CN117625457A - Lactobacillus plantarum CCFM1354 with targeted glucose resistance, aging resistance and skin health improvement functions and metayuan thereof - Google Patents
Lactobacillus plantarum CCFM1354 with targeted glucose resistance, aging resistance and skin health improvement functions and metayuan thereof Download PDFInfo
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Abstract
The invention discloses a target glucose-resistant, anti-aging and skin health-improving lactobacillus plantarum CCFM1354 and its progeny, belonging to the technical field of microorganisms and medicines. The invention screens and obtains a lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1354, and provides application of the lactobacillus plantarum CCFM1354 and the metagen prepared by the same in the aspects of anti-saccharification, anti-aging and skin health improvement. The strain of the invention can be used for preparing targeted anti-sugar anti-aging external or oral products for improving skin health, and has great application prospect.
Description
Technical Field
The invention relates to a plant lactobacillus CCFM1354 for targeting, resisting sugar and aging and improving skin health and a progeny thereof, belonging to the technical field of microorganisms and medicines.
Background
Glycosylation, also known as maillard reaction, occurs widely in humans, and refers to the non-enzymatic condensation reaction between carbonyl groups on reducing sugars (e.g., glucose) and free amino groups on macromolecules such as proteins, lipids, or nucleic acids, which irreversibly produce stable adducts, i.e., advanced glycation end products (Advanced glycation end products, AGE). The reactive nature of AGE is very prone to cause subsequent glycation lesions in the body, where AGE captures and crosslinks adjacent proteins to affect normal functional properties of the protein on the one hand, and binds to AGE-specific Receptors (RAGE) on the cell surface to activate various signaling pathways, inducing further oxidative stress, inflammatory responses, etc.
As the largest organ in the body, the skin is most severely damaged by glycation; the aging is most pronounced in the adverse effects of glycation on the skin. AGE also gradually accumulates in the skin with AGE, affecting long-lived skin proteins such as collagen and elastin, causing post-translational modification of extracellular matrix proteins to be disturbed. The link between glycation and skin health is very tight, and as a result of glycation damage, skin is more likely to exhibit problems of increased dryness, reduced elasticity, wrinkles and pigmentation, etc., so there is increasing attention to anti-glycation relief of skin aging and sub-health.
Currently, the anti-glycation representative ingredients are mainly carnosine, lipoic acid, nicotinamide and plant extracts rich in flavonoids and polyphenols. Their anti-glycation efficacy is exerted more by the way AGE is reduced, with limited anti-glycation efficacy for the subsequent sustained glycation lesions of AGE in the body. CN116350531a discloses an anti-glycation application of opal D, which verifies that the opal D, an extracted component of crowndaisy chrysanthemum, has an anti-glycation effect. CN116570544a discloses an anti-saccharification emulsion and a preparation method thereof, wherein plant extracts such as raspberry, turmeric and grape seeds are added into the emulsion to realize the anti-saccharification effect of the skin care product emulsion. CN116459172a provides anti-glycation applications of vitexin. Although these patents relate to the anti-sugar efficacy of different plant extracts, the main focus is on reducing the AGE content, and the anti-sugar means for other processes of sugar damage are under-utilized, and for this purpose, the following probiotic bacteria and their metagen anti-glycation functional pathways are proposed: the probiotics and the metagen inhibit the generation of AGEs and prevent saccharification reaction; probiotics and their metagens inhibit direct biochemical reactions caused by AGEs by inhibiting AGE-RAGE binding, reducing cross-linking of AGEs with proteins; the probiotics and its metazoans inhibit the activation of the subsequent signal path of saccharification and reduce the continuous cascade reaction in the body downstream.
Disclosure of Invention
The invention provides lactobacillus plantarum (Lactiplantibacillus plantarum) and a metagen prepared by the same, which can target to resist sugar and aging and improve skin health.
The invention provides a lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1354, which is named Lactiplantibacillus plantarum in taxonomy and is preserved in the microorganism strain collection of Guangdong province at 10 months 25 in 2023, wherein the preservation number is GDMCC No:63924, the preservation address is Guangzhou Mr. first 100 college No. 59 building.
The lactobacillus plantarum CCFM1354 is separated from healthy human body excrement, and the 16S rRNA sequence of the strain is shown as SEQ ID NO.1 through sequencing analysis.
The lactobacillus plantarum CCFM1354 forms colony with medium diameter, bulges, rough surface and curled edge under a microscope, and the colony is generally yellowish, smooth bulges and round with the diameter of 3mm after being inoculated on MRS culture medium for 48 hours.
The lactobacillus plantarum CCFM1354 is a gram positive bacterium, is facultative anaerobic and is warm, the optimal growth temperature is 35-40 ℃, and the optimal growth pH is 6.0-7.0.
The invention also provides a metagen prepared by using the lactobacillus plantarum CCFM 1354.
In one embodiment, the metazoan comprises inactivated or inactivated cells, fermentation supernatant, bacterial lysate, or a dried powder of any of the foregoing.
In one embodiment, the inactivated or inactivated cells are prepared as follows: culturing the lactobacillus plantarum CCFM1354 in a culture medium for a period of time, collecting the bacterial cells in a cell culture solution, and carrying out heat treatment or freeze drying to obtain the inactivated bacterial 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 CCFM1354 in a culture medium for a period of time, collecting bacterial cells, homogenizing under high pressure, centrifuging, and collecting the supernatant after centrifuging to obtain bacterial lysate.
In one embodiment, the fermentation supernatant is a supernatant obtained by culturing lactobacillus plantarum CCFM1354 in a medium for a period of time and centrifuging.
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 CCFM1354 and/or the 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 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 profile of the individual, including but not limited to blood biochemical indicators, skin appearance.
(4) Preventing abnormal degradation of collagen of skin fibroblasts (HSF) under high glucose culture;
(5) Preventing abnormal expression of EGR2 mRNA of a key growth regulation target point of skin fibroblasts (HSF) under high glucose culture;
(6) Reducing AGE content in serum, skin, liver, kidney, brain tissue of aging individuals;
(7) Reducing the content of inflammatory markers IL-6 and TNF-alpha in serum of aged individuals;
(8) Reducing the water content of the skin horny layer at the back of an aged individual caused by saccharification injury;
(9) Reducing the content of III type collagen in the back skin of an aged subject caused by saccharification injury.
The invention provides application of lactobacillus plantarum CCFM1354 and/or metazoan thereof in preparing a product for resisting sugar and aging and improving skin health problems caused by saccharification.
In one embodiment, the anti-glycation and anti-aging comprises inhibiting the levels of glycation senescence markers AGE in blood, skin, liver, and/or kidney, inhibiting the levels of inflammatory injury markers TNF-alpha and IL-6 in serum, inhibiting muscle strength decline during aging, and inhibiting skin aging characterization.
In one embodiment, the improvement of glycation-induced skin health problems includes alleviating a decrease in skin stratum corneum moisture, alleviating a decrease in skin elasticity state in multiple dimensions, and alleviating skin type III collagen loss.
In one embodiment, the product includes, but is not limited to, a food product or a topical skin care product stock.
In one embodiment, the product is used in a manner that includes, but is not limited to, topical, oral use.
In one embodiment, the product has a content of Lactobacillus plantarum CCFM1354 of not less than 1×10 6 CFU/mL or 1X 10 6 CFU/g。
In one embodiment, the post-natal prepared by lactobacillus plantarum CCFM1354 is used in a dosage of not less than 10mg/kg body weight.
In one embodiment, the food product contains the lactobacillus plantarum CCFM1354 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 CCFM1354 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 pharmaceutical product contains the lactobacillus plantarum CCFM1354 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 contains the lactobacillus plantarum CCFM1354 and/or its metazoan, as well as 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 provides application of the composition in preparing a product for preventing and/or relieving skin aging related symptoms.
In one embodiment, the symptoms associated with skin aging include dry skin, reduced elasticity, sagging, wrinkling, oxidative damage, collagen loss.
The invention also provides application of the lactobacillus plantarum CCFM1354 or the metazoan in preparing food.
The beneficial effects are that:
the invention screens and obtains a lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1354, and the lactobacillus plantarum CCFM1354 and the metazoan prepared by the lactobacillus plantarum CCFM1354 have the functions of anti-saccharification, anti-aging and improving skin health, and are specifically expressed in the following steps:
(1) Inhibiting the generation of fluorescent AGE in an in vitro fructose-bovine serum albumin system;
(2) Preventing a decrease in cell viability in a methylglyoxal to skin fibroblast (HSF) injury model;
(3) Preventing abnormal degradation of collagen of skin fibroblasts (HSF) under high glucose culture;
(4) Preventing abnormal expression of EGR2 mRNA of a key growth regulation target point of skin fibroblasts (HSF) under high glucose culture;
(5) Reducing AGE content in serum, skin, liver, kidney, brain tissue of aging individuals;
(6) Reducing the content of inflammatory markers IL-6 and TNF-alpha in serum of aged individuals;
(7) Reducing the water content of the skin horny layer at the back of an aged individual caused by saccharification injury;
(8) Reducing the content of III type collagen in the back skin of an aged subject caused by saccharification injury.
Therefore, the lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1354 and the metazoan prepared by the same have great application prospect in targeted products for resisting sugar and aging and improving skin health.
Preservation of biological materials
Lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1354, taxonomic designation Lactiplantibacillus plantarum, was deposited on the microorganism strain collection in Guangdong province at day 25 of 10 in 2023 under the accession number GDMCC No:63924, the preservation address is Guangzhou Mr. first 100 college No. 59 building.
Drawings
Fig. 1: influence of different metagens on the generation inhibition of fluorescent AGE in an in vitro fructose-bovine serum albumin system;
fig. 2: influence of different metants on HSF cell proliferation;
fig. 3: different metagens prevent the influence of HSF cell viability under the action of methylglyoxal which is a saccharification reaction intermediate;
fig. 4: different metagens prevent the effects of MMP-9mRNA and EGR 2mRNA expression in HSF cells under high glucose culture;
fig. 5: a mouse experiment flow chart;
fig. 6: influence of Lactobacillus plantarum CCFM1354 and its prepared metagen on AGE, IL-6 and TNF-alpha content in mouse blood;
fig. 7: the effect of lactobacillus plantarum CCFM1354 and its prepared metazoan on AGE content in multiple organs of aging mice (skin, liver, brain);
fig. 8: effect of lactobacillus plantarum CCFM1354 and its prepared metagen on moisture content of skin stratum corneum;
fig. 9: effect of lactobacillus plantarum CCFM1354 and its prepared metagen on skin type III collagen content;
fig. 10: the effect of Lactobacillus plantarum CCFM1354 and its prepared metagen on the expression of target genes (RAGE mRNA, DDOST mRNA, MMP-2mRNA, COL3A1 mRNA) related to skin saccharification loss;
fig. 11: influence of lactobacillus plantarum CCFM1354 fermented peanut coat on improvement of in-vitro anti-saccharification capability of peanut coat;
Fig. 12: the effect of lactobacillus plantarum CCFM1354 fermented peanut coat on improving the capacity of peanut coat for inhibiting AGE accumulation by oral administration;
fig. 13: the effect of lactobacillus plantarum CCFM1354 fermented peanut coat on improving the oral anti-skin saccharification damage of peanut coat.
"" 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 CCFM1354, 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; the commercial peanut coat extract was purchased from Shaanxi Cheng Heng Biotechnology Inc., 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: screening and identification of lactobacillus plantarum
1. Screening
The method comprises the steps of (1) pretreating a sample from healthy human body excrement, storing the sample in a refrigerator at the temperature of minus 80 ℃ in 20% glycerol, 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 the physiological saline, selecting proper gradient diluent to be coated on an MRS solid culture medium, culturing for 48 hours at the temperature of 37 ℃, picking a typical colony of lactobacillus plantarum, streaking and purifying the colony on the MRS solid culture medium, picking a single colony, transferring the single colony to the MRS liquid culture medium for enrichment, and storing 30% glycerol to obtain a strain, wherein the strain is named as CCFM1354; wherein, the typical colony of the lactobacillus plantarum is round, faint yellow and smooth.
2. Authentication
The genome of the strain CCFM1354 is extracted, the 16S rDNA of the strain CCFM1354 is amplified and sequenced (the nucleotide sequence of the 16S rDNA of the strain CCFM1354 is shown as SEQ ID NO.1 by Jin Weizhi biotechnology Co., ltd., suzhou) and the sequence is subjected to nucleic acid sequence alignment in NCBI, so that the strain is shown to be lactobacillus plantarum and named as lactobacillus plantarum (Lactiplantibacillus plantarum) CCFM1354.
Example 2: cell resuscitation and culture
Firstly taking out frozen Human Skin Fibroblast (HSF), 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 medium containing 5% CO 2 Culturing in a 37 ℃ incubator, and carrying out cell passage when the cell is recovered and grown for 1-2 d and reaches 70% -80% fusion.
Example 3: preparation of Lactobacillus plantarum CCFM1354 metazoan
(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; 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 2-5% (v/v) 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 (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 CCFM1354 fermentation supernatant (designated CCFM 1354_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 at 8000r/min for 30min after homogenizing, collecting supernatant, collecting bacterial lysate (marked as CCFM 1354_Z), and freeze-drying to obtain the metagen freeze-dried powder for later use.
The preparation method of the lactobacillus plantarum CCFM1354 viable bacteria is the same as that of the metaplasia, except that bacterial liquid b is subjected to centrifugation under the condition of 8000r/min and 30min to obtain bacterial mud, the bacterial mud is subjected to reselection according to the proportion of 1g to 2mL of freeze-drying protective agent, and then the bacterial mud is directly freeze-dried to obtain lactobacillus plantarum CCFM1354 viable bacteria powder, which is marked as CCFM1354.
The preparation method comprises the following steps: lactobacillus plantarum CCFM1354 metazoan (cell lysate CCFM1354_Z and fermentation supernatant CCFM 1354_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 4: preparation of peanut coat fermentation liquid by fermenting peanut coat with lactobacillus plantarum CCFM1354
(1) Activation of Lactobacillus plantarum CCFM1354
Dipping lactobacillus plantarum CCFM1354 bacterial liquid by an inoculating loop, 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.
(2) Preparation of culture solution for peanut coat fermentation
5g/L of commercial peanut coat extract, 4g/L of glucose, 5g/L of yeast powder and 4g/L of calcium carbonate are added, the pH is adjusted to 6.8-7.2, and sterilization is carried out at 115 ℃ for 20min, so as to prepare a culture solution (abbreviated as hsp) for peanut coat fermentation.
(3) Fermentation of Lactobacillus plantarum CCFM1354
Inoculating the activated bacterial liquid obtained in the step (1) into the culture liquid for fermenting the treated peanut skin obtained in the step (2) with an inoculum size of 2% (v/v), and carrying out shake culture at 200rpm for 72 hours at 37 ℃.
(4) Preparation of lactobacillus plantarum CCFM1354 fermented peanut coat supernatant
Collecting fermentation broth for 72h, centrifuging the bacterial broth at 8000r/min for 30min to obtain supernatant, heat treating the supernatant (65 deg.C for 30 min), and lyophilizing to obtain powder for use, and preparing into the final product: lactobacillus plantarum CCFM1354 ferments peanut coat supernatant (noted ccfm1354_h).
Example 5: effect of the metazoan produced by Lactobacillus plantarum CCFM1354 on the inhibition of the production of fluorescent AGE in vitro fructose-bovine serum Albumin System
10mg/mL bovine serum albumin and 0.5. 0.5M d- (+) -fructose are mixed according to the final concentration in 0.1M phosphate buffer (pH 7.4), and a filter membrane with a water system of 0.22 mu M is used for obtaining a fructose-bovine serum albumin saccharification system under aseptic conditions. Metagen samples with a concentration of 5% were prepared with PBS, respectively, and incubated with a fructose-bovine serum albumin saccharification system at 37℃for 7 days. The incubated samples were used to detect the formation of fluorescent AGEs 7 days after incubation.
The grouping is as follows:
CCFM1354_Z: using a lactobacillus plantarum CCFM1354 thallus lysate;
CCFM1354_M: the supernatant was fermented using lactobacillus plantarum CCFM 1354.
JCM1132_z, using lactobacillus plantarum JCM1132 cell lysate;
JCM1132_m, fermenting the supernatant using lactobacillus plantarum JCM 1132;
FFJND7L5_Z is prepared from lactobacillus plantarum FFJND7L5 bacterial lysate; the method comprises the steps of carrying out a first treatment on the surface of the
FFJND7L5_M using Lactobacillus plantarum FFJND7L5 cell supernatant;
FSHXBX32L130_Z is prepared from lactobacillus plantarum FSHXBX32L130 thallus lysate;
FSHXBX32L 130-use Lactobacillus plantarum FSHXBX32L130 cell supernatant.
After the incubation, fluorescent AGEs in glucose-modified BSA were detected with an excitation wavelength of 340nm and an emission wavelength of 440nm, and the percent inhibition of fluorescent AGEs generation was calculated as 1 minus the difference in fluorescence intensity of the sample compared to the control (bsa+/glucose+).
The effect of the inhibition of fluorescent AGE production in the in vitro fructose-bovine serum albumin system is shown in FIG. 1, and the inhibition of AGE by CCFM1354_Z and CCFM1354_M is 54.46% and 35.66%, respectively, which is obvious compared with other Lactobacillus plantarum.
Example 6: effect of the metazoan produced by Lactobacillus plantarum CCFM1354 on HSF cell proliferation
The method comprises the following specific steps:
(1) 100. Mu.L of HSF cells in logarithmic growth phase was taken at 3X 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: resuspension of metazoan with cell culture medium (amount of metazoan 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. 2, the effect on cell proliferation was compared with the control group (cell proliferation rate 100%), and the concentration of the cells inactivated was 5.0X10 by adding the metagens (CCFM 1354_M and CCFM 1354_Z) of Lactobacillus plantarum CCFM1354, the metagens (FXJCJ22M3_M and FXJCJ22M3_Z) of Lactobacillus plantarum FJCJY 22M3, the metagens (FSCDJY 93L1 (FSCDJY 93L1_M and FSCDJY93 L1_Z)) of Lactobacillus plantarum FXJCJ26M6 (FXJCJ26 M6_M and FXJCJ26 M6_Z) of Lactobacillus plantarum CCFM1354, and the like 7 The cell proliferation rates at CFU/mL were 106.05%, 115.63%, 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 7: the metagen prepared from lactobacillus plantarum CCFM1354 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 concentration of individual cells/holes is inoculated in a 96-well plate, wherein the outermost ring is filled with PBS solution to prevent edge effect, and after the cells/holes are cultured for 24 hours and are attached to the wall, a blank group, a control group 1 and a treatment group 1 are arranged;
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 CCFM1354, 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 change of the control group 1 in the step (1), the control group contains a cell culture medium and HSF cells, and does not contain a methylglyoxal molding agent after being subjected to metaplasia treatment;
model group: changing the liquid of the control group 1 in the step (1) into a cell culture medium containing a methylglyoxal molding compound, containing original HSF cells without post-metaplasia treatment,
cell culture medium containing a methylglyoxal molding composition: uniformly mixing methylglyoxal in a common cell culture medium, sterilizing by using a 0.22 mu m water-based filter membrane, wherein the final concentration of methylglyoxal in the cell culture medium is 400 mu mol/L;
treatment group: the treatment group 1 in the step (1) is changed into a cell culture medium containing a methylglyoxal molding agent, and the cell culture medium contains original HSF cells and is subjected to metaplasia treatment.
(3) And (3) respectively carrying out the steps of (2) preparing the pore plate at the temperature of: incubation was performed in an incubator at 37℃for 24h, and after incubation, 10. Mu.L of CCK8 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. 3, 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 groups added with CCFM1354_M and CCFM1354_Z is 68.80% and 79.54% respectively, wherein compared with the treated groups, the CCFM1354_Z remarkably improves the activity of HSF cells compared with the treated groups, which proves that the metagen of the lactobacillus plantarum CCFM1354 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) were treated, and the HSF cell viability was 37.99%, 43.54%, 42.92%, 35.04%, 49.72%,36.02%, respectively, i.e. the post-metants of other lactobacillus plantarum did not have the ability to prevent damage to HSF cells exhibited by the post-metants of lactobacillus plantarum CCFM 1354.
Example 8: the metagen prepared by the lactobacillus plantarum CCFM1354 prevents the influence of MMP-9mRNA and EGR2 mRNA expression in HSF cells under high glucose culture.
The method comprises the following specific steps:
(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;
control group 1: the group without metagen;
the treatment components are lactobacillus plantarum CCFM1354 lysate (CCFM 1354_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 used for resuspension of metazoan after treatment (the amount and the fermentation of metazoan after resuspension until the concentration is 5.0x10) 7 The amount of metagen prepared by CFU/mL bacterial liquid is equivalent).
2mL of a Lactobacillus plantarum CCFM1354 lysate (CCFM 1354_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:
control group: after the liquid is changed in the control group 1, the original HSF cells are not treated by metaplasia and are added with 2mL of common cell culture medium;
model group: step (1), changing the liquid of the control group 1 into a cell culture medium containing 35mmol/L glucose, wherein the cell culture medium contains original HSF cells and is not subjected to metaplasia treatment;
treatment group: and (3) changing the liquid of the control group 1 into a cell culture medium containing 35mmol/L glucose, and carrying out metaplasia treatment on the cell culture medium containing the original HSF cells.
(3) Will step by stepIncubating the above-mentioned well plate in step (2) in an incubator at 37 ℃ for 24 hours, discarding culture supernatant, rapidly washing 3 times with PBS per well, adding 1mL of cell lysate per well, repeatedly blowing, sucking the 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 MMP-9mRNA and EGR2 mRNA were calculated using the formula, wherein the internal reference was beta-actin, and the primers are described in Table 1 below, and the results are shown in FIG. 4.
TABLE 1 primer sequences
As a result, as shown in FIG. 4, the expression level of MMP-9mRNA in the control group was about 1, and the expression level in the model group was increased to 2.54 after intervention with the high sugar medium (cell culture medium containing 35mmol/L glucose); the metagen (CCFM 1354_Z) prepared by the lactobacillus plantarum CCFM1354 obviously reduces the MMP-9mRNA expression level in HSF cells to 0.75; in contrast, the expression levels of MMP-9mRNA after the treatment of the metazoan of other Lactobacillus plantarum (FXJCJ22M3_ Z, FSCDJY93L1_ Z, FXJCJ M6_Z) were about 5.19, 2.30 and 1.22, and none of them showed significant downregulation of MMP-9mRNA expression compared with the model group. The expression quantity of the EGR2 mRNA in the HSF cells is obviously increased to 2.59 by using a control group, wherein the expression quantity of the EGR2 mRNA is about 1, the dry prognosis expression quantity of a high sugar culture medium is reduced to 0.75, and the metagen (CCFM 1354_Z) prepared by the lactobacillus plantarum CCFM1354 is obviously increased; other group treatments did not achieve similar regulatory effects on EGR2 mRNA from cells that were subject to saccharification loss.
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. Early growth reaction protein 2 (Early growth response protein, EGR 2) is induced by T cell receptor, is a substance necessary for inducing T cell disability, and participates in the regulation of inflammatory pathways; in hyperglycemic conditions, EGR2 expression is reduced, inhibiting anti-inflammatory Socs1 and increasing the pro-inflammatory gene. The results show that the metagen (thallus lysate) prepared by the lactobacillus plantarum CCFM1354 can down regulate the expression of MMP-9mRNA in high glucose culture, up regulate the expression of EGR2 mRNA and prevent sugar damage to HSF cells caused by high sugar culture conditions.
Example 9: lactobacillus plantarum CCFM1354 and its prepared metazoan effect on blood biochemical index level (AGE, TNF-alpha, IL-6 content) of aging mice
The preparation of the metagens (CCFM 1355_M and CCFM 1354_Z) of Lactobacillus paracasei CCFM1354 referred to in the following examples is the same as that of example 3; the CCFM1354_Z group is bacterial mud obtained by centrifuging the bacterial liquid b obtained in the group at 8000r/min and 30min, re-suspending the bacterial mud with physiological saline with the volume of 75% of the original bacterial liquid, carrying out heat treatment (65 ℃ for 30 min) on the re-suspension, carrying out no high-pressure homogenization to obtain inactivated bacterial cells (marked as CCFM 1354_Z), and carrying out freeze-drying to obtain the metagen freeze-dried powder for later use.
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;
CCFM1354 group: the lactobacillus plantarum CCFM1354 viable bacteria are used, and the dosage is as follows: 5X 10 9 CFU/kg mouse body weight;
CCFM1354_Z group: lactobacillus plantarum CCFM1354 metazoan (inactivated thallus) was used at the following doses: 500mg/kg mouse body weight;
CCFM1354_M group: lactobacillus plantarum CCFM1354 metabolite (fermentation supernatant) was used at the dose: 500mg/kg mouse body weight;
wherein, the fermentation supernatant or the inactivated bacteria in each group are as follows: the inactivated bacteria or metabolites are prepared by the bacterial liquid fermented with the bacterial amount 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. 5.
Mice were sacrificed to obtain eyeball blood after the experiment was completed, and serum was obtained by centrifugation at 3000r/min for 20min after standing for 40 min. The AGE, IL-6, TNF- α content of skin detected by ELISA kit is shown in figure 6:
(1) AGE content: the post-natal comparison model group, prepared from lactobacillus plantarum CCFM1354 orally, significantly reduced the level of the saccharification senescence marker AGE in mouse serum compared to the control group (262.70 ng/L), the AGE levels in model group serum were significantly increased to 406.22ng/L, and the ccfm1354_z group and the ccfm1354_m group reduced the AGE levels in serum to 305.51ng/L and 286.87ng/L, respectively. (24.8% and 29.4% decrease compared to model group, respectively). Oral administration of Lactobacillus plantarum CCFM1354 live bacteria has the best effect in reducing AGE content in serum of aged mice, and the AGE content is reduced to 178.67ng/L (56% compared with model group).
(2) IL-6 content: the IL-6 content in the serum of the model group was significantly increased to 126.79ng/L compared to the control group (80.20 ng/L), and the IL-6 content in the serum of mice was significantly reduced by the oral administration of the metazoan prepared from Lactobacillus plantarum CCFM1354, and the IL-6 content in the serum was 89.18ng/L, 114.167ng/L and 97.19ng/L by the CCFM 1354-Z group, the CCFM 1354-Z group and the CCFM1354 viable bacteria group, respectively, and the reduction of the IL-6 content of the inflammatory factor of the model group by 29.66% was most remarkable.
(3) TNF-alpha 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 CCFM1354 orally, and the TNF-alpha content in the serum is respectively 324.40ng/L, 379.53ng/L and 374.67ng/L by using the CCFM1354_Z group, the CCFM1354_M group and the CCFM1354 viable bacteria group, and the inflammatory factor TNF-alpha content of the model group is obviously reduced by 15.1 percent only by using the CCFM 1354_Z.
The result synthesis of related biochemical indexes in animal serum shows that lactobacillus plantarum CCFM1354 and the prepared metaplasia (inactivated thallus) thereof can resist saccharification and relieve inflammation by obviously reducing the AGE content and the inflammatory factors IL-6 and TNF-alpha content to resist the overall aging condition of a host.
Example 10: effect of Lactobacillus plantarum CCFM1354 and metazoan prepared from same on AGE content in aged mouse skin
The method for establishing the animal model in the following example is the same as that in example 9, except that after the mice are sacrificed after the experiment is completed, back skin tissues are sheared and ground according to the weight-to-volume ratio of PBS1:10 to prepare homogenate, the homogenate is centrifuged at 3000r/min for 20min, and the supernatant is taken and used for detecting the AGE content in the skin through an ELISA kit.
The results of detecting the AGE content of the marker for glycation reaction in each organ are shown in fig. 7:
skin: skin is the largest organ of the body, the accumulation of AGE in skin causes skin conditions, and an increase in AGE content causes a significant change in the signs of skin aging. The AGE levels in the skin of the model group increased significantly to 463.21ng/L compared to the control group (336.76 ng/L), the post-natal comparison model group, prepared orally from lactobacillus plantarum CCFM1354, reduced the levels of the glycation senescence markers AGE in the skin of mice, the ccfm1354_z group and the ccfm1354_m group reduced the AGE levels in the skin to 406.50ng/L and 421.13ng/L, respectively (12.2% and 9.1% compared to the model group); the oral CCFM1354 viable bacteria can reduce the skin AGE content to 350.13ng/L, and compared with a model group, the oral CCFM1354 viable bacteria can reduce the skin AGE content by 24.4%, and the effect is obvious.
From the results, the lactobacillus plantarum CCFM1354 and the metazoan (inactivated thallus and fermentation supernatant) prepared by the lactobacillus plantarum CCFM1354 can not only remarkably reduce the AGE content in serum, but also relieve the accumulation of AGE in skin tissues, and achieve the purposes of host anti-saccharification, anti-aging and skin health improvement.
Example 11: effect of Lactobacillus plantarum CCFM1354 and its prepared metazoan on anti-glycation damage ability of aged mouse skin
The method for establishing an animal model in the following example was the same as in example 9, and the skin elasticity tester MPA580 (Corneometer CM 825) from CK company in Germany was used at the end of the experiment to measure the moisture content of the horny layer at the back of each mouse, and the results are shown in FIG. 8.
The skin tissue homogenate prepared by the method for detecting Elisa kit in mice was the same as in example 10, and the type III collagen content in the skin tissue homogenate was detected by the skin type III collagen Elisa kit, and the results are shown in FIG. 9.
Extracting skin RNA by Trizol method, and reversely transcribing into cDNA to detect gene expression of related target points in saccharification loss process. Genes directly related to sugar damage in the skin tested included COL3A1, MMP-2, DDOST, RAGE, and the primers are described in Table 2 below, and the results of gene expression assays are shown in FIG. 10.
TABLE 2 primer sequences
(1) As can be seen from fig. 8, compared with 66.78% of the blank group, the moisture content of the horny layer of the model group is significantly reduced to 47.95%, the moisture content (62.75%) of the horny layer of the lactobacillus plantarum CCFM1354_z group is increased by about 30.87% compared with the model group, the moisture content (55.62%) of the lactobacillus plantarum CCFM1354_m group is increased by about 15.60% compared with the model group, and the moisture content (62.34%) of the lactobacillus plantarum CCFM1354 group is increased by 29.97% compared with the model group; that is, it is known from the experimental results that the metazoan prepared from Lactobacillus plantarum CCFM1354, particularly its inactivated cells (Lactobacillus plantarum CCFM 1354_Z), can increase the moisture content of the back of the aged mice after saccharification injury.
In the aging process, the AGE concentration is gradually increased, and the AGE is crosslinked with surrounding longevity proteins or the expression of the metalloproteinase is enhanced through an AGE-RAGE approach, so that the skin structure is loose, the water holding capacity of the skin stratum corneum is reduced, and exogenous supplementation of the metagen with the anti-saccharification function can relieve the skin moisture loss in the aging process; lactobacillus plantarum CCFM1354 and the metaplasia prepared by the same have the function of preventing skin dryness in the aging process after being orally taken. The metagen (CCFM 1354_Z) prepared by the lactobacillus plantarum CCFM1354 has more obvious effect of relieving the reduction of the moisture content of the horny layer caused by saccharification injury than the pure fermentation supernatant CCFM1354_M group, and the CCFM1354 viable bacteria have the strongest effect of up-regulating the moisture content of the horny layer.
(2) The type III collagen content in the back skin of mice is shown in FIG. 9, the skin elasticity compactness of the model group is significantly reduced to 6.09 μg/L compared to the blank group (7.17 μg/L), the skin elasticity of the Lactobacillus plantarum CCFM1354_Z group (8.16 μg/L) is increased by 33.93% compared to the model group, and the skin elasticity of the CCFM1354_M group (7.60 μg/L) is increased by 24.78% compared to the model group. The metazoan prepared from lactobacillus plantarum CCFM1354 significantly increased the medium collagen content in the backs of aging mice.
(3) In order to explore the influence mechanism of metaplasia specifically blocking saccharification damage on collagen synthesis, skin gene expression is detected. The results are shown in fig. 10, wherein: (1) the metagen CCFM1354_Z prepared from the lactobacillus plantarum CCFM1354 can significantly reduce the expression of the AGE receptor RAGE mRNA to 0.93 (68.4 percent lower than the model group 2.95); at the same time, up-regulates the expression of the RAGE competitive receptor AGE 1, DDOST mRNA, to 0.95, (129.3% higher than 0.41 in model group), thereby blocking AGE-RAGE binding in both ways. The viable bacteria of the lactobacillus plantarum CCFM1354 can reduce RAGE mRNA to 1.01 (65.6 percent lower than that of a model group), and the effect is slightly lower than that of the CCFM1354_Z; however, the effect of the viable bacteria on enhancing the expression of the competitive receptor DDOST mRNA of RAGE was not obvious, and the expression level was only 0.45 (model group 0.41). (2) The metagen CCFM1354_Z prepared from the lactobacillus plantarum CCFM1354 can significantly reduce the expression of the metalloproteinase MMP-2mRNA to 0.95 (67.6 percent lower than the model group 2.77); the lactobacillus plantarum CCFM1354 viable bacteria can reduce the MMP-2mRNA expression level to 0.48 (83.8 lower than the model). Namely, the Lactobacillus plantarum CCFM1354 and the metaplasia prepared by the same can prevent adverse effects on protein functions under saccharification reaction. (3) The Lactobacillus plantarum CCFM1354 and the metagen CCFM1354_Z prepared by the same can obviously up-regulate the expression of III type collagen synthase COL3A1 mRNA, the expression levels of the two are respectively 2.04 and 1.89 (which are respectively increased by 362.3 percent and 314.2 percent compared with the model group 0.44), the abnormal decrease of III type collagen synthesis caused by saccharification loss is relieved, and the normal function of collagen is maintained.
It is demonstrated that lactobacillus plantarum CCFM1354 and its prepared metagen can reduce the continuous proceeding of downstream saccharification reaction by down-regulating RAGE mRNA expression and up-regulating DDOST mRNA expression to prevent AGE-RAGE binding, target anti-glucose anti-aging, and improve host skin health level by down-regulating MMP-2mRNA expression and up-regulating COL3A1 mRNA expression to alleviate protein function attenuation caused by glucose injury in aging process and increase collagen synthesis process.
Example 12: effect of Lactobacillus plantarum CCFM1354 fermented peanut coat on improving anti-saccharification capability of peanut coat
(1) The lactobacillus plantarum CCFM1354 fermented peanut coat supernatant improves the in-vitro anti-saccharification capacity of the peanut coat:
preparation of peanut coat fermentation broth (hsp) and lactobacillus plantarum CCFM1354 fermented peanut coat supernatant (CCFM 1354_H) as in example 4; in vitro construction of fructose-bovine serum albumin system and detection of fluorescent AGE production as shown in example 5, detection of prevention of glycation damage by methylglyoxal to HSF cells is shown in example 7.
The peanut coat extract mainly contains flavonoid compounds such as proanthocyanidin, resveratrol, quercetin and the like, 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 since 2017, a few papers report that the peanut coat has the effect of inhibiting AGE from generating in vitro, 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, low bioavailability and deep red blackening of the high concentration peanut coat extract, and in order to better exert the anti-glycation function property of the peanut coat, the aim of preparing lactobacillus plantarum CCFM1354 fermented peanut coat supernatant (CCFM 1354_H) by fermenting peanut coats with lactobacillus plantarum CCFM1354 is tried, and the influence of the lactobacillus plantarum CCFM1354 on the glycation resistance of the peanut coats is verified by using 100 mug/mL peanut coat fermentation culture solution and fermented peanut coat supernatant concentration.
As shown in fig. 11, the inhibition rate of the original peanut coat on fluorescent AGE production of fructose-bovine serum albumin system is 61.70%, the inhibition rate of the supernatant after fermentation of lactobacillus plantarum CCFM1354 reaches 69.77%, and the inhibition ability of AGE production is remarkably improved by 13.1% compared with that of the unfermented group; in addition, after the original peanut coat is used for preventing the methylglyoxal injury, the cell activity of the HSF cells is only 56.16 percent, is relieved by 5.5 percent compared with that of a model group (53.23 percent), and after the lactobacillus plantarum CCFM1354 is fermented, the activity of the HSF cells is increased to 76.07 percent, so that the cell activity of the HSF cells after the methylglyoxal injury is obviously improved (42.9 percent compared with that of the model group). Thus, lactobacillus plantarum CCFM1354 can improve the original in-vitro anti-saccharification capacity of the peanut coat by fermenting the peanut coat.
(2) The effect of lactobacillus plantarum CCFM1354 fermented peanut coat supernatant on improving peanut coat oral administration to alleviate serum AGE levels in aging mice:
methods for experimental construction and serum and skin AGE detection in aging mice are as in example 9 and example 10, with the addition of a peanut coat synthetic formulation lavage group comprising: live lactobacillus plantarum CCFM1354, peanut coat fermentation broth (designated CCFM 1354+hsp).
As can be seen from FIG. 12, 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 CCFM1354 has better effect, and the AGE content is reduced by 48.7 percent compared with a model group by 208.42 ng/L; the peanut coat synthesis formulation group was not as effective as the other two groups, but only reduced 27.1% of the model group.
The experiment result combines with the in vitro experiment result, and proves that the lactobacillus plantarum CCFM1354 fermented peanut coat not only improves the anti-saccharification function of the unfermented peanut coat for inhibiting the generation of AGE in vitro, but also exerts the synergistic effect in vivo after being orally taken, namely, the anti-saccharification capability of the peanut coat is further improved by utilizing the lactobacillus plantarum CCFM 1354.
(3) The lactobacillus plantarum CCFM1354 fermented peanut coat supernatant improves the effect of oral administration of peanut coat to relieve skin saccharification damage of aged mice:
skin collagen content is also a direct biochemical index for detecting skin conditions of aging mice, aging modeling can obviously reduce III type collagen content in the skin of the mice, a model group is 6.09 mug/mL (7.17 mug/mL of a control group), an hsp group can obviously increase III type collagen content to 7.43 mug/mL (22.0% higher than the model), CCFM1354_H can obviously increase III type collagen content to 8.42 mug/mL (38.3% higher than the model), and a synthetic preparation group can increase III type collagen content but only to 6.73 mug/mL (10.5% higher than the model).
The results comprehensively show that the lactobacillus plantarum CCFM1354 fermented peanut coat supernatant relieves the influence of saccharification injury by improving the oral peanut coat content to relieve the skin III type collagen content of the aging mice.
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) CCFM1354, deposited at the collection of microorganisms and cell cultures of Guangdong province under the accession number GDMCC No:63924.
2. a metazoan prepared using the Lactobacillus plantarum CCFM1354 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 CCFM1354 and/or lactobacillus plantarum CCFM1354 metants according to claim 1.
5. The composition of claim 4, wherein the composition comprises a food, pharmaceutical, or nutraceutical.
6. The composition of claim 4, which is the product of the lactobacillus plantarum CCFM1354 after fermentation in peanut coat-containing medium.
7. Use of the lactobacillus plantarum CCFM1354 of claim 1, the metazoan of claim 2 or 3, or the composition of any of claims 4-6 for the preparation of a product for combating sugar, anti-aging, improving skin health problems caused by glycation.
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 forming intermediate;
(3) Reducing the aging profile of the individual;
(4) Preventing abnormal degradation of collagen of skin fibroblasts under high glucose culture;
(5) Preventing abnormal expression of EGR2 mRNA which is a key target point for growth regulation and control of skin fibroblasts under high glucose culture;
(6) Reducing AGE content in serum, skin, liver, kidney, brain tissue of aging individuals;
(7) Reducing the content of inflammatory markers IL-6 and TNF-alpha in serum of aged individuals;
(8) Reducing the water content of the skin horny layer at the back of an aged individual caused by saccharification injury;
(9) Reducing the content of III type collagen in the back skin of an aged subject caused by saccharification injury.
9. Use according to claim 7 or 8, whichCharacterized in that the product is a drug, and the content of the lactobacillus plantarum CCFM1354 in the product is not less than 1 multiplied by 10 6 CFU/mL or 1X 10 6 CFU/g。
10. Use of the lactobacillus plantarum CCFM1354 of claim 1, the metazoan of claim 2 or 3 in the preparation of a food product.
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