as in example 12.
(1) Skin elasticity:
as can be seen from fig. 12, although peanut skin itself can raise the elastic performance R2 of the back skin of the aging mice to 63.98% (74.90% in the control group and 53.7% in the model group), it is significantly improved by 19.13% compared with the model group; however, the effect of peanut coat fermentation supernatant (CCFM 1283_H) fermented by lactobacillus plantarum CCFM1283 is better, and the skin elasticity R2 reaches 76.03 percent (41.6 percent improvement); the effect of the peanut coat synthetic preparation group is also improved, and the elastic performance reaches 74.96 percent (39.6 percent higher than that of a model).
(2) Skin glycation corresponding target point:
as can be seen from FIG. 13, the control group had 1.20% of the gastric lavage DDOST mRNA and the hsp group had 0.66% of the DDOST mRNA (17.87% higher than the model group 0.56), but the oral administration of CCFM 1283-H and CCFM1283+hsp resulted in 1.18 and 1.47% of the skin DDOST mRNA (109.9% higher than the model group and 161.0%) after the participation of Lactobacillus plantarum CCFM 1283.
The type III collagen content in the back skin of the mice shows that compared with a blank group (7.17 mug/L), the type 3 collagen content of a model group is obviously reduced to 6.09 mug/L, the type 3 collagen content of the back skin of the mice after the gastric lavage hsp group reaches 7.43 mug/L (22.0 percent higher than the model group), and after the fermentation synergy of lactobacillus plantarum CCFM1283, the type III collagen content of the skin after the gastric lavage CCFM1283_H and CCFM1283+hsp is respectively 7.51 mug/L and 7.49 mug/L (23.2 percent higher than the model group and 22.9 percent higher than the model group).
It can be seen from the two aspects, the lactobacillus plantarum CCFM1283 fermented peanut coat has positive influence on improving the skin elasticity caused by the saccharification of the peanut coat released by oral administration and corresponding targets.
Example 16: effects of Lactobacillus plantarum CCFM1283 fermented peanut coat on improving skin elasticity and oxidative stress caused by saccharification of peanut coat oral administration and relieving
Experimental construction for the aging mice is shown in example 8; skin IL-6 content determination the determination of skin TNF-alpha content was imitated as in example 9; the method for detecting Cu/Zn-SOD mRNA is shown in example 12. Preparation of peanut coat fermentation broth (hsp) and lactobacillus plantarum CCFM1283 fermented peanut coat supernatant (CCFM 1283_H) as described above; the peanut coat synthesis preparation is added into the mixture for gastric lavage (viable bacteria lactobacillus plantarum CCFM1283 and peanut coat fermentation culture solution (hsp) which is marked as CCFM 1283+hsp).
As can be seen from FIG. 14, the expression level of Cu/Zn-SOD mRNA was increased to 0.67 (36.94% higher than that of model group 0.49) in the gastric lavage hsp group, but after participation of Lactobacillus plantarum CCFM1283, i.e. after oral administration of CCFM1283_H and CCFM1283+hsp, the expression levels of Cu/Zn-SOD mRNA in the skin were 0.86 and 1.04 (76.4% and 113.2% higher than that of model group), respectively. The lactobacillus plantarum CCFM1283 improves the original capacity of the peanut skin for relieving oxidative stress after skin saccharification through fermentation.
Skin IL-6 levels As shown in FIG. 14, the IL-6 levels in the skin of the model group increased significantly to 106.01ng/L compared to the control group (74.84 ng/L), and the oral hsp group reduced IL-6 levels in the skin of mice to 85.94ng/L (18.93% lower than the model group), but the IL-6 levels after oral CCFM1283+ hsp were reduced to 85.06ng/L, 19.76% lower than the model group. The CCFM1283_H group also has the function of orally improving AGE accumulation in the skin, and the AGE content in the skin reaches 88.04ng/L (16.95% lower than that of the model group).
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. Lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1283 was deposited at the Cantonese microorganism strain collection at 10 and 14 days 2022 under the accession number: GDMCC No:62885.
2. a metazoan prepared using the Lactobacillus plantarum CCFM1283 strain of claim 1.
3. The metant of claim 2, wherein the metant comprises an inactivated or inactivated cell, cell culture and/or cell lysate.
4. A composition comprising lactobacillus plantarum CCFM1283 and/or lactobacillus plantarum CCFM1283 metants according to claim 1.
5. The composition of claim 4, wherein the composition comprises a food, a pharmaceutical, a nutraceutical, or a cosmetic.
6. The composition of claim 4, wherein the composition is the product of fermentation of the lactobacillus plantarum CCFM1283 in a peanut coat-containing medium.
7. Use of a lactobacillus plantarum CCFM1283 according to claim 1, a metazoan according to claim 2 or 3, or a composition according to any of claims 4 to 6 for the preparation of an anti-glycation, anti-aging product.
8. The use according to claim 7, characterized by comprising at least one of the following actions:
(1) Inhibiting the generation of fluorescent AGE;
(2) Preventing injury and function decrease of skin fibroblast caused by high glucose and AGE formed intermediate;
(3) Reducing the aging profile of the individual;
(4) Reducing AGE content in blood and skin tissue of an individual;
(5) Reducing AGE-RAGE binding in skin tissue of an individual;
(6) Reducing inflammatory responses and oxidative stress in skin tissue of an individual;
(7) Relieving skin elasticity and collagen decrease of aging individuals.
9. The use according to claim 7 or 8, wherein the product is a pharmaceutical, and wherein the content of lactobacillus plantarum CCFM1283 in the product is not less than 1 x 10 6 CFU/mL or 1X 10 6 CFU/g。
10. Use of the lactobacillus plantarum CCFM1283 of claim 1, the metazoan of claim 2 or 3 in the preparation of a food product.
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