CN117771152A - Skin-tendering anti-wrinkle bifidobacterium longum SF-B-60 fermentation product and preparation method and application thereof - Google Patents
Skin-tendering anti-wrinkle bifidobacterium longum SF-B-60 fermentation product and preparation method and application thereof Download PDFInfo
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- CN117771152A CN117771152A CN202410205851.3A CN202410205851A CN117771152A CN 117771152 A CN117771152 A CN 117771152A CN 202410205851 A CN202410205851 A CN 202410205851A CN 117771152 A CN117771152 A CN 117771152A
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- bifidobacterium longum
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Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of microorganisms, in particular to a bifidobacterium longum SF-B-60 fermented product capable of tendering skin and resisting wrinkle, and a preparation method and application thereof. Bifidobacterium longumBifidobacterium longum) SF-B-60 with preservation number of CGMCC No.29022 is prepared by fermenting substrate to be fermented including Chinese herbal medicine extract and supplementing material by using Bifidobacterium longum SF-B-60 to obtain the final productThe herbal materials comprise the following components: the feed supplement comprises snail protein peptide. The fermented product of Bifidobacterium longum SF-B-60 has antioxidant, skin caring and wrinkle resisting effects.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a bifidobacterium longum SF-B-60 fermented product capable of tendering skin and resisting wrinkle, and a preparation method and application thereof.
Background
Skin is the largest organ of human body, and damage to skin can be caused by various factors such as sun exposure, air pollution, electronic product radiation, living habit and the like, so that elastic fibers of the skin are damaged. Collagen and spandex in the skin gradually decrease, and at the same time, as the age increases, the self-repair mechanism of the skin also decreases, and wrinkles gradually appear. Wrinkle formation is mainly due to internal causes, and somatic aging is the most fundamental cause of wrinkles. With the increase of age, the cell activity gradually decreases, the metabolic capacity decreases, the number of new cells increases to be lower than the aging speed of the cells, the hydration capacity of the aged cells decreases, the photoaging effect is enhanced, the skin water-tightness and the moisture retention are reduced, the weight-added fiber tissue is deteriorated, and the skin is dry and easy to form fine lines. In addition, skin aging is accompanied by deterioration of sweat gland system, insufficient sebum secretion, and increases skin damage and dryness, and causes wrinkles.
The anti-wrinkle skin care products on the market at present have uneven effects, and the common cosmetic active ingredient additives mainly comprise arbutin, nicotinamide, tretinoin, azelaic acid and the like, have single action mechanism and have toxic and side effects such as cytotoxicity, skin irritation and the like. From modern medical research, chinese herbal medicines contain abundant proteins and various amino acids, lipids, polysaccharides, pectin, vitamins, trace elements, organic acids, alkaloids, saponins and other nutrient substances, and if the deep research is carried out, the formula is precisely combined, the functional activity is enhanced, and the cell proliferation and metabolism can be better promoted, the skin microcirculation is improved, and the oxidation resistance of an organism is enhanced.
Disclosure of Invention
Aiming at the technical problems of single action mechanism and skin sensitivity caused by long-term use of the existing anti-wrinkle skin care product, the invention provides a bifidobacterium longum SF-B-60 fermented product for tendering skin and resisting wrinkle, and a preparation method and application thereof. The bifidobacterium SF-B-60 ferment has the advantages that through the combined action of a plurality of Chinese herbal medicine skin tendering and wrinkle resisting components and snail protein peptide, the cell regeneration can be promoted, the skin is plump and compact, the fine lines are eliminated, the skin quality is improved, the skin tendering and wrinkle resisting effects are achieved, and no toxic or side effect is caused after long-term use.
In a first aspect, the invention provides a skin-tendering and wrinkle-resisting bifidobacterium longum SF-B-60 ferment, which is prepared from a substrate to be fermented through bifidobacterium longumBifidobacterium longum) SF-B-60 is fermented to obtain the product; the fermented substrate comprises Chinese herbal medicine extract and feed; the Chinese herbal medicine raw materials for preparing the Chinese herbal medicine extract comprise the following components in parts by weight: 5-30 parts of radix scutellariae, 2-15 parts of angelica sinensis, 2-15 parts of astragalus membranaceus, 2-10 parts of ginseng, 1-10 parts of medlar, 0-10 parts of aloe, 2-10 parts of chrysanthemum, 2-10 parts of dandelion, 2-10 parts of honeysuckle and 1-10 parts of grape seeds; mixing the bifidobacterium longum SF-B-60 bacterial liquid and the Chinese herbal medicine extract according to the weight ratio of 1:1 to obtain a mixed liquid, and adding the following components in each 1000mL of the mixed liquid: 20g of glucose, 10g of starch, 20g of peptone, 5g of yeast extract powder, 20g of acetylated sodium hyaluronate, 20g of salmon nasal cartilage, 15g of type I collagen peptide, 10g of elastin peptide and 10g of snail protein peptide; bifidobacterium longum SF-B-60 has been deposited in China general microbiological culture Collection center (China Committee for culture Collection) for 11 months and 15 days, with a deposit address of Hospital No. 1 and 3 in the North Chen West Lu of the Chao area of Beijing, and a deposit number of CGMCC No.29022.
The bifidobacterium longum SF-B-60 ferment provided by the invention is rich in skin-tendering anti-wrinkle Chinese herbal medicine extract components, and the Chinese herbal medicine extract is prepared from ten Chinese herbal medicine raw materials of radix scutellariae, angelica sinensis, astragalus membranaceus, ginseng, medlar, aloe, chrysanthemum, dandelion, honeysuckle and grape seeds. The Chinese herbal medicine extract can promote cell proliferation, enhance skin metabolism, and promote collagen and elastin generation; the Chinese herbal medicine extract contains natural antioxidant active ingredients, and can be used for resisting oxidation by activating the activity of oxidoreductase in human body to prevent skin aging; the Chinese herbal medicine extract has strong ultraviolet absorption effect and plays a role in inhibiting skin photoaging; according to the synergistic effect, compatibility and the like of different Chinese herbal medicine extracts, the Chinese herbal medicine raw materials are selected for scientific compounding, and the Chinese herbal medicine extract without preservative, pigment and essence is obtained.
The strain is Bifidobacterium longum SF-B-60, and the fermentation product obtained by fermenting the substrate to be fermented with Bifidobacterium longum SF-B-60 contains organic acid (lactic acid, citric acid, succinic acid, etc.), and vitamins (B) 1 、B 2 、B 6 、B 12 Pantothenic acid, folic acid, etc.), bacteriocins, polysaccharides, proteins, amino acids, etc., wherein lactic acid is a natural moisturizing factor, and is present in human skin, which can effectively remove fine lines and wrinkles, and accelerate the turnover of cutin; folic acid can activate skin epidermis cells, promote nutrient absorption, and reduce skin keratinization speed, thereby achieving the purposes of maintaining beauty, nourishing skin and tendering skin; the active ingredients such as amino acid, lipid, polysaccharide, adenosine, vitamins, trace minerals and the like can moisten and nourish skin, help the skin resist the damage to the skin caused by external environment and pressure, and simultaneously can promote the expression of various protein genes related to the epidermal differentiation and the skin barrier function, thereby achieving the effects of removing wrinkles, repairing, preserving moisture and enhancing skin elasticity.
The feed provided by the invention comprises snail protein peptide, wherein the snail protein peptide has extremely high collagen content and has obvious wrinkle-removing nutrition function. When it permeates into the wrinkled skin groove and the concave skin lesion, it rearranges to approximate the natural collagen fiber in the body after dewatering and shrinking, and at the same time, the fiber cell, capillary vessel and fat cell in the body migrate to grow to the permeated part to synthesize the collagen protein of the user, finally form normal connective tissue, and eliminate skin wrinkles fundamentally.
In a second aspect, the invention also provides a preparation method of the skin-tendering and wrinkle-resisting bifidobacterium longum SF-B-60 fermented product, which comprises the following steps:
(1) Preparing a Chinese herbal medicine extract: weighing Chinese herbal medicine raw materials, decocting the Chinese herbal medicine raw materials with water, concentrating decoction, and filtering to obtain a Chinese herbal medicine extract with the dry weight of 10 times of that of the Chinese herbal medicine raw materials;
(2) Preparing bifidobacterium longum SF-B-60 bacterial liquid: inoculating the bifidobacterium longum SF-B-60 to an improved MRS culture medium, and culturing to obtain bifidobacterium longum SF-B-60 bacterial liquid;
(3) Preparing a Chinese herbal medicine fermentation broth rich in bacterial mud: mixing the bifidobacterium longum SF-B-60 bacterial liquid with the Chinese herbal medicine extract to obtain a mixed liquid, adding the supplementary material into the mixed liquid, uniformly mixing, and fermenting to obtain a Chinese herbal medicine fermentation liquid rich in bacterial mud;
(4) Preparation of Bifidobacterium longum SF-B-60 fermentate: filtering the Chinese herbal medicine fermentation liquor rich in bacterial mud to remove bacterial mud, and obtaining clear and transparent bifidobacterium longum SF-B-60 fermentation product.
Further, the Chinese herbal medicine raw materials in the step (1) are decocted for three times, the water adding amount of each time is 20 times of the mass of the Chinese herbal medicine raw materials, and the time of each time is 40 minutes.
Further, in the step (2), the inoculation amount of the bifidobacterium longum SF-B-60 is 2 percent, and the bifidobacterium longum is subjected to anaerobic culture for 14 hours at the temperature of 37 ℃ under the anaerobic condition that the volume ratio is N 2 :CO 2 Mixed gas=4:1, modified MRS medium included the following components: glucose 20g, peptone 20g, yeast extract 10g, K 2 HPO 4 ·7H 2 O5 g, ammonium citrate 3g, CH 3 COONa·3H 2 O4.5 g, sodium chloride 2g and MgSO 4 ·7H 2 O 1g、MnSO 4 ·4H 2 O0.1 g, tween 80 1g, and distilled water to 1000mL.
Further, the fermentation condition in the step (3) is anaerobic fermentation at 37 ℃ for 18 hours.
In a third aspect, the invention also provides an application of the bifidobacterium longum SF-B-60 fermented product in preparing a skin care product for tendering skin and resisting wrinkle.
In a fourth aspect, the invention also provides a skin care product for tendering and resisting wrinkle, which comprises the bifidobacterium longum SF-B-60 fermented product; the skin care product for tendering and resisting wrinkle comprises the following components in parts by weight: 20-40 parts of bifidobacterium longum SF-B-60 fermented product, 10-30 parts of emulsifying agent, 10-30 parts of humectant, 5-30 parts of softener, 0-1 part of thickening agent and 0-1 part of chelating agent.
In a fifth aspect, the invention also provides a preparation method of the skin-tendering anti-wrinkle skin care product, which comprises the steps of adding a humectant and a thickener into a bifidobacterium longum SF-B-60 fermentation product, standing and heating to obtain a water phase; mixing the emulsifier and the softener, and heating to the same temperature as the water phase to obtain an oil phase; adding the oil phase and chelating agent into the water phase, stirring and cooling to obtain the skin care product.
Further, the emulsifier is one or more than two of polyglycerol-4 oleate, hydrogenated lecithin, sorbitan laurate, sorbitan oleate, stearamide AMP, coco-caprylate, carnosine, polyglycerol-3 diisostearate and methyl serine; the humectant is one or more of acetylated sodium hyaluronate, glycerol, butanediol, arginine, panthenol, tremella polysaccharide, glycosyl trehalose, lysine, serine, glycine and algae extract; the softening agent is one or more of dioctyl carbonate, butter fruit, cyclomethicone, stearyl alcohol, methyl glucose ether and lanolin alcohol; the thickener is one or more of xanthan gum, carbomer, dextrin, disodium cocoyl amphodiacetate and sodium acrylate; the chelating agent is one or more of disodium EDTA, trisodium EDTA, tetrasodium EDTA and pentasodium pentetate.
Further, the emulsifier is a composition of sorbitan oleate, stearamide AMP and methyl serine in a mass ratio of 3:3:1; the humectant is an acetylated sodium hyaluronate, glycosyl trehalose and algae extract composition with a mass ratio of 3:3:2; the softening agent is a butter and lanolin alcohol composition with a mass ratio of 2:1; the thickener is xanthan gum and carbomer composition with the mass ratio of 1:1; the chelating agent is disodium EDTA.
The invention has the beneficial effects that:
after the Chinese herbal medicine extract is fermented by bifidobacterium SF-B-60, the effective components are easier to be absorbed by skin, and the effects of tendering skin and resisting wrinkle can be better exerted. The invention takes the principle of traditional Chinese medicine theory as the principle, takes skin science as the basis, takes microbial fermentation as the basis, and takes bifidobacterium longum SF-B-60 as the basis for fermentation, and the contained skin tendering anti-wrinkle active ingredients can be organically combined, so that the metabolism of the stratum corneum is enhanced in an omnibearing and multilayer way, the cell regeneration is promoted, damaged tissues are repaired, the elasticity and the vitality of skin are aroused, the skin is further compacted, fine lines are reduced, the skin moisture is maintained, and the skin elasticity is improved. The skin tendering anti-wrinkle skin care product containing the bifidobacterium longum SF-B-60 fermentation product provided by the invention has no stimulation to skin and no toxic or side effect, and care tender skin.
Detailed Description
In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
The bifidobacterium longum SF-B-60 used in the invention is obtained by separation of the inventor and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in the year 2023, 11 and 15, and the preservation address is the number 3 of North Chen Xiyu No. 1 in the Korean region of Beijing, and the preservation number is CGMCC No.29022.
The improved MRS culture medium used in the invention comprises the following components: glucose 20g, peptone 20g, yeast extract 10g, K 2 HPO 4 ·7H 2 O5 g, ammonium citrate 3g, CH 3 COONa·3H 2 O4.5 g, sodium chloride 2g and MgSO 4 ·7H 2 O 1g、MnSO 4 ·4H 2 O0.1 g, tween 80 1g, and distilled water to 1000mL.
The sodium hyaluronate mentioned in the following examples was purchased from the division of Biotechnology, kashire, salmon nasal cartilage was purchased from Japan, type I collagen peptide, elastin peptide was purchased from the division of Hubei Jian peptide biotechnology, and snail protein peptide was purchased from the division of Shanxi Taike biotechnology.
EXAMPLE 1 isolation of seed
(1) Bacterial source: yoghurt, 11 months 2022, was collected in the inner Mongolian municipality of China.
(2) Isolation of strains:
sampling the yoghurt by using a sterilized sampling spoon, avoiding pollution in the sampling process, immediately preserving the yoghurt in a refrigerating box after sampling, numbering and recording, weighing 1g of the yoghurt sample, dissolving in 10mL of physiological saline, uniformly mixing, carrying out ten-time gradient dilution on the sample liquid, uniformly coating 100 mu L of each gradient on an improved MRS flat plate culture medium, then taking out the sample, placing the sample in an anaerobic tank, and carrying out anaerobic culture at 37 ℃ for overnight;
after the cultivation is finished, different colonies with different forms and sizes grow on the flat plate, the colony features are selected to be round, convex, milky white or white, opaque and smooth and soft, mycelium is not formed, microscopic examination is carried out, after the microscopic examination is finished, the mycelium of bifidobacterium is selected as a microscopic examination result, further scribing and purifying treatment are carried out, and the flat plate anaerobic tank is activated for 1 day, so that the culture is obtained. And then strain identification is carried out.
Example 2 identification of species
(1) Authentication unit
Bioengineering (Shanghai) Co., ltd.
(2) Primer(s)
27F：5'-AGAGTTTGATCCTGGCTCAG-3'；
1492R：5'-GGTTACCTTGTTACGACTT-3'。
(3) Identification of sequences
The identification sequences are shown below:
AGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGGATCCATCAAGCTTGCTTGGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATACACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGTTCCAGTTGATCGCATGGTCTTCTGGGAAAGCTTTCGCGGTATGGGATGGGGTCGCGTCCTATCAGCTTGACGGCGGGGTAACGGCCCACCGTGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTATCGGGGAGCAAGCGAGAGTGAGTTTACCCGTTG-AATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACGGTCGTAGAGATACGGCTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTATGCCGGGAACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACGCGGCGACGCGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAAGCCGGTGGCCTAACCCCTTGTGGGATGGAGCCGTCTAAGGTGAGGCTCGTGATTGGGACTAAGTCGTA
(4) Identification result
The strain is identified as bifidobacterium genus%Bifidobacterium) It is presumed that bifidobacterium longum is [ ]Bifidobacterium longum) The strain is named as bifidobacterium longum SF-B-60 and is sent to China general microbiological culture Collection center for preservation, and the preservation information is as follows:
the bifidobacterium longum is%Bifidobacterium longum) SF-B-60 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 11 and 15 days 2023, and has a preservation address of CGMCC No.29022, namely, north Chen West Lu No. 1, no. 3, of the Korean region of Beijing city.
EXAMPLES 3-8 preparation of Bifidobacterium longum SF-B-60 fermentate
(1) Preparing a Chinese herbal medicine extract: weighing Chinese herbal medicine raw materials according to the proportion of the table 1, decocting the Chinese herbal medicine raw materials with water for three times, wherein the water added each time is 20 times of the mass of the Chinese herbal medicine raw materials, the time of each decoction is 40min, and combining, concentrating and filtering the decoction liquid obtained from the three times to finally obtain the Chinese herbal medicine extract with the dry weight of 10 times of the Chinese herbal medicine raw materials;
(2) Preparing bifidobacterium longum SF-B-60 bacterial liquid: inoculating bifidobacterium longum SF-B-60 into an improved MRS culture medium according to an inoculum size of 2 percent, and performing anaerobic culture for 14 hours at 37 ℃ to obtain bifidobacterium longum SF-B-60 bacterial liquid; anaerobic condition is volume ratio N 2 :CO 2 Mixed gas=4:1;
(3) Preparing a Chinese herbal medicine fermentation broth rich in bacterial mud: the bifidobacterium longum SF-B-60 bacterial liquid and the Chinese herbal medicine extract are mixed according to the weight ratio of 1:1, mixing to obtain a mixed solution, and adding the following supplementary materials into each 1000mL of the mixed solution: 20g of glucose, 10g of starch, 20g of peptone, 5g of yeast extract powder, 20g of acetylated sodium hyaluronate, 20g of salmon nasal cartilage, 15g of type I collagen peptide, 10g of elastin peptide and 10g of snail protein peptide; uniformly mixing, and fermenting, wherein anaerobic fermentation is carried out at 37 ℃ for 18 hours to obtain a Chinese herbal medicine fermentation broth rich in bacterial mud;
(4) Preparation of Bifidobacterium longum SF-B-60 fermentate: filtering the Chinese herbal medicine fermentation liquor rich in bacterial mud by a cross-flow membrane filter to remove bacterial mud, and obtaining clear and transparent bifidobacterium longum SF-B-60 fermentation product.
Examples 3-8 of the present invention prepared herbal extracts as shown in Table 1:
table 1 examples 3-8 weight of herbal materials used for preparing herbal extracts
Example 9 determination of antioxidant Activity
Using 10mL test tubes to establish a sample tube (T2), a sample background (T1), a blank control tube (C2) and a solvent background (C1), setting up 3 parallel tubes in the sample tube (T2) and the blank control tube (C2) of each sample, sequentially adding samples of the bifidobacterium longum SF-B-60 fermented products of the examples 3-8 as sample solutions according to the reference table 2, fully mixing by a vortex oscillator, placing the mixture in a 37 ℃ water bath for incubation for 15 minutes, taking out the reaction solutions of the tubes, transferring the reaction solutions into a cuvette, measuring the absorbance at 510nm, and calculating the antioxidant activity of the bifidobacterium longum SF-B-60 fermented products according to the following formula:
in the formula A T1 Is the absorbance of the sample background (T1), A T2 Is the absorbance of the sample tube (T2), A C1 Is the absorbance of the solvent background (C1), A C2 Is the absorbance of the blank control tube (C2).
TABLE 2 sample addition requirement
Note that: ferrous sulfate concentration of 9mmol/L and salicylic acid concentration of 9mmol/L, H 2 O 2 The concentration was 8.8mmol/L.
Experimental results show that the antioxidant activity of the bifidobacterium longum SF-B-60 fermentation products in the embodiments 3-8 is 44.2%, 63.7%, 75.4%, 58.1%, 68.5% and 84.9%, respectively, which shows that the Chinese herbal medicine fermentation liquid provided by the invention has high antioxidant activity and a certain skin tendering and wrinkle resisting effect. Wherein, the bifidobacterium longum SF-B-60 ferment of the embodiment 8 has the highest antioxidant activity, and the Chinese herbal medicine formula is the optimal Chinese herbal medicine formula.
Examples 10 to 17 preparation of skin-rejuvenating and anti-wrinkle skin care products
Skin care products for tendering and anti-wrinkle are prepared according to the component proportions in Table 3. Adding humectant and thickener into the fermented product of Bifidobacterium longum SF-B-60, standing for 12 hr, and heating to 80deg.C to obtain water phase. Mixing the emulsifier and the softener, and heating and dissolving at 80 ℃ to obtain an oil phase. Then adding the oil phase and the chelating agent into the water phase, and fully stirring and cooling to 30 ℃ through a homogeneous mixer to prepare the skin tendering anti-wrinkle skin care product.
Wherein the emulsifier is a composition of sorbitan oleate, stearamide AMP and methyl serine according to a mass ratio of 3:3:1; the humectant is a composition of acetylated sodium hyaluronate, glycosyl trehalose and algae extract according to a mass ratio of 3:3:2; the softening agent is a composition of butter fruit tree fruit fat and lanolin alcohol according to a mass ratio of 2:1; the thickener is a composition of xanthan gum and carbomer according to a mass ratio of 1:1; the chelating agent is disodium EDTA.
Table 3 the composition ratio of skin care products for tendering and anti-wrinkle
Example 18 determination of fibroblast proliferation Activity of skin rejuvenating anti-wrinkle skin care products on human skin
(1) Experimental materials: human skin fibroblasts, purchased from synthetic fertilizer, all-round biotechnology limited; DMEM culture solution, MTT solution, and preserving in a refrigerator at 4 ℃;0.25% trypsin (Sigma); dimethyl sulfoxide; skin care products for skin rejuvenation and anti-wrinkle prepared in examples 10 to 17.
(2) Experimental method
A. The skin care products prepared in examples 10 to 17 were dissolved in dimethyl sulfoxide, sterilized by filtration through a 0.2mm sterile filter head, and then prepared into a culture medium containing 10% of the sample with DMEM medium.
B. Obtaining human skin fibroblast by tissue block culture method, adding 0.25% trypsin 1mL for digestion, centrifuging, adding DMEM culture solution for resuspension, counting, and diluting to cell concentration of 1×10 5 Per mL, 100mL per well in 96-well plates. After culturing for 48h, observing the cell wall adhesion under a mirror, sucking the culture medium in the holes, dividing the culture medium into 9 groups (12 holes in each group), wherein the first 8 groups are the example groups, adding 100mL of culture medium containing 10% of the example 10-17, the 9 th group is the blank group, and adding 100mL of DMEM culture medium. At 37℃5% CO 2 After 24h, 48h and 72h of static culture in an incubator, 4 holes are respectively taken from each group, 20mL of MTT solution is added into each hole, and then the mixture is treated by CO 2 Incubation was continued for 4h in an incubator, 150mL of dimethyl sulfoxide was added to each well, and after shaking at low speed on a constant temperature shaker for 10min, the absorbance value of each well was measured at 490nm on an microplate reader. The results are shown in the following table.
TABLE 4 absorbance values for human skin fibroblasts at various times for examples 10-17
The results show that the light absorption value of the example group is obviously increased compared with that of the blank control group, so that the skin tendering and anti-wrinkle skin care product has obvious effect of promoting cell regeneration, can promote the repair of injured tissues, and achieves the purposes of tendering and anti-aging. From the light absorption value, the best effect of example 14 can be seen, and the skin care product of example 14 is selected for subsequent safety evaluation.
Example 19 safety evaluation-skin irritation test
(1) Animal and feeding environment: the Netherlands rabbits (pass number: QZTF 2023-0002), common grade, are provided by Qingzhou market, tongfu farms for a total of 4. The weight is 2.1-2.2kg. The animals are kept in a single cage in a common animal house, the temperature of the animal house is 20-22 ℃, and the humidity is 45-55% RH. Animal feed was purchased from Fulika pet products Inc., and drinking water was tap water.
(2) The test method comprises the following steps: about 24 hours before the experiment, the two sides of the spine of the dorsum of the Holland rabbit are sheared, and the dehairing range is about 3cm multiplied by 3cm on the left and right. The skin care product for caring skin prepared in example 14 was applied to the left skin in an amount of about 1g, with an application range of about 2.5cm×2.5cm, 1 time per day, and 14 days continuously, to give a test group; the right skin served as a control and was not smeared with any substance. The skin care product was removed by washing with warm water after the next day, and removing hair before each application. The results were observed after 1 hour of application, and scored according to the skin irritation test criteria of the cosmetic hygiene Specification, and the test results are shown in Table 5.
TABLE 5 skin tendering anti-wrinkle skin care products results of skin irritation experiments on Holland rabbits
Note that: skin irritation intensity classification: no irritation is caused between 0 and less than 0.5; light irritation of 0.5 to less than 2.0; irritation in 2.0 to less than 6.0; strong irritation of 6.0 to less than 8.0.
As is clear from the results in Table 5, the skin-rejuvenating and anti-wrinkle skin care product prepared in example 14 was non-irritating to the skin of Dutch rabbits.
Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (9)
1. A skin-tendering and wrinkle-resisting bifidobacterium longum SF-B-60 ferment is characterized in that a substrate to be fermented is subjected to bifidobacterium longum fermentationBifidobacterium longum) SF-B-60 is fermented to obtain the product; the fermented substrate comprises Chinese herbal medicine extract and feed; the Chinese herbal medicine raw materials for preparing the Chinese herbal medicine extract comprise the following components in parts by weight: 5-30 parts of radix scutellariae, 2-15 parts of angelica sinensis, 2-15 parts of astragalus membranaceus, 2-10 parts of ginseng, 1-10 parts of medlar, 0-10 parts of aloe, 2-10 parts of chrysanthemum, 2-10 parts of dandelion, 2-10 parts of honeysuckle and 1-10 parts of grape seeds; mixing the bifidobacterium longum SF-B-60 bacterial liquid and the Chinese herbal medicine extract according to the weight ratio of 1:1 to obtain a mixed liquid, and adding the following components in each 1000mL of the mixed liquid: 20g of glucose, 10g of starch, 20g of peptone, 5g of yeast extract powder, 20g of acetylated sodium hyaluronate, 20g of salmon nasal cartilage, 15g of type I collagen peptide, 10g of elastin peptide and 10g of snail protein peptide; bifidobacterium longum SF-B-60 has been deposited in China general microbiological culture Collection center (China Committee for culture Collection) for 11 months and 15 days, with a deposit address of Hospital No. 1 and 3 in the North Chen West Lu of the Chao area of Beijing, and a deposit number of CGMCC No.29022.
2. A method for preparing a skin rejuvenating and anti-wrinkle bifidobacterium longum SF-B-60 ferment as claimed in claim 1, comprising the steps of:
(1) Preparing a Chinese herbal medicine extract: weighing Chinese herbal medicine raw materials, decocting the Chinese herbal medicine raw materials with water, concentrating decoction, and filtering to obtain a Chinese herbal medicine extract with the dry weight of 10 times of that of the Chinese herbal medicine raw materials;
(2) Preparing bifidobacterium longum SF-B-60 bacterial liquid: inoculating the bifidobacterium longum SF-B-60 to an improved MRS culture medium, and culturing to obtain bifidobacterium longum SF-B-60 bacterial liquid;
(3) Preparing a Chinese herbal medicine fermentation broth rich in bacterial mud: mixing the bifidobacterium longum SF-B-60 bacterial liquid with the Chinese herbal medicine extract to obtain a mixed liquid, adding the supplementary material into the mixed liquid, uniformly mixing, and fermenting to obtain a Chinese herbal medicine fermentation liquid rich in bacterial mud;
(4) Preparation of Bifidobacterium longum SF-B-60 fermentate: filtering the Chinese herbal medicine fermentation liquor rich in bacterial mud to remove bacterial mud, and obtaining clear and transparent bifidobacterium longum SF-B-60 fermentation product.
3. The method for preparing the skin-tendering anti-wrinkle bifidobacterium longum SF-B-60 fermentation product according to claim 2, wherein the Chinese herbal medicine raw materials in the step (1) are decocted for three times, the water adding amount of each time is 20 times of the mass of the Chinese herbal medicine raw materials, and the time of each time is 40 minutes.
4. The method for producing a skin-rejuvenating and wrinkle-resistant fermentation broth of Bifidobacterium longum SF-B-60 according to claim 2, wherein the inoculum size of Bifidobacterium longum SF-B-60 in step (2) is 2%, anaerobic culture is carried out at 37℃for 14 hours, and anaerobic conditions are a volume ratio N 2 :CO 2 Mixed gas=4:1, modified MRS medium included the following components: glucose 20g, peptone 20g, yeast extract 10g, K 2 HPO 4 ·7H 2 O5 g, ammonium citrate 3g, CH 3 COONa·3H 2 O4.5 g, sodium chloride 2g and MgSO 4 ·7H 2 O 1g、MnSO 4 ·4H 2 O0.1 g, tween 80 1g, and distilled water to 1000mL.
5. The method for preparing skin-tendering and wrinkle-resistant bifidobacterium longum SF-B-60 fermented product according to claim 2, wherein the fermentation condition in the step (3) is anaerobic fermentation at 37 ℃ for 18 hours.
6. Use of a bifidobacterium longum SF-B-60 ferment as claimed in claim 1 in the preparation of a skin rejuvenation anti-wrinkle skin care product.
7. A skin care product for tendering and anti-wrinkle, comprising the bifidobacterium longum SF-B-60 ferment of claim 1; the skin care product for tendering and resisting wrinkle comprises the following components in parts by weight: 20-40 parts of bifidobacterium longum SF-B-60 fermented product, 10-30 parts of emulsifying agent, 10-30 parts of humectant, 5-30 parts of softener, 0-1 part of thickening agent and 0-1 part of chelating agent.
8. A method for preparing the skin care product for tendering and anti-wrinkle according to claim 7, wherein the humectant and the thickener are added into the fermentation product of bifidobacterium longum SF-B-60, and the mixture is stood and heated to obtain an aqueous phase; mixing the emulsifier and the softener, and heating to the same temperature as the water phase to obtain an oil phase; adding the oil phase and chelating agent into the water phase, stirring and cooling to obtain the skin care product.
9. The method for preparing a skin-rejuvenating and anti-wrinkle skin care product according to claim 8, wherein the emulsifier is a composition of sorbitan oleate, stearamide AMP and methyl serine in a mass ratio of 3:3:1; the humectant is an acetylated sodium hyaluronate, glycosyl trehalose and algae extract composition with a mass ratio of 3:3:2; the softening agent is a butter and lanolin alcohol composition with a mass ratio of 2:1; the thickener is xanthan gum and carbomer composition with the mass ratio of 1:1; the chelating agent is disodium EDTA.
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