CN113088473B - Bifidobacterium animalis with effect of relieving oxidative damage of HaCaT cells - Google Patents
Bifidobacterium animalis with effect of relieving oxidative damage of HaCaT cells Download PDFInfo
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Abstract
The invention discloses bifidobacterium animalis with an effect of relieving oxidative damage of HaCaT cells, and belongs to the technical field of microorganisms and medicines. The invention provides a Bifidobacterium animalis (CCFM 1155), which can relieve the oxidative damage of human cells and obviously improve H 2 O 2 The survival rate of the induced HaCaT cells and the antioxidant capacity of the HaCaT cells; significantly improve H 2 O 2 The induced oxidative damage degree of the HaCaT cells can activate an antioxidant signal channel of Nrf2 and improve the capability of secreting Nrf2, thereby obviously relieving and repairing H 2 O 2 Induced damage caused by HaCaT cell oxidation, therefore, the bifidobacterium animalis CCFM1155 has great application prospect in preparing products for preventing or treating skin oxidative damage.
Description
Technical Field
The invention relates to bifidobacterium animalis with a function of relieving oxidative damage of HaCaT cells, belonging to the technical field of microorganisms and medicines.
Background
The skin is the largest organ of the human body and is the first line of defense against various external mechanical, chemical, physical and biological stimuli. And UV irradiation or pollutants such as ozone and benzopyrene induce cells to generate a large amount of Reactive Oxygen Species (ROS) to generate lipid peroxides, induce oxidative stress injury and inflammatory reaction, break the balance of an oxidation/oxidation resistance system in vivo, decline body tissues and physiological functions, damage skin barriers and cause skin aging.
Excessive active oxygen in a human body can break the dynamic balance of active oxygen generation and removal, an in-vivo antioxidant system cannot be removed in time, cells are in an oxidative stress state, excessive free radicals damage DNA in the cells, protein and lipid structures are damaged, and the cells are reduced in activity and even die. Skin, if exposed to this condition for a long period of time, can develop a darkened skin tone, sunburn, inflammatory diseases of the skin and even skin cancer.
With the increasing occurrence of skin diseases, the demand and more intensive research on skin care products has increased. Conventional sunscreens contain inorganic compounds, such as titanium dioxide (TiO) 2 ) And zinc oxide (ZnO), these minerals being capable of reflecting UVA and UVB radiation. However, the safety issues of these metal-based particles also raise human concerns.
Therefore, there is a need for the development of a natural product that does not cause side effects to patients, and that can enhance the endogenous antioxidant capacity of the skin to neutralize oxidative damage of ROS to the skin and alleviate various skin diseases caused by oxidative damage of the skin, and that can be applied to various types of patients.
Disclosure of Invention
The technical scheme of the invention is as follows:
in order to solve the problems, the invention provides Bifidobacterium animalis (Bifidobacterium animalis) CCFM1155, which is deposited in the Guangdong province collection center of microbial strains in 2021, 2 and 4 days, and the deposit number is GDMCC No: 61495, the preservation address is No. 59 building 5 of No. 100 Dazhong Jie-Lu-100 Guangzhou city.
The bifidobacterium animalis is derived from a human body excrement sample in a stannum-free area of Jiangsu, the strain is subjected to sequencing analysis, the 16S rDNA sequence of the strain is shown as SEQ ID NO.1, the sequence obtained by sequencing is subjected to nucleic acid sequence comparison in GeneBank, and the result shows that the strain is the bifidobacterium animalis and is named as bifidobacterium animalis CCFM 1155.
The microbial morphological characteristics of the bifidobacterium animalis CCFM1155 are as follows: the cells are milky white, irregular, round, convex and smooth.
The invention also provides application of the cells, fermentation supernatant or intracellular substances of the animal bifidobacterium in preparing products for preventing and/or treating skin oxidative damage.
In one embodiment of the present invention, the intracellular material is a supernatant of the disrupted bifidobacterium animalis.
In one embodiment of the invention, the preparation method of the intracellular material comprises the steps of centrifuging the bacterial liquid of the bifidobacterium animalis to collect bacterial sludge, adding a PBS solution for dissolving, adding liquid nitrogen for grinding and crushing, collecting bacterial sludge suspension, adding the PBS solution for dissolving, performing ultrasonic crushing, centrifuging and collecting supernate; 0.22 μm disposable filter was sterilized by filtration.
The invention also provides a product which is characterized by containing one or more of the cells, fermentation supernatant or intracellular substances of the bifidobacterium animalis.
In one embodiment of the present invention, the intracellular material is a supernatant of the disrupted bifidobacterium animalis.
In one embodiment of the invention, the preparation method of the intracellular material comprises the steps of centrifuging the bacterial liquid of the bifidobacterium animalis to collect bacterial sludge, adding a PBS solution for dissolving, adding liquid nitrogen for grinding and crushing, collecting bacterial sludge suspension, adding the PBS solution for dissolving, performing ultrasonic crushing, centrifuging and collecting supernate; 0.22 μm disposable filter for sterile filtration.
In one embodiment of the invention, the product comprises a daily chemical or pharmaceutical product.
In one embodiment of the present invention, the daily chemical product is a skin care product or a washing product.
In one embodiment of the present invention, the daily chemical product contains one or more of the cells, fermentation supernatant or intracellular material of bifidobacterium animalis described above.
In one embodiment of the invention, the adjunct comprises a humectant, an emollient and/or an emulsifier.
In one embodiment of the invention, the humectant comprises glycerin, propylene glycol, butylene glycol, pentylene glycol, hexylene glycol, sorbitol, and/or ethanol.
In one embodiment of the invention, the emollient comprises caprylic/capric triglyceride, isopropyl myristate, isononyl isononanoate, ethylhexyl palmitate and/or ethylene glycol distearate.
In one embodiment of the invention, the emulsifier comprises coco glucoside, polyglyceryl-2 dipolyhydroxystearate and/or polysorbate-60.
In one embodiment of the invention, the drug is a smear-type drug.
In one embodiment of the present invention, the smear-type medicine contains one or more of the cells, fermentation supernatant or intracellular material of the bifidobacterium animalis.
In one embodiment of the present invention, the application-type medicine further contains an additive.
In one embodiment of the invention, the dosage form of the smearing type medicine is cream, milky lotion, toner and ointment.
In one embodiment of the invention, the additive comprises an ointment, a preservative and/or an antioxidant.
In one embodiment of the invention, the ointment comprises monoglycerides, stearic acid, stearyl alcohol, glycerol, white petrolatum and/or ethylparaben.
In one embodiment of the invention, the preservative comprises benzoic acid and/or ethanol.
In one embodiment of the invention, the antioxidant is sodium sulfite.
In one embodiment of the invention, the dosage form of the smearing type medicine is paste, film or gel.
In one embodiment of the present invention, the method for preparing bifidobacterium animalis comprises: inoculating animal bifidobacterium into a proliferation culture medium added with a carbon source, a nitrogen source, inorganic salt and trace elements for fermentation to obtain animal bifidobacterium fermentation liquor.
In one embodiment of the present invention, the preparation method of the bifidobacterium animalis fermentation supernatant comprises: inoculating animal bifidobacterium into a proliferation culture medium for fermentation, wherein the viable count reaches 4-6 multiplied by 10 9 After cfu/mL, 8000-10000g of the mixture is centrifuged for 15-20 min, and the mixture is filtered and sterilized by a filter membrane of 0.22-0.45 mu m to obtain fermentation supernatant.
In one embodiment of the present invention, the animal bifidobacterium cell is prepared by the following method: the bifidobacterium animalis prepared by the method is centrifuged under the conditions of 8000-10000g for 15-20 min, a proper amount of PBS is added, the mixture is repeatedly ground by liquid nitrogen until the mixture is powdered, the mixture is ultrasonically treated under the conditions of 100-500W for 5-15 min after the proper amount of PBS is added, then the mixture is centrifuged under the conditions of 8000-10000g for 15-20 min, and the required bifidobacterium animalis intracellular substances are obtained after filtration and sterilization through a filter membrane of 0.22-0.45 mu m.
In one embodiment of the invention, the carbon source in the enrichment medium is 10-40 g/L glucose, the nitrogen source is 5-25 g/L yeast extract powder, tryptone or soybean peptone, the inorganic salt is 0.1-0.5 g/L magnesium sulfate, 0.1-0.5 g/L manganese sulfate, the trace elements are 1-5 g/L anhydrous sodium acetate, 1-5 g/L diammonium hydrogen citrate, 1-5 g/L potassium dihydrogen phosphate, 0.25-0.75 g L-cysteine hydrochloride and Tween 800.5-1.5 mL/L.
Has the beneficial effects that:
the invention provides a Bifidobacterium animalis (Bifidobacterium animalis) CCFM1155, the Bifidobacterium animalis CCFM1155 can relieve and relieve oxidative damage of skin cells, and is specifically embodied in that:
(1) increase H 2 O 2 Survival of induced HaCaT cells;
(2) significantly improve H 2 O 2 Degree of oxidative damage of induced HaCaT cells;
(3) can increase H 2 O 2 The antioxidant capacity of induced HaCaT cells;
(4) can activate the antioxidant signal channel of Nrf2 and improve the ability of secreting Nrf2, thereby obviously relieving and repairing H 2 O 2 Damage caused by induced HaCaT cell oxidation;
therefore, the bifidobacterium animalis CCFM1155 has a huge application prospect in preparing products for preventing or treating skin oxidative damage.
Biological material preservation
A strain of Bifidobacterium animalis (Bifidobacterium animalis) CCFM1155, which is taxonomically named as Bifidobacterium animalis, has been deposited in Guangdong province collection of microorganisms at 2.4.2021 with the deposit number GDMCC No: 61495, the preservation address is No. 59 building 5 of No. 100 Dazhong Jie-Lu-100 Guangzhou city.
Drawings
FIG. 1: different groups promote H 2 O 2 Effect of induced HaCaT cell proliferation was compared.
FIG. 2: increase of H in different groups 2 O 2 And comparing the effect of the T-AOC antioxidant capacity of the induced HaCaT cells.
FIG. 3: increase of H in different groups 2 O 2 Comparison of the effects of the levels of SOD enzyme activity in induced HaCaT cells.
FIG. 4: increase of H in different groups 2 O 2 Comparison of the effects of induced ability of HaCaT cells to secrete Nrf 2.
Detailed Description
The skin covers the surface of the human body and is one of the largest organs of the human body. The skin is composed of two parts, namely epidermis and dermis, wherein the key function of the epidermis is to form a barrier between the organism and the outside, and the epidermis is important to the human bodyAnd (5) protection. Skin Keratinocytes (KCs) are the main components of epidermis, while human immortalized cortical forming cells (HaCaT cells) belong to the cell line transformed from adult epidermal cells, have the same proliferation, differentiation and genetic stability as normal human keratinocytes, and are commonly used in the biomedical field as cell lines for in vitro studies, such as skin repair and healing, sun protection and anti-aging. Furthermore, keratinocytes are an important component in the study of oxidative damage to the skin. Thus, warp H is used in the examples below 2 O 2 Induced human immortalized cortical forming cells were used as a model of oxidative damage cells for cellular experiments.
The human immortalized cortical forming cells (HaCaT cells) referred to in the examples below were purchased from the chinese type culture collection; the DMEM medium referred to in the examples below was purchased from siermer femtoler (suzhou) instruments ltd; fetal Bovine Serum (FBS), penicillin, streptomycin and trypsin as referred to in the examples below were purchased from sigma aldrich trade ltd; the MTT referred to in the examples below was purchased from Beijing Solebao technologies, Inc.; the human factor E2-related factor 2(Nrf2) ELISA kit (cat # SBJ-M0807) referred to in the examples below was purchased from Shanghai Senega Biotech Ltd; the kits of T-AOC (cat # S0119) and SOD (cat # S0101S) referred to in the examples below were purchased from Shanghai Bin Yuntian science and technology, Inc.
The media involved in the following examples are as follows:
MRS liquid culture medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate 2 PO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 0 0.1g/L、MnSO 4 0.05g/L, Tween-801 ml/L, pH 7.0.
The bifidobacterium No.1 fermentation medium formula comprises: glucose: 20g of the total weight of the mixture; soaking yeast into powder: 10g of a mixture; na (Na) 2 HPO 4 ：3.5g；MgSO 4 -7H 2 O：0.25g；MnSO 4 -H 2 O: 0.1 g; diammonium hydrogen citrate: 2g of the total weight of the mixture; KH (Perkin Elmer) 2 PO 4 : 3.5 g; l-cysteineHydrochloride of a salt: 0.5 g; tween 801 mL; the pH was adjusted to 6.8. (initial osmotic pressure was around 325).
The bifidobacterium No. 2 fermentation medium formula comprises: glucose: 20g of the total weight of the mixture; tryptone (tryptone): 15g of the total weight of the mixture; na (Na) 2 HPO 4 ：3g；MgSO 4 -7H 2 O：0.25g；MnSO 4 -H 2 O: 0.1 g; diammonium hydrogen citrate: 2g of the total weight of the mixture; KH (Perkin Elmer) 2 PO 4 : 3g of the total weight of the mixture; l-cysteine hydrochloride: 0.5 g; tween 801 mL; the pH was adjusted to 6.8.
The following examples relate to methods for preparing bifidobacterium animalis culture fermentation broth and intracellular materials as follows:
1. activation and culture of strains
All containers and tools are sterilized. And (3) after the strain preservation pipe is taken and vibrated uniformly, a small amount of bacterial liquid is dipped by an aseptic inoculating loop and is streaked and purified in an MRS solid culture medium. After standing for a while, the plate was placed upside down in a 37 ℃ constant temperature anaerobic incubator for 48 hours. After the culture is finished, a single colony is selected and inoculated in 5mL of MRS liquid culture medium, and is statically placed in a constant-temperature anaerobic incubator at 37 ℃ for culture for 12-18 h. Obtaining the seed liquid of the strain. Two successive generations were activated with an inoculum size of 2% (v/v). Inoculating the seed solution into MRS liquid culture medium at an inoculation amount of 2% (v/v), and culturing in a constant-temperature anaerobic incubator at 37 deg.C for 12-18 h. The operation was repeated 1 time.
The MRS culture medium can be replaced by a bifidobacterium No.1 fermentation culture medium or a bifidobacterium No. 2 fermentation culture medium, and the culture conditions are the same as the method.
2. Preparation of probiotic samples for external use
(1) And (3) fermenting supernatant by using probiotics: centrifuging cultured probiotic bacteria at 8000r for 15min, and collecting supernatant; adjusting the pH value to 7.2-7.4; filtering with 0.22 μm disposable filter, sterilizing, packaging, and storing in refrigerator at-20 deg.C.
(2) Probiotic endosomes: taking 10mL of cultured probiotics, centrifuging and collecting bacterial sludge, and adding 1mL of PBS solution for dissolution; adding liquid nitrogen, grinding and crushing, and collecting bacterial sludge suspension; adding 1mL of PBS solution for dissolving, and carrying out ultrasonic crushing; centrifuging at 8000r for 10min, and collecting supernatant; 0.22 μm disposable filter for filtration and sterilization, subpackaged and stored in a refrigerator at-20 ℃ (0.1g wet bacterial sludge gives 1mL intracellular supernatant).
Complete medium: 5% Fetal Bovine Serum (FBS), 100U/mL penicillin, 100mg/mL streptomycin, 95% DMEM medium.
Example 1: screening and identification of strains
The method comprises the following specific steps:
1. screening
Taking human excrement from Jiangsu Wuxi area as a sample, pretreating the sample, storing the pretreated sample in 20% glycerol in a refrigerator at the temperature of-80 ℃, taking out and unfreezing the sample, uniformly mixing the sample, sucking 0.5mL of the sample, adding the sample into 4.5mL of physiological saline, carrying out gradient dilution by using 9g/L of physiological saline containing 0.05g/L of cysteine, selecting a proper gradient diluent to coat the gradient diluent on an MRS solid culture medium, culturing the mixture at the temperature of 37 ℃ for 48 hours, selecting a typical colony of animal bifidobacterium, streaking and purifying the typical colony on the MRS solid culture medium, selecting a single colony, transferring the single colony to the MRS liquid culture medium (containing 0.05g/L of cysteine) for enrichment, and preserving the single colony by using 30% glycerol to obtain a strain; wherein, the typical colony of the animal bifidobacterium is milky white, irregular, round, convex and smooth.
2. Identification
The genome of the strain CCFM1155 is extracted, the 16S rDNA of the strain CCFM1155 is amplified and sequenced (the 16S rDNA nucleotide sequence of the strain CCFM1155 is shown in SEQ ID NO.1 by Jinzhi Biotechnology Limited, Suzhou), and the sequence is subjected to nucleic acid sequence comparison in NCBI, so that the strain CCFM1155 is animal Bifidobacterium and is named as Bifidobacterium animalis (Bifidobacterium animalis) CCFM 1155.
Example 2: bifidobacterium animalis pair H 2 O 2 Effect of induced HaCaT cell survival
The method comprises the following specific steps:
(1) cell culture
Rewarming the frozen HaCaT cells at 37 ℃, centrifuging at 1000rpm for 5min, and taking the precipitate; washing the precipitate once with complete culture medium, re-suspending the cells with the complete culture medium and counting to obtain cell re-suspension; the cell suspension was inoculated into a 10cm dish containing 5% (v/v) CO in gas phase at 37 ℃ 2 Culturing in a cell culture box, replacing the complete culture medium the next day, continuing to culture at 37℃,The gas phase contains 5% (v/v) CO 2 The cell culture box of (2). And (4) carrying out passage when the cells grow to 70-80% of the density of the culture dish.
Selecting HaCaT cells in a good growth state, digesting the HaCaT cells by trypsin with the concentration of 2.5g/L, centrifuging, using a complete culture medium for resuspension, and counting the cells to obtain a cell resuspension solution; resuspending the cells at 5X 10 3 One well was inoculated into a 96-well plate at 100. mu.L per well, and the 96-well plate was placed at 37 ℃ in a gas phase containing 5% (v/v) CO 2 Culturing for 24 hours in a cell culture box; replacement of 50 mu M H 2 O 2 The cells were treated for 1 h.
(2) Fermentation broth, viable bacteria or intracellular pair H 2 O 2 Effect of induced HaCaT cell survival
A normal control group (blank control), a bifidobacterium animalis MRS culture medium fermentation liquid experimental group D1, a bifidobacterium fermentation culture medium fermentation supernatant experimental group D2, a bifidobacterium fermentation culture medium fermentation liquid experimental group D3, a bifidobacterium animalis viable bacteria experimental group D4, a bifidobacterium animalis intracellular substance experimental group D5, a lactobacillus casei experimental group L1 and a pediococcus pentosaceus fermentation supernatant experimental group L2 are arranged (the lactobacillus paracasei and the pediococcus pentosaceus are other bacteria obtained by screening the same batch as the bifidobacterium animalis CCFM 1155).
Culturing animal Bifidobacterium with MRS culture medium, Bifidobacterium No.1 fermentation culture medium or Bifidobacterium No. 2 fermentation culture medium to concentration of 5 × 10 9 CFU/mL, centrifugation and collection of supernatant, respectively noted in experimental groups D1, D2 and D3 incubation supernatant; selecting fermentation liquor for culturing animal bifidobacteria by using MRS culture medium, centrifuging to obtain live bacteria, and using complete culture medium to resuspend the bacteria-inducing concentration to 5X 10 9 CFU/mL, recorded as the incubation supernatant of panel D4; selecting fermentation liquor obtained by culturing animal bifidobacteria by using an MRS culture medium, centrifuging to obtain live bacteria, preparing an intracellular substance, and recording the intracellular substance as an incubation supernatant of an experimental group D5; respectively culturing lactobacillus casei experimental group L1 and pediococcus pentosaceus experimental group L2 by using MRS culture medium until the bacterial concentration reaches 5 multiplied by 10 9 CFU/mL, centrifuged and the supernatant collected and recorded as the incubation supernatants of experimental groups L1 and L2, respectively.
Mixing D1, D2 and D3The incubation supernatants of D4, D5 and L1, L2 were mixed with complete medium (5%: 95%) and 100. mu.L of each was pipetted through H in the cultivation step (1) 2 O 2 Treated HaCaT cells were for 24 h. The control group was added with 100. mu.L of complete medium, each group was set with 5 duplicate wells, and cell viability was measured by MTT method.
As can be seen from fig. 1, the proliferation survival rate of the normal control group is 61.80%, while bifidobacterium animalis significantly promotes the proliferation of cells (P < 0.001), the cell survival rate of the experimental group D1 of fermentation broth of bifidobacterium animalis MRS medium is 82.74%, the cell survival rate of the experimental group D2 of fermentation broth of bifidobacterium No.1 is 76.52%, the cell survival rate of the experimental group D3 of fermentation broth of bifidobacterium No. 2 is 77.91%, the cell survival rate of the experimental group D4 of viable bifidobacterium animalis is 78.46%, and the cell survival rate of the experimental group D5 of bifidobacterium animalis is 85.36%, and the proliferation effect is better than that of lactobacillus paracasei (cell survival rate is 52.09%) and pediococcus pentosaceus (cell survival rate is 45.22%).
Therefore, compared with lactobacillus paracasei and pediococcus pentosaceus, bifidobacterium animalis CCFM1155 can remarkably promote growth of HaCaT cells and relieve oxidative damage of the cells.
Example 3: bifidobacterium animalis pair H 2 O 2 Induced effects of T-AOC antioxidant capacity in HaCaT cells
The method comprises the following specific steps:
selecting HaCaT cells in a good growth state, digesting the HaCaT cells by trypsin with the concentration of 2.5g/L, centrifuging, using a complete culture medium for resuspension, and counting the cells to obtain a cell resuspension solution; cell resuspension was as follows 10 6 One well was inoculated into 8mL per well of 10cm dish, and the 10cm dish was placed at 37 ℃ in a gas phase containing 5% (v/v) CO 2 Culturing for 24 hours in a cell culture box; replacement of the container with 50 mu M H 2 O 2 The cells were treated for 1 h.
The control group was supplemented with 100. mu.L of complete medium, the experimental group used the incubation supernatant of the same concentration as in example 2, the incubation supernatants of D1, D2, D3, D4, D5 and L1, L2 were mixed with the complete medium (5%: 95%) respectively, and 100. mu.L of each aliquot was aspirated and culturedH 2 O 2 Treated HaCaT cells were 24 h. The cells were washed twice with PBS solution and then scraped into 4 ℃ PBS solution. Sonicate to break the cells sufficiently and release the intracellular material, centrifuge at 12000g for 5min at 4 ℃ and store the supernatant at-80 ℃ for testing. AOC was determined using an assay kit.
As can be seen from fig. 2, bifidobacterium animalis can improve the antioxidant capacity of cells. The T-AOC method is also called ABTS method. ABTS + Is a free radical that can exist in aqueous solution relatively stably, and can be scavenged by electron transfer. ABTS is not easy to be interfered by external factors + The method can accurately reflect the antioxidant activity of the sample. FIG. 2 shows that ABTS pairs are selected from Bifidobacterium animalis MRS culture medium fermentation broth experimental group D1, Bifidobacterium No.1 fermentation culture medium fermentation broth experimental group D2, Bifidobacterium No. 2 fermentation culture medium fermentation broth experimental group D3, Bifidobacterium animalis viable bacteria experimental group D4 and Bifidobacterium animalis intracellular material experimental group D5 + The scavenging effect of free radicals is higher than that of a positive control group, and the lactobacillus paracasei and pediococcus pentosaceus do not improve the antioxidant capacity of cells.
Therefore, bifidobacterium animalis CCFM1155 has a good antioxidant effect, and can protect HaCaT cells in an antioxidant way.
Example 4: bifidobacterium animalis pair H 2 O 2 Effect of induced SOD enzyme Activity levels in HaCaT cells
The method comprises the following specific steps:
cultivation with H 2 O 2 The cells were treated in the same manner as in example 3. The control group was supplemented with 100. mu.L of complete medium, the experimental group used the incubation supernatant of the same concentration as in example 2, the incubation supernatants of D1, D2, D3, D4, D5 and L1, L2 were mixed with the complete medium (5%: 95%) respectively, and 100. mu.L of each of them was aspirated 2 O 2 Treated HaCaT cells were 24 h. The cells were washed twice with PBS solution and then scraped into 4 ℃ PBS solution. Sonicating to break the cells sufficiently and release the oxidized material, centrifuging at 12000g for 5min at 4 deg.C, and storing the supernatant at-80 deg.C. SOD was measured using a measurement kit.
As can be seen from FIG. 3, the SOD enzyme activity of the conventional HaCaT cells was 2.63U/mg. After bifidobacterium animalis MRS culture medium fermentation broth, bifidobacterium No.1 fermentation culture medium fermentation broth, bifidobacterium No. 2 fermentation culture medium fermentation broth, bifidobacterium animalis live bacteria, bifidobacterium animalis intracellular substances, lactobacillus paracasei and pediococcus pentosaceus are added and treated for 24 hours respectively, the SOD enzyme activity is respectively 6.47, 6.25, 6.28, 5.46, 7.04, 2.68 and 1.91U/mg, and the data analysis result shows that the SOD enzyme activity is improved by 108.42 percent compared with the conventional HaCaT cell after the bifidobacterium animalis is added into the cells and cultured for 24 hours, the antioxidant activity of the cells is further tested after the antioxidant enzyme activity of the cells is improved, and the SOD enzyme activity of the cells is not improved by the lactobacillus paracasei and the pediococcus pentosaceus.
Example 5: bifidobacterium animalis pair H 2 O 2 Induced secretion of Nrf2 by HaCaT cells
The method comprises the following specific steps:
cultivation with H 2 O 2 The cells were treated in the same manner as in example 3, and the cells were washed twice with PBS solution and then scraped into 4 ℃ PBS solution. The cell suspension is treated by repeated freeze-thawing to destroy the cells. Centrifuging at 1000 Xg for 5min at 4 deg.C, and storing the supernatant at-80 deg.C.
Nrf2/HO-1 was assayed using an ELISA assay kit. The cells are divided into a normal control group, a positive control group, an animal bifidobacterium MRS culture medium fermentation broth experimental group D1, a bifidobacterium fermentation medium fermentation broth experimental group D2, a bifidobacterium fermentation medium fermentation broth experimental group D3, an animal bifidobacterium viable bacteria experimental group D4, an animal bifidobacterium intracellular material experimental group D5, a lactobacillus casei experimental group L1 and a pediococcus pentosaceus experimental group L2 (the lactobacillus paracasei and the pediococcus pentosaceus are other bacteria obtained by screening the same batch as the animal bifidobacterium CCFM 1155).
As shown in FIG. 4, after 24 hours of action of Bifidobacterium animalis CCFM1155, the quantity of Nrf2 secreted by HaCaT cells can be obviously promoted, and the difference (P) is very significant compared with a normal control group and a positive control group<0.05). Therefore, the bifidobacterium animalis CCFM1155 can activate the quantity of Nrf2 secreted by HaCaT cells and can also improve the Nrf2 secretion capacity, compared with a control groupThe secretion of Nrf2 is improved by 40%, thereby obviously relieving and repairing H 2 O 2 Induced damage from HaCaT cell oxidation, while lactobacillus paracasei, pediococcus pentosaceus did not promote the secretion of Nrf2 by HaCaT cells.
Example 6: application of animal bifidobacterium
(1) Bifidobacterium animalis CCFM1155 can be used for preparing body milk, and the specific preparation process of the body milk comprises the following steps:
mixing 10 parts of culture supernatant of bifidobacterium animalis CCFM1155, 0.3 part of humectant, 0.3 part of emollient, 0.1 part of emulsifier, 0.5 part of preservative, 30 parts of oil phase matrix and 60 parts of water phase matrix to obtain the body lotion.
(2) The bifidobacterium animalis CCFM1155 can be used for preparing shower gel, and the specific preparation process of the shower gel is as follows:
mixing 10 parts of bifidobacterium animalis CCFM1155 culture supernatant, 10 parts of surfactant fatty alcohol-polyoxyethylene ether sulfate, 5 parts of emulsifier octenyl succinic acid starch sugar, 5 parts of thickener polyethylene glycol, 10 parts of detergent lauryl sodium sulfate (sodium lauryl sulfate), 0.3 part of preservative hydantoin, 0.5 part of essence and 60 parts of aqueous phase matrix to obtain the body lotion.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that 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.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> animal bifidobacterium with effect of relieving oxidative damage of HaCaT cells
<130> BAA210308A
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1386
<212> DNA
<213> Artificial sequence
<400> 1
ggtcgggcca ccggcttcgg gtgctaccca ctttcatgac ttgacgggcg gtgtgtacaa 60
ggcccgggaa cgcattcacc gcggcgttgc tgatccgcga ttactagcga ctccgccttc 120
acgcagtcga gttgcagact gcgatccgaa ctgagaccgg ttttcagcga tccgccccac 180
gtcaccgtgt cgcaccgcgt tgtaccggcc attgtagcat gcgtgaagcc ctggacgtaa 240
ggggcatgat gatctgacgt catccccacc ttcctccgag ttgaccccgg cggtcccaca 300
tgagttcccg gcatcacccg ctggcaacat gcggcgaggg ttgcgctcgt tgcgggactt 360
aacccaacat ctcacgacac gagctgacga cgaccatgca ccacctgtga accggccccg 420
aagggaaacc gtgtctccac ggcgatccgg cacatgtcaa gcccaggtaa ggttcttcgc 480
gttgcatcga attaatccgc atgctccgcc gcttgtgcgg gcccccgtca atttctttga 540
gttttagcct tgcggccgta ctccccaggc gggatgctta acgcgttggc tccgacacgg 600
gacccgtgga aagggcccca catccagcat ccaccgttta cggcgtggac taccagggta 660
tctaatcctg ttcgctcccc acgctttcgc tcctcagcgt cagtgacggc ccagagacct 720
gccttcgcca ttggtgttct tcccgatatc tacacattcc accgttacac cgggaattcc 780
agtctcccct accgcactcc agcccgcccg tacccggcgc agatccaccg ttaggcgatg 840
gactttcaca ccggacgcga cgaaccgcct acgagccctt tacgcccaat aaatccggat 900
aacgctcgca ccctacgtat taccgcggct gctggcacgt agttagccgg tgcttattcg 960
aacaatccac tcaacacggc cgaaaccgtg ccttgccctt gaacaaaagc ggtttacaac 1020
ccgaaggcct ccatcccgca cgcggcgtcg ctgcatcagg cttgcgccca ttgtgcaata 1080
ttccccactg ctgcctcccg taggagtctg ggccgtatct cagtcccaat gtggccggtc 1140
accctctcag gccggctacc cgtcaacgcc ttggtgggcc atcaccccgc caacaagctg 1200
ataggacgcg accccatccc atgccgcaaa agcatttccc accccaccat gcgatggagc 1260
ggagcatccg gtattaccac ccgtttccag gagctattcc ggtgcacagg gcaggttggt 1320
cacgcattac tcacccgttc gccactctca ccccgacagc aagctgccag ggatcccgtt 1380
cgactg 1386
Claims (7)
1. Bifidobacterium animalis strain (b)Bifidobacterium animalis) CCFM1155, deposited at the Guangdong province culture Collection on 2021, 2/4, with the deposit number GDMCC No: 61495, the preservation address is No. 59 building 5 of No. 100 Dazhong Jie-Lu-100 Guangzhou city.
2. Use of cells, fermentation supernatant or intracellular material of bifidobacterium animalis as claimed in claim 1 in the manufacture of a product for the prevention and/or treatment of oxidative damage to the skin; the product is a daily chemical product or a medicine; the intracellular material is the supernatant of the crushed animal bifidobacterium of claim 1.
3. A product comprising one or more of cells, fermentation supernatant or intracellular material of bifidobacterium animalis according to claim 1; the product is a daily chemical product or a medicine; the intracellular material is the supernatant of the crushed animal bifidobacterium of claim 1.
4. The product of claim 3, wherein the commodity is a skin care product or a laundry product.
5. A product according to claim 3, wherein the pharmaceutical product is a spreadable pharmaceutical product.
6. A product according to claim 5, further comprising an additional agent which is an ointment, a preservative and/or an antioxidant.
7. The product of claim 5, wherein the form of the application-type medicine is an ointment, a film or a gel.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112159778A (en) * | 2020-09-30 | 2021-01-01 | 江南大学 | Bifidobacterium animalis capable of relieving psoriasis and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Ethanol Extract of Yak-Kong Fermented by Lactic Acid Bacteria from a Korean Infant Markedly Reduces Matrix Metallopreteinase-1 Expression Induced by Solar Ultraviolet Irradiation in Human Keratinocytes and a 3D Skin Model;Heanim Park等;《Antioxidants》;20210215;第10卷(第2期);第291页 * |
| 植物乳杆菌C88对H2O2诱导氧化损伤的Caco-2细胞的保护作用;李盛钰等;《中国农业科学》;20130201;第46卷(第3期);第606-613页 * |
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