Facial mask containing bifidobacterium lactis fermented active extract
Technical Field
The invention relates to a facial mask containing a bifidobacterium lactis fermentation active extract, belonging to the field of cosmetics.
Background
At present, global industrialization and urbanization have great influence on environmental protection, and people in various countries generally pay attention to the pollution problem. The expression is in the beauty skin care products, that is, people find the biotechnology skin care products which have high technological content, are beneficial to skin health and are not polluted by chemicals. In addition, as people's safety awareness increases, people tend to select skin care products with high safety in order to avoid adverse effects on the skin.
In the prior art, the lysate of the yeast fermentation product of the second crack is used by various brands, and data show that the yeast fermentation filtrate can really reduce the oxidative damage of skin and promote the skin repair under the action of ultraviolet rays. The material consists of a bifidobacterium lysate synthesized by biotechnology. The data published by the manufacturers comprise metabolites, cytoplasm, cell walls and polysaccharide complexes, active substances obtained by means of biotechnology being able to support specifically the protective and reparative matrix of the skin itself. Can provide preventive care for normal skin and urinary skin, protect the damage caused by ultraviolet rays by enhancing the endogenous enzyme of cells to promote a repair system, and prevent the skin from aging in advance.
The prior art also discloses a bifidobacterium lactis fermented active extract which can effectively promote skin renewal and reduce melanin synthesis, has the effects of whitening skin, effectively removing free radicals and improving physiological functions of dermal tissues, has the efficacy of resisting aging and has higher safety, a preparation method and application thereof.
Bifidobacterium lactis is an obligate anaerobe, has strict requirements on environmental conditions and nutrition, and has a plurality of factors influencing the survival and growth of the bifidobacterium lactis. The invention aims to provide a mask containing a bifidobacterium lactis fermentation active extract.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a mask containing bifidobacterium lactis fermentation active extract, which obtains the anti-allergy effect with breakthrough exceeding hydrocortisone effect and the effect of stimulating collagen regeneration by creatively optimizing the fermentation process and combining with scientific proportioning.
The Okinra brevicaulis, a compound of Okinra brevicornus saponin R, which has been reported by Yangylon et al to be isolated from Okinra brevicornus, and has a structure of 3-O-beta-D-glucopyranosyl medicogenic acid 28-O-beta-D-allopyranosyl- (1 → 3) -beta-D-glucopyranosyl- (1 → 4) - [ beta-D-allopyranosyl- (1 → 3) ] -alpha-L-rhamnopyranosyl- (1 → 2) -alpha-L-arabinopyranosyl ester, and has a structural formula:
the synthesis of exopolysaccharides in bifidobacterium lactis can be divided into 2 classes, the synthesis of homologous polysaccharides and the synthesis of heterologous polysaccharides. Researchers have pointed out that strains with high extracellular polysaccharide yield generally have slow growth and reproduction speed and weak fragrance producing capacity and acidifying capacity. Homologous polysaccharides, such as dextran, fructan, are synthesized extracellularly. Heterologous polysaccharides are synthesized on the cell membrane.
The production of bifidobacterium lactis exopolysaccharide is a very complex process, and the bifidobacterium lactis exopolysaccharide has large difference by using different substrates and fermentation conditions. Different carbon sources, nitrogen sources and inorganic salts have an effect on the synthesis of exopolysaccharides of bifidobacterium lactis, and different bifidobacterium lactis have different requirements for these substances. It is considered that the proper control of the content of the nitrogen source in the medium is advantageous for the synthesis of the extracellular polysaccharide of Bifidobacterium lactis. The inventor tries to add the brevicoside R in the fermentation stage, and combines other optimized process conditions (such as cooperation of various carbon sources) to find that the physiological performance of the bifidobacterium lactis can be changed, and active substances such as biological polysaccharide and the like with extremely strong activity, anti-allergy and regeneration-promoting biological activity are generated.
The purpose of the invention is realized by the following technical scheme:
a facial mask containing Bifidobacterium lactis fermented active extract is prepared by the following method:
(1) preparing a bifidobacterium lactis fermentation active extract:
a. expanding and culturing the bifidobacterium lactis in a seed culture medium;
the seed culture medium comprises the following components in parts by weight: 30g of glucose, 10g of peptone, 10g of acerola cherry powder, 5g of yeast extract, 5g of sodium chloride, 801 ml of tween-801, 1g of dipotassium phosphate, 0.65g of magnesium sulfate heptahydrate, 0.19g of manganese sulfate monohydrate, 0.5g of baijiu grass saponin R, adding water to 1000ml, adjusting the pH value to 6.5, and sterilizing for later use;
b. inoculating the expanded bifidobacterium lactis seeds into a fermentation culture medium for fermentation to obtain a fermentation liquid;
fermentation medium: 1.8% of glucose, 1% of trehalose, 0.5% of mannose, 1% of peptone, 0.5% of acerola cherry powder, 1.5% of yeast extract, 800.1% of tween-2, 0.2% of dipotassium phosphate, 0.058% of magnesium sulfate heptahydrate, 0.019% of manganese sulfate monohydrate, 0.1% of L-cysteine hydrochloride and 0.2% of baijiu grass saponin R;
fermentation time: 9h, tank-out OD: 10;
fermentation temperature: controlling the initial pH value to be 7.0, the fermentation pH value to be 5.0, and the stirring speed to be: 80 r/min;
c. and (3) centrifugal treatment:
centrifuging the fermentation liquor at 7000r/min for 20min, pouring out the supernatant, and collecting bacterial sludge;
d. emulsification:
according to the mass of the bacterial sludge, adding a trehalose protective agent according to the ratio of 1:5, and fully emulsifying by using a high-speed stirrer to obtain an emulsion;
e. and (3) disinfection:
pasteurizing the emulsion to obtain active extract of fermented Bifidobacterium lactis;
(2) preparing other pure plant components:
preparing other plant extracts and additives, and making into facial mask liquid.
Preferably, the other plant extracts and additives are: water, sorbitol, glycerol, betaine, butylene glycol, rhizoma Polygoni Cuspidati (Polygonum CUSPIDATUM) extract, herba Centellae (CENTELLA ASIATICA) extract, radix Scutellariae (Scutellaria BAICALENSIS) root extract, folium Camelliae sinensis (CAMELLIA SINENSIS) extract, radix Glycytthizae (Glycyrrhiza GLABRA) root extract, flos Matricariae Chamomillae (Chamomilla RECUTITA) flower extract, herba Rosmarini OFFICINALIS (Rosmarinus OFFICINALIS) extract, propylene glycol, xanthan gum, carbomer, triethanolamine, hydroxyethyl cellulose, PEG-40 hydrogenated castor oil, phenoxyethanol, methylparaben, ethylparaben, and ethylhexylglycerol.
The invention has the advantages that:
1) the inventor tries to add the brevicoside R in the fermentation stage, and combines other optimized process conditions (such as cooperation of various carbon sources) to find that the physiological performance of the bifidobacterium lactis can be changed, and active substances such as biological polysaccharide and the like with extremely strong activity, anti-allergy and regeneration-promoting biological activity are generated.
2) The bifidobacterium lactis fermented active extract has stronger anti-allergy effect than hydrocortisone, and is a great breakthrough in the utilization of bifidobacterium lactis.
3) The bifidobacterium lactis fermented active extract has a strong effect of stimulating skin collagen regeneration.
Description of the drawings:
FIG. 1 shows control cells;
FIG. 2 shows the appearance of degranulation of cells after C48/80 stimulation;
FIG. 3 shows the cell morphology after C48/80 stimulation and simultaneous administration of 25g/L of Bifidobacterium lactis fermented active extract.
Detailed Description
Example 1:
preparing a bifidobacterium lactis fermentation active extract:
a. expanding and culturing the bifidobacterium lactis in a seed culture medium;
the seed culture medium comprises the following components in parts by weight: 30g of glucose, 10g of peptone, 10g of acerola cherry powder, 5g of yeast extract, 5g of sodium chloride, 801 ml of tween-801, 1g of dipotassium phosphate, 0.65g of magnesium sulfate heptahydrate, 0.19g of manganese sulfate monohydrate, 0.5g of baijiu grass saponin R, adding water to 1000ml, adjusting the pH value to 6.5, and sterilizing for later use;
b. inoculating the expanded bifidobacterium lactis seeds into a fermentation culture medium for fermentation to obtain a fermentation liquid;
fermentation medium: 1.8% of glucose, 1% of trehalose, 0.5% of mannose, 1% of peptone, 0.5% of acerola cherry powder, 1.5% of yeast extract, 800.1% of tween-2, 0.2% of dipotassium phosphate, 0.058% of magnesium sulfate heptahydrate, 0.019% of manganese sulfate monohydrate, 0.1% of L-cysteine hydrochloride and 0.2% of baijiu grass saponin R;
fermentation time: 9h, tank-out OD: 10;
fermentation temperature: controlling the initial pH value to be 7.0, the fermentation pH value to be 5.0, and the stirring speed to be: 80 r/min;
c. and (3) centrifugal treatment:
centrifuging the fermentation liquor at 7000r/min for 20min, pouring out the supernatant, and collecting bacterial sludge;
d. emulsification:
according to the mass of the bacterial sludge, adding a trehalose protective agent according to the ratio of 1:5, and fully emulsifying by using a high-speed stirrer to obtain an emulsion;
e. and (3) disinfection:
and (3) carrying out pasteurization on the emulsion to obtain the bifidobacterium lactis fermented active extract.
Example 2:
control 1, without brevicoside R:
preparing a bifidobacterium lactis fermentation active extract:
a. expanding and culturing the bifidobacterium lactis in a seed culture medium;
the seed culture medium comprises the following components in parts by weight: 30g of glucose, 10g of peptone, 10g of acerola cherry powder, 5g of yeast extract, 5g of sodium chloride, 801 ml of tween-801, 1g of dipotassium phosphate, 0.65g of magnesium sulfate heptahydrate and 0.19g of manganese sulfate monohydrate, adding water to 1000ml, adjusting the pH value to 6.5, and sterilizing for later use;
b. inoculating the expanded bifidobacterium lactis seeds into a fermentation culture medium for fermentation to obtain a fermentation liquid;
fermentation medium: 1.8% of glucose, 1% of trehalose, 0.5% of mannose, 1% of peptone, 0.5% of acerola cherry powder, 1.5% of yeast extract, 800.1% of tween-2, 0.2% of dipotassium phosphate, 0.058% of magnesium sulfate heptahydrate, 0.019% of manganese sulfate monohydrate and 0.1% of L-cysteine hydrochloride;
fermentation time: 9h, tank-out OD: 10;
fermentation temperature: controlling the initial pH value to be 7.0, the fermentation pH value to be 5.0, and the stirring speed to be: 80 r/min;
c. and (3) centrifugal treatment:
centrifuging the fermentation liquor at 7000r/min for 20min, pouring out the supernatant, and collecting bacterial sludge;
d. emulsification:
according to the mass of the bacterial sludge, adding a trehalose protective agent according to the ratio of 1:5, and fully emulsifying by using a high-speed stirrer to obtain an emulsion;
e. and (3) disinfection:
and (3) carrying out pasteurization on the emulsion to obtain the bifidobacterium lactis fermented active extract.
Example 3:
control 2, fermentation medium used a single glucose carbon source:
preparing a bifidobacterium lactis fermentation active extract:
a. expanding and culturing the bifidobacterium lactis in a seed culture medium;
the seed culture medium comprises the following components in parts by weight: 30g of glucose, 10g of peptone, 10g of acerola cherry powder, 5g of yeast extract, 5g of sodium chloride, 801 ml of tween-801, 1g of dipotassium phosphate, 0.65g of magnesium sulfate heptahydrate, 0.19g of manganese sulfate monohydrate, 0.5g of baijiu grass saponin R, adding water to 1000ml, adjusting the pH value to 6.5, and sterilizing for later use;
b. inoculating the expanded bifidobacterium lactis seeds into a fermentation culture medium for fermentation to obtain a fermentation liquid;
fermentation medium: 2.8% of glucose, 1% of peptone, 0.5% of acerola cherry powder, 1.5% of yeast extract, 800.1% of tween-800, 0.2% of dipotassium phosphate, 0.058% of magnesium sulfate heptahydrate, 0.019% of manganese sulfate monohydrate, 0.1% of L-cysteine hydrochloride and 0.2% of baijiu grass saponin R;
fermentation time: 9h, tank-out OD: 10;
fermentation temperature: controlling the initial pH value to be 7.0, the fermentation pH value to be 5.0, and the stirring speed to be: 80 r/min;
c. and (3) centrifugal treatment:
centrifuging the fermentation liquor at 7000r/min for 20min, pouring out the supernatant, and collecting bacterial sludge;
d. emulsification:
according to the mass of the bacterial sludge, adding a trehalose protective agent according to the ratio of 1:5, and fully emulsifying by using a high-speed stirrer to obtain an emulsion;
e. and (3) disinfection:
and (3) carrying out pasteurization on the emulsion to obtain the bifidobacterium lactis fermented active extract.
Example 4:
testing the anti-inflammatory effect:
the active extract of the bifidobacterium lactis is fermented into 10 percent solution, the solution is directly diluted into required concentration by a DMEM high-sugar culture medium containing 10 percent fetal calf serum during the experiment, and the positive control drug is hydrocortisone sodium succinate.
Experimental Material
1. Cell: rat basophilic leukemia cell RBL-2H3, Shanghai Life sciences research institute of Chinese academy of sciences cell resource center; human keratinocyte cell line HaCaT cell, cell resource center of institute of basic medicine of Chinese academy of medical sciences.
2. Reagent: DMEM high-glucose medium and 0.25 pancreatin (basic medical research institute of chinese medical science institute cell resource center); fetal bovine serum (Gibco); hydrocortisone sodium succinate for injection (Tianjin biochemicals, Inc.); sodium lauryl sulfate, Compound48/80(C48/80) and 4-nitrophenyl-N-acetyl-beta-D-glucosaminide (Sigma Co.); histamine ELISA kit (Elabscience); IL-1 alpha ELISA kit (Shanghai Eikesai Biometrics, Inc.); neutral red (national drug group chemical reagent corporation); CCK-8 (Tortoise chemical Co., Ltd., Japan).
The experimental method comprises the following steps:
in the research, sodium lauryl sulfate is adopted to stimulate HaCaT cells, an in vitro inflammation model is established, and the influence of the bifidobacterium lactis fermentation activity extract on the release of IL-1 alpha induced by the sodium lauryl sulfate under the concentration without cytotoxicity is detected.
The appropriate concentration of the stimulant sodium lauryl sulfate was first investigated. At concentrations of 3.9063-62.5. mu.g/mL, sodium lauryl sulfate had no effect on the proliferation and survival of HaCaT cells. After 125 mu g/mL-1000 mu g/mL of sodium lauryl sulfate acts for 24 hours, the proliferation and the activity of HaCaT cells can be obviously inhibited. Thus, in subsequent experiments, HaCaT cells will be stimulated with a concentration of 50. mu.g/mL without cytotoxic effect.
Experiments prove that after 25, 50 and 100g/L of the bifidobacterium lactis fermentation active extract and 0.25, 0.5 and 1g/L of hydrocortisone sodium succinate act on RBL-2H3 cells for 24 hours, no significant difference is generated compared with a normal control group, and the bifidobacterium lactis fermentation active extract and the hydrocortisone sodium succinate with different concentrations have no obvious toxicity on the RBL-2H3 cells.
Mast cell degranulation underlies pathological reactions such as immediate allergy (type I hypersensitivity) and inflammation. The RBL-2H3 cell is a rat basophilic leukemia cell strain, has many biological characteristics of mast cells, has a plurality of receptors and adhesion molecules expressed on the surface which are very similar to the mast cells matured in vivo, can generate degranulation reaction after being activated, releases active substances such as histamine, beta-hexosaminidase and the like, and is a good model for evaluating anaphylactic reaction/anaphylactoid reaction. In the study, C48/80 is used as a stimulant to stimulate RBL-2H3 cells, an in vitro sensitization model is established, and the influence of the bifidobacterium lactis fermentation activity extract on cell degranulation and histamine and beta-hexosaminidase release at a concentration without cytotoxicity is detected.
The IL-1 alpha level of the sodium lauryl sulfate-stimulated group of cells was significantly increased compared to the normal control group of cells. And simultaneously, 25g/L of bifidobacterium lactis fermentation active extract or 0.25g/L of hydrocortisone sodium succinate can obviously reduce the release of IL-1 alpha induced by sodium lauryl sulfate.
The specific data are shown in the following table:
the method is repeated for 5 times, has no significant difference every time, and has statistical significance.
Therefore, 25g/L of the bifidobacterium lactis fermentation active extract has a remarkable anti-inflammatory effect, and the baijiu grass saponin R and the carbon source control have a remarkable synergistic effect.
Example 5:
and (3) testing the anti-allergy effect:
the measurement of mast cell released bioactive substances (such as histamine, beta-hexosaminidase, etc.) is a common method for detecting allergy/anaphylactoid reactions and evaluating the anti-allergic effect of drugs. As shown in the following table, the histamine release from the cells of the C48/80-stimulated group was significantly increased compared to the cells of the normal control group. After 25g/L of the bifidobacterium lactis fermentation active extract or 1g/L of hydrocortisone sodium succinate is simultaneously given with C48/80 stimulation, the histamine release level is obviously reduced.
The method is repeated for 5 times, has no significant difference every time, and has statistical significance.
Therefore, the 25g/L bifidobacterium lactis fermentation active extract has a remarkable anti-allergy effect, and the control of the baijiu grass saponin R and the carbon source has a remarkable synergistic effect.
Example 6:
cell morphology by electron microscopy:
RBL-2H3 is used as basophil, the cytoplasm is large, and the cytoplasm is full of coarse basophil granules, so that the basophil granules have heterochromous property, and therefore, the degranulation condition can be observed from the cell morphology by a staining method. As shown in FIG. 1, after staining with neutral red, the cells of the normal control group grew well, were spindle-shaped or polymorphic, had smooth edges, had intact cell membranes, and had uniformly distributed, uniformly sized, and uniformly stained red particles in the cells.
As shown in FIG. 2, the degranulation of cells after C48/80 stimulation was observed: the cells become round and swell, the cell membranes are broken, the particles are discharged outside, and the cells are scattered outside the cell membranes and are more red-stained particles. As shown in FIG. 3, the C48/80 stimulation was accompanied by 25g/L of the Bifidobacterium lactis fermented active extract, and then normal spindle cells were increased to a level close to the normal level.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.