CN111759793B - Application of microbial polysaccharide as additive in preparation of daily cosmetics - Google Patents
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
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Abstract
The invention discloses a microbial polysaccharide and application thereof in daily cosmetics. The microbial polysaccharide is prepared by fermenting, separating and purifying Pantoea camel (Pantoea alhagi), has moisturizing, whitening and antioxidant functions, has different effects due to the difference of molecular weight, and can be used for being added into conventional daily cosmetics. Meanwhile, the Pantoea camelina (Pantoea alhagi) is a kind of endophyte, and the extracellular polysaccharide has various biological activities and no pathogenicity.
Description
Technical Field
The invention relates to the field of microbial polysaccharide innovation application, in particular to application of microbial polysaccharide in daily cosmetics.
Background
Polysaccharide is an important bioactive macromolecule, and is widely concerned nowadays in three fields, namely protein and gene, and life science, and hyaluronic acid becomes a new pet of the international cosmetic world with excellent moisturizing function as early as the last century, and scientists at home and abroad predict: "the coming decades will be the era of polysaccharides". The polysaccharides are classified according to their sources and can be classified into five major groups, i.e., fungal polysaccharides, bacterial polysaccharides, algal lichen polysaccharides, higher plant polysaccharides, and animal polysaccharides.
With the development of modern science and technology and the deepening of scientific research, daily cosmetics are developed from basic skin care products aiming at cleaning and moistening skin to functional daily cosmetics aiming at delaying aging and beautifying skin color. In recent years, researches show that the polysaccharide has various pharmacological effects, such as antitumor, antiviral, anti-aging, anti-hypoxia, anti-injury, antioxidant activity, anti-inflammation, immunity enhancement and the like, and has the advantages of safe use, mild effect, obvious effect and no side effect. In addition, a large number of hydrophilic hydroxyl groups linked with the polysaccharide enable the polysaccharide to have strong water absorption, emulsifying property, high viscosity and film forming property, the exclusivity is low, high affinity with human skin can be guaranteed when the polysaccharide is added into daily cosmetics, and compared with chemically synthesized cosmetics on the market, the microbial polysaccharide daily cosmetics have wider development and utilization space. The pharmacological activity and physicochemical property of the polysaccharide provide basis for the application of the polysaccharide in cosmetics, and the polysaccharide can be applied to the cosmetics to achieve the effects of moisturizing, repairing damaged skin, resisting inflammation, delaying aging, resisting oxidation and the like.
Therefore, the application of the bioactive microbial polysaccharide as an effective daily cosmetic additive in daily cosmetics is a research hotspot and direction in the future.
Disclosure of Invention
The purpose of the invention is as follows: the invention discovers that the addition of the pantoea polysaccharide in the daily cosmetics has various good effects for the first time, and provides the application of a novel microbial polysaccharide as an additive in the daily cosmetics. The invention aims to provide a functional microbial polysaccharide for a daily cosmetic raw material, which not only widens the application field of the microbial polysaccharide, but also supplements the limitation of the source of the daily cosmetic raw material.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the application of microbial polysaccharide in the preparation of daily cosmetics is characterized in that the microbial polysaccharide is prepared by fermenting Pantoea camelina. The Pantoea camelina taxonomic name of the Pantoea camelina is Pantoea alhagi which is a type of endophyte and has no pathogenicity.
Preferably, the microbial polysaccharide has a molecular weight of 1000Da to 2000 kDa.
Further, applicants have found that differences in the molecular weight of microbial polysaccharides have an effect on their performance.
Specifically, the applicant found that when the molecular weight range of the microbial polysaccharide is 1000kDa to 2000kDa, relatively excellent moisturizing performance is exhibited, and therefore the present invention further proposes the use of the above microbial polysaccharide as a moisturizing agent for the preparation of a daily cosmetic product, when the moisturizing performance is emphasized, the molecular weight range of the microbial polysaccharide is 1000kDa to 2000 kDa.
Further, the applicant found that whitening performance is outstanding when the molecular weight region of the microbial polysaccharide is 100kDa to 1000kDa, and thus further proposed the use of the above microbial polysaccharide as a whitening agent for the preparation of a daily cosmetic, and when emphasis is placed on whitening performance, the molecular weight region of the microbial polysaccharide is 100kDa to 1000 kDa.
In addition, the applicant found that the antioxidant property is outstanding when the molecular weight region of the microbial polysaccharide is 1000Da to 100kDa, and thus proposed the use of the above microbial polysaccharide as an antioxidant in the preparation of a daily cosmetic, and when the whitening property is emphasized, the molecular weight region of the microbial polysaccharide is 1000Da to 100 kDa.
Preferably, the addition amount of the microbial polysaccharide in the daily cosmetics is 0.1-0.5% (g/g), namely 0.001-0.005g of microbial polysaccharide is added in each g of daily cosmetics.
Specifically, the preparation method of the microbial polysaccharide comprises the following steps:
a) fermentation preparation: the fermentation medium comprises carbon sources, nitrogen sources, inorganic salts and oxygen carriers, wherein the concentration of the carbon sources is 30-50 g/L, preferably 35-45 g/L; the concentration of the nitrogen source is 2-8 g/L, and the preferred scheme is 3-5 g/L; the total concentration of the inorganic salt is 0.5-2.5 g/L, preferably 1.5-2 g/L; the total concentration of the oxygen carriers is 0.5-10 g/L; in the fermentation process, the temperature is 25-32 ℃, the aeration intensity is 0.1-0.8 vvm, the fermentation period is 16-48 h, and the pH is 6.5-7.5;
b) extraction and purification: sterilizing the fermentation liquor collected in the step a) at high temperature, preferably, sterilizing at 121 ℃ for 20min, precipitating with 2-3 times of ethanol, collecting floccule, drying, redissolving the dry matter, filtering and removing impurities by a plate-and-frame filter, precipitating with 2-3 times of ethanol again, collecting floccule precipitate, and drying to obtain the purified extracellular polysaccharide.
Preferably, in the step a), the polysaccharide content in the fermentation liquor after the fermentation is finished is not lower than 20 g/L.
Specifically, the carbon source is sucrose or glucoseOne or a combination of glucose, glycerol, starch and molasses; the nitrogen source is any one or a combination of more of ammonium sulfate, corn steep liquor, soybean meal and peptone; the inorganic salt is K2HPO4、KH2PO4、MgSO4、MnSO4、CaCl2Any one or a combination of several of the above, preferably 1.5-2 g/L; the oxygen carrier is any one or combination of n-hexane, n-heptane and n-hexadecane, and the preferable scheme is 2.33g/L of n-hexane, 5.36g/L of n-heptane and 9.06g/L of n-hexadecane.
Has the advantages that: compared with the prior art, the method has the following advantages:
(1) the invention discovers that the endophyte Pantoea camelina (Pantoea alhagi) which is nonpathogenic to human bodies can be used as an additive for preparing daily cosmetics, and extracellular products of the Pantoea camelina are fermented, separated and purified, so that various excellent biological activities of the Pantoea camelina are discovered, and the application field of the Pantoea camelina is widened;
(2) the invention discovers that the pantoea polysaccharide has good moisturizing, whitening and antioxidant effects for the first time, provides the application of the polysaccharide in preparing cosmetics, widens the application field of the pantoea camelina extracellular product, supplements moisturizing, whitening and antioxidant raw materials of the cosmetics, and widens the variety of the existing microbial polysaccharide.
Drawings
FIG. 1 is a graph showing a comparison of moisturizing properties of Pantoea polysaccharide;
FIG. 2 is a comparison of the whitening efficacy of Pantoea polysaccharide;
FIG. 3 shows the radical scavenging ability of pantoea polysaccharide DPPH.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The examples will help to understand the present invention given the detailed embodiments and the specific operation procedures, but the scope of the present invention is not limited to the examples described below.
Example 1 preparation of microbial polysaccharides.
The applicant successfully finds a plant endophyte Pantoea camelina which is nonpathogenic to human bodies in previous researches, the taxonomic name of the Pantoea alhagi XK-11 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation time is 2018, 3 and 29 days, the preservation address is No. 3 of West Lu No. 1 of the Chaoyang district in Beijing, the preservation number is CGMCC 15526, and the Pantoea camelina is a kind of plant endophyte which is nonpathogenic.
Preparing exopolysaccharide by further performing high-density fermentation on the pantoea camel, wherein the fermentation medium comprises a carbon source, a nitrogen source, inorganic salt and an oxygen carrier, wherein the carbon source is one or a combination of sucrose, glucose, glycerol, starch and molasses, and the concentration of the carbon source is 40 g/L; the nitrogen source is one or a combination of ammonium sulfate, corn steep liquor, soybean meal and peptone, and the concentration of the nitrogen source is 6 g/L; the inorganic salt is K2HPO4、KH2PO4、MgSO4、MnSO4、CaCl2One or a combination of (1) and (3), wherein the total concentration of the inorganic salt is 1.3 g/L; the oxygen carrier is one or a combination of n-hexane, n-heptane and n-hexadecane, and the total concentration of the oxygen carrier is 2.6 g/L; in the fermentation process, the temperature is 30 ℃, the aeration intensity is 0.6vvm, the fermentation period is 36h, and the pH is 6.8.
And after the fermentation is finished, extracting and purifying the collected fermentation liquor to obtain the purified extracellular polysaccharide. The steps comprise a series of steps of high-temperature sterilization, alcohol precipitation, impurity removal of a redissolution plate frame, secondary precipitation and drying. Firstly, sterilizing the collected fermentation liquor at high temperature (121 ℃, 20min), then precipitating with 2-3 times of ethanol, collecting floccules, drying, re-dissolving the dry matter, filtering and removing impurities by a plate-and-frame filter, secondarily precipitating with 2-3 times of ethanol, collecting floccules, and drying to obtain the purified extracellular polysaccharide.
The determination method of the polysaccharide is an HPLC method, a GPC gel chromatographic column is adopted as the chromatographic column, an evaporative light scattering detector is adopted as the detector, pure water is adopted as the mobile phase, the separation temperature is 60 ℃, and the flow rate is 1 mL/min.
The purified exopolysaccharide obtained by the preparation method is neutral polysaccharide, wherein monosaccharide components of the neutral polysaccharide comprise glucose, galactose and mannose, and the proportion of the monosaccharide components is about 0.48-0.59: 0.24-0.33: 0.087-0.18, the monosaccharide ratio is not constant, but slightly fluctuates due to different components and concentrations of the fermentation medium.
The monosaccharide components are derived by a sugar nitrile acetate method and then are measured by GC-MS.
Example 2 preparation of polysaccharides of different molecular weight ranges.
The polysaccharides with different molecular weights are prepared by an acidolysis method, and the specific implementation steps are as follows:
weighing the obtained purified exopolysaccharide, preparing into 2% polysaccharide solution, adding sulfuric acid solution to adjust pH to 3.0, heating at 80 deg.C for hydrolysis, sampling at different time points, and detecting to determine molecular weight region.
The detection method is high performance liquid chromatography detection, the chromatographic column adopts GPC gel chromatographic column, the detector is an evaporative light scattering detector, the mobile phase is pure water, the separation temperature is 60 ℃, and the flow rate is 1 mL/min.
The results of the hydrolysis experiments are shown in Table 1.
TABLE 1 Effect of different hydrolysis times on polysaccharide molecular weight
Example 3 moisturizing performance examination of microbial polysaccharides.
The moisture retention was investigated by the following experiment:
respectively weighing the purified pantoea polysaccharide with different molecular weight regions prepared in example 2 and 0.5g of sodium alginate, chitosan and glycerol which are dried to constant weight at room temperature, respectively placing the pantoea polysaccharide and the sodium alginate, the chitosan and the glycerol into a culture dish, adding 0.2g of deionized water, slowly shaking to enable the sample to fully absorb moisture, placing the sample into a dryer filled with allochroic silica gel, standing the sample for 1, 2, 3, 4, 5 and 6 hours, weighing the sample, and obtaining the moisture retention ratio (%) according to the formula (1) (H)n/H0) X 100, obtaining the moisture retention rate, wherein HnAnd H0The moisture mass before and after standing, respectively, is shown in FIG. 1.
As can be seen from FIG. 1, in a dry silica gel environment, the moisturizing sequence for 1-6 hours is as follows: glycerol is 1000kDa to 2000kDa pantoea polysaccharide, chitosan is 100kDa to 1000kDa pantoea polysaccharide, sodium alginate is 1kDa to 100kDa pantoea polysaccharide; although the moisture retention rate of the 1000kDa-2000kDa pantoea polysaccharide is slightly lower than that of glycerol, compared with chitosan and sodium alginate, the pantoea polysaccharide has obvious moisture retention advantages.
Example 4 whitening effect of microbial polysaccharides.
To examine the whitening efficacy of the microbial polysaccharide of the present invention and its application in cosmetics for daily use, we know that tyrosinase is a major rate-limiting enzyme in the process of melanin formation, and this enzyme activity determines the amount of melanin. It is a copper-containing metalloenzyme, synthesized by melanocytes, which plays a central role in the production of melanin. The tyrosinase activity is increased, and the melanin production capacity of melanocytes is correspondingly enhanced; and vice versa decreases accordingly.
The preparation of the reagent in the tyrosinase inhibition experiment and the sample pretreatment are as follows:
PBS phosphate buffer solution, namely accurately weighing 17.71g of dodecahydrate disodium hydrogen phosphate into a 500mL volumetric flask, and diluting with distilled water to constant volume to obtain solution a; accurately weighing 7.8g of sodium dihydrogen phosphate dihydrate into a 500mL volumetric flask, and diluting with distilled water to a constant volume to obtain a solution b; 92.6 mL and 107.4mL of solutions a and b were prepared into 200mL of PBS phosphate buffer with pH 6.8.
L-tyrosine solution: accurately weighing 0.05g tyrosine, dissolving in 35mL 0.1 mol.L-1Then, 65mL of PBS phosphate buffer (pH 6.8) was added thereto.
Sample liquid: the purified polysaccharides with different molecular weights prepared in example 2 were weighed out accurately and prepared into 20, 60, 100, 140, 180mg/L polysaccharide solutions for further use.
The above solutions were mixed, and the results are shown in table 2.
TABLE 2 compounding ratio of each solution
A, B, C, D tubes were filled with the corresponding reagents according to the formulation in Table 1, A and C were placed in a 37 ℃ water bath for 10min before the enzyme was added, then the enzyme was added, and A, B, C, D was placed in a 37 ℃ water bath at the same time. After 10min, the absorbance value of each tube at 475nm was measured and recorded as A1、B1、C1And D1(B1And D1Zero adjustment), tyrosinase inhibition (I) was calculated. The calculation formula is as follows:
I＝(A1-C1)/A1×100％
the inhibition rate of tyrosinase was taken as the ordinate, the concentration of the sample to be measured was taken as the abscissa, and the inhibition rate curve was plotted, and the result is shown in fig. 2. As can be seen from fig. 2, both pantoea polysaccharide (polysaccharide) and vitamin C have tyrosinase inhibitory effects, and the inhibitory effects increase with increasing mass concentration. As can be seen from the figure, the rise tendency of Pantoea polysaccharide with molecular weight range of 100kDa-1000kDa is larger than that of vitamin C, has potential tyrosinase inhibition effect, and has mass concentration of more than 100 mg.L-1It is higher than vitamin C.
At the same time, the inhibition rate is plotted against the inhibitor concentration, and the IC of pantoea polysaccharide and Vc is obtained according to the inhibition curve50The value is obtained. Results show IC for Vc50The value was (152. + -. 2.8) mg/L; example 2 IC of Pantoea polysaccharide prepared with a molecular weight range of 100kDa to 1000kDa50The value is (126 +/-3.5) mg/L, and the inhibition effect on tyrosinase is similar to the intensity level of Vc.
Example 5 in vitro antioxidant activity study of microbial polysaccharides.
The DPPH method is a rapid, simple, convenient, sensitive and feasible method for evaluating the antioxidant activity of the natural antioxidant, and is widely applied at home and abroad. The mechanism by which DPPH scavenges free radicals is the reduction of DPPH to DPPH-H in the presence of a hydrogenation antioxidant, resulting in the discoloration of violet, thereby inhibiting the propagation of oxidation reactions.
The experimental procedure is as follows:
the polysaccharide in different molecular weight regions prepared in example 2 is accurately weighed, and prepared into polysaccharide solutions of 0.2, 0.4, 0.6, 0.8 and 1.0mg/mL for later use, a Vc solution with the same concentration is used as a control, 1mL of the polysaccharide solution is added into 2mL of a 0.2mmol/mL DPPH ethanol solution respectively, the mixture is uniformly mixed, the mixture is kept stand for 30min at room temperature in a dark place, the absorbance is measured at the wavelength of 517nm, and the Vc is used as a positive control, and the calculation is carried out according to the formula (2).
DPPH radical scavenging ratio (%) - [ A [ ]0-(A1-A2)]/A0×100 (2)
Wherein A is1: adding a sample to be detected and a DPPH ethanol solution; a. the2: adding absolute ethyl alcohol into a sample to be detected; a. the0: absolute ethanol + DPPH ethanol solution.
As shown in FIG. 3, the scavenging ability of pantoea polysaccharide to DPPH radical increases with increasing concentration, and particularly, when the molecular weight of pantoea polysaccharide is in the range of 1kDa to 100kDa, the scavenging rate of pantoea polysaccharide reaches about 79.63% when the concentration reaches 1mg/mL, and it can be seen from the results of the figure that the scavenging ability of pantoea polysaccharide to DPPH radical varies greatly with concentration, and the difference between the scavenging ability of pantoea polysaccharide to scavenge radicals and vitamin C is gradually reduced when the concentration is greater than 1 mg/mL.
The invention provides the idea and method of application of microbial polysaccharide in daily cosmetics, and the method and way for implementing the technical scheme are many, the above description is only the preferred embodiment of the invention, it should be noted that, for those skilled in the art, without departing from the principle of the invention, several improvements and modifications can be made, and these improvements and modifications should be regarded as the protection scope of the invention. All the components not specified in this embodiment can be implemented by the prior art.
Claims (7)
1. The application of the microbial polysaccharide as the humectant for preparing the daily cosmetics is characterized in that the microbial polysaccharide is prepared by fermenting Pantoea camelina which is classified and named asPantoea alhagi，Said Camellia sinensis can be preserved in Chinese microbial strainThe preservation number of the general microorganism center of the Tibetan management Committee is CGMCC 15526, and the molecular weight range of the microbial polysaccharide is 1000kDa-2000 kDa.
2. The application of the microbial polysaccharide as the whitening agent for preparing the daily cosmetics is characterized in that the microbial polysaccharide is prepared by fermenting Pantoea camelina which is classified and named asPantoea alhagi，The Pantoea alhagi is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, the preservation number is CGMCC 15526, and the molecular weight range of the microbial polysaccharide is 100kDa-1000 kDa.
3. The application of microbial polysaccharide as antioxidant in preparing daily cosmetics is characterized in that the microbial polysaccharide is prepared by fermenting Pantoea camelina which is classified and named as Pantoea camelinaPantoea alhagi，The Pantoea alhagi is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, the preservation number is CGMCC 15526, and the molecular weight range of the microbial polysaccharide is 1000Da-100 kDa.
4. Use according to any one of claims 1 to 3, characterized in that the microbial polysaccharide is added in an amount of 0.1-0.5% by weight in the daily cosmetics.
5. Use according to any one of claims 1 to 3, characterized in that the microbial polysaccharide is prepared as follows:
a) fermentation preparation: the fermentation medium comprises carbon sources, nitrogen sources, inorganic salts and oxygen carriers, wherein the concentration of the carbon sources is 30-50 g/L; the concentration of the nitrogen source is 2-8 g/L; the total concentration of the inorganic salt is 0.5-2.5 g/L; the total concentration of the oxygen carriers is 0.5-10 g/L; in the fermentation process, the temperature is 25-32 ℃, the aeration intensity is 0.1-0.8 vvm, the fermentation period is 16-48 h, and the pH is 6.5-7.5;
b) extraction and purification: sterilizing the fermentation liquor collected in the step a) at high temperature, precipitating with ethanol, collecting floccule, drying, redissolving dry matter, filtering with a plate-and-frame filter to remove impurities, precipitating with ethanol again, collecting floccule precipitate, and drying to obtain purified extracellular polysaccharide.
6. The use according to claim 5, wherein in step a), the polysaccharide content in the fermentation broth after the fermentation is finished is not less than 20 g/L.
7. The use according to claim 6, wherein the carbon source is one or a combination of sucrose, glucose, glycerol, starch, molasses; the nitrogen source is any one or a combination of more of ammonium sulfate, corn steep liquor, soybean meal and peptone; the inorganic salt is K2HPO4、KH2PO4、MgSO4、MnSO4、CaCl2Any one or a combination of several of them; the oxygen carrier is any one or combination of n-hexane, n-heptane and n-hexadecane.
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Address after: 210000 No. 85, constellation Road, Jiangbei new area, Nanjing, Jiangsu Applicant after: Nanjing xuankai Biotechnology Co.,Ltd. Address before: Floor 10, building a, phase I, Zhongdan Life Science Industrial Park, No. 3-1, xinjinhu Road, Jiangbei new area, Nanjing, Jiangsu 210008 Applicant before: NANJING SHINEKING BIOTECH Co.,Ltd. |
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