CN110840781B - Preparation method of tremella spore fermentation extract and product thereof - Google Patents
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Abstract
The invention provides a preparation method of a tremella spore fermentation extract, which comprises the following steps: adjusting the pH value of the fermentation liquor to 7.0-11.0, and centrifuging to obtain supernatant; then adjusting pH to 5.5-7.0, adding active carbon for adsorption, and filtering to obtain filtrate I; then adjusting the pH value to 8.5-9.5, heating, cooling to room temperature, and filtering to obtain a filtrate II; and adding activated carbon into the filtrate II for adsorption and then filtering to obtain a tremella spore fermentation extract. The invention can ensure that the fermentation liquor is easier to purify by selecting proper fermentation liquor viscosity and pH value during thallus removal, the fermentation extracting solution has no sticky feeling, the content of amino acid is relatively higher, and the invention is a natural plant high-quality skin care and moisture preservation product.
Description
Technical Field
The invention relates to the field of cosmetic raw materials, in particular to a preparation method of a tremella spore fermentation extracting solution for cosmetics.
Background
Tremella is commonly called white fungus or white fungus and is recorded in traditional Chinese medicine ancient books (Taiping Jinghui Fang), and the white fungus has been used for bath therapy in Tang dynasty and has the effects of moistening and smoothing skin. The tremella is like transparent petals in appearance, can extract mucopolysaccharide rich in glucose, trehalose, pentose polyacid, mannitol and other components, and has excellent moisture retention.
The main active component of the tremella fermentation extract is tremella polysaccharide which is an acidic heteropolysaccharide, the main chain structure is mannan connected by alpha- (1 → 3) glycosidic bond, the branched chain is tremella heteroglycan consisting of glucuronic acid and xylose, the tremella fermentation extract has excellent moisture-preserving and water-locking capabilities, is applied to skin-care cosmetics, and has the functions of repairing epidermis, increasing the moisture content of the epidermis, improving the integral value of the skin conductivity, enhancing the skin protection barrier and preventing the water from evaporating from the skin. Moreover, the tremella polysaccharide has good lubricity and film forming property, and when products such as skin care products or hair care products added with the tremella polysaccharide are used, a layer of uniform film can be formed on the surface of skin or hair, so that the tremella polysaccharide plays roles in moisturizing, lubricating and eliminating static electricity. In addition, the tremella polysaccharide also has certain capability of removing hydroxyl free radicals, and can be used as an anti-aging and anti-oxidation component to be applied to cosmetics.
When tremella polysaccharide is prepared by a tremella spore fermentation method, other various active products such as various small molecule essential amino acids, sterol, phytic acid, vitamins and the like can be metabolized by tremella spores, so that the nutrient substances, the nutrient density and the physiological activity of the tremella fermentation extract are greatly improved, and the value of the tremella polysaccharide is far higher than that of tremella polysaccharide.
The tremella extract and tremella polysaccharide have been widely added into cosmetics, for example, patent CN 105002254A, a preparation method of tremella fermentation extract and its application, in this patent, the liquid of saccharomyces cerevisiae, streptococcus thermophilus, etc. is inoculated into the solution made of tremella powder for fermentation, and the tremella extract prepared by this method is natural and stable in quality. However, the method still needs a large amount of tremella sporophore, and compared with the direct extraction of tremella spore fermentation liquid, the method has high cost and is not easy for large-scale production.
The existing tremella spore fermentation is mainly used for preparing tremella polysaccharide, for example, patent CN 107446825A provides an application of tremella spore strain HX5-1 in production of tremella polysaccharide, barley peptone, yeast peptone, glucose, potassium dihydrogen phosphate, magnesium sulfate and VB1 are used as fermentation medium components, and a high molecular weight tremella polysaccharide product is obtained through three-stage aeration fermentation, alcohol precipitation, decolorization, protein removal, refining and drying. The patent obtains high molecular tremella polysaccharide pure product and does not contain other active products metabolized by tremella spores.
At present, no relevant preparation method of the tremella spore fermentation extract is reported. Therefore, in order to improve the utilization rate of tremella spore fermentation and obtain cosmetic raw materials with better activity, a preparation method of tremella spore fermentation extract needs to be developed.
Disclosure of Invention
Aiming at the problem that no preparation method of tremella spore fermentation extract exists in the prior art, the invention provides the preparation method of the tremella spore fermentation extract, which has the advantages of short production period, high content of active substances tremella polysaccharide and amino acid in fermentation liquor, stable product and benefit for later purification by selecting proper fermentation viscosity and controlling the pH value during thallus removal.
In order to achieve the purpose, the invention adopts the following technical scheme.
A preparation method of a tremella spore fermentation extract comprises the following steps:
(1) And (3) removing thalli: adjusting the pH value of the fermentation liquid, and centrifuging to obtain a supernatant;
(2) Decoloring and removing impurities: adjusting the pH value of the supernatant, adding activated carbon for adsorption, and filtering to obtain a filtrate I;
(3) Protein removal: adjusting the pH value of the filtrate I, heating, cooling to room temperature, and filtering to obtain a filtrate II;
(4) Adsorbing and removing impurities: and adding activated carbon into the filtrate II for adsorption, and filtering to obtain a tremella spore fermentation extracting solution.
The fermentation liquor is obtained by inoculating tremella spores to a fermentation medium for fermentation, and the viscosity of the fermentation liquor is 6000-12000mpa & s, preferably 8000-10000mpa & s.
The fermentation medium comprises the following components: barley protein powder or soybean protein powder 3-8 g/L, yeast extract 3-8 g/L, glucose 10-20 g/L, potassium dihydrogen phosphate 1-5 g/L, magnesium sulfate 1-3 g/L and V B1 10-20 mg/L。
In step (1), the pH is adjusted to 7.0 to 11.0, preferably 9.0 to 11.0.
In the step (1), the centrifugation speed is 10000-15000rpm, and the centrifugation time is 15-30min.
In the step (2), the pH is 5.5-7.0.
In the step (2), the adding amount of the activated carbon is 0.2-1% (w/v), the adsorption temperature is 60-70 ℃, and the adsorption time is 0.5-1h.
In the step (2), the filtration pore size is 1.2 μm.
In the step (3), the pH is 8.5-9.5.
In the step (3), the heating temperature is 80-90 ℃, and the heating time is 0.5-1h.
In the step (3), the filtration pore size is 0.65 μm.
In the step (4), the adding amount of the activated carbon is 0.2-1% (w/v), the adsorption temperature is 60-70 ℃, and the adsorption time is 0.5-1h.
In the step (4), the filtration pore size is 0.45 μm.
A Tremella spore fermentation extractive solution obtained by the above preparation method. The content of tremella polysaccharide in the tremella spore fermentation extracting solution is 3-5 g/L; the total amino acid content is 70-90mg/L.
A cosmetic containing the above Tremella spore fermentation extract is provided.
The invention has the following advantages:
the invention ensures that the fermentation liquor prepared by the process is easier to purify by controlling the final viscosity of the fermentation liquor and controlling the pH value during thallus removal, the content of active substances in the extract is relatively higher, and the prepared fermentation extract has no sticky feeling and is easier to use in cosmetics.
Drawings
FIG. 1 is a standard curve of the determination of sugar content by the phenol-sulfuric acid method;
FIG. 2 is a graph showing the effect of the fermented extract of Tremella spores on cell migration.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
Example 1 preparation of Tremella spore fermentation broth
1. Seed liquid preparation
Inoculating Tremella fuciformis strain HX5-1 (preservation number is CCTCC M2017271) into liquid culture medium (barley protein powder 5g/L, yeast powder 5g/L, glucose 20 g/L, potassium dihydrogen phosphate 3 g/L, magnesium sulfate 1.5 g/L and V B1 15 mg/L; pH value of 6.0), fermenting in a shaking flask at 30 ℃ for 3 days at 200 r/min to obtain seed liquid;
2. three-stage aeration fermentation
(1) Inoculating the seed liquid into a 10L fermentation tank by 3 percent of inoculation amount, wherein the formula of a culture medium is as in the step (1), the culture is performed at the temperature of 30 ℃ under the condition of aeration and stirring, the rotating speed is 200 r/min, and the aeration amount is as follows: initially 10L/min, 15L/min after 24 h, 20L/min after 48 h until the end of fermentation, adjusting the pH value to 6.3-6.5 by using 40% NaOH in the fermentation process, intermittently feeding materials in a feeding mode, ending the fermentation when the viscosity is 10000mPa · s, and obtaining a fermentation liquid F1, wherein the content of the tremella polysaccharide is 10.2 g/L;
(2) Fermentation conditions in the step (1) are adopted to respectively ferment so as to obtain fermentation liquor F2-F6, and the viscosity and the tremella polysaccharide content are shown in the table 1:
TABLE 1 viscosity of Tremella spore fermentation broth and Tremella polysaccharide content
As can be seen from the data in Table 1, the higher the viscosity of the tremella spore fermentation broth, the higher the content of tremella polysaccharide.
Example 2 preparation of Tremella spore fermentation extract
An extract was prepared from the fermentation broth F1 prepared in example 1 by the following method:
(1) And (3) removing thalli: adjusting the pH value of the fermentation liquor to 7.0, and centrifuging at 15000rpm for 20 min to obtain a supernatant;
(2) Decoloring and removing impurities: adjusting the pH of the supernatant to 6.5, adding 0.5% w/v activated carbon (200 mesh) to adsorb 1h at 65 deg.C, and filtering to obtain filtrate I;
(3) Protein removal: adjusting the pH value of the filtrate I to 9.5, heating to 0.75 h at 90 ℃, cooling to room temperature, and filtering to obtain filtrate II;
(4) Adsorbing and removing impurities: adding 0.5% w/v active carbon (200 mesh) into the filtrate II, adsorbing at 60 deg.C for 1 hr, cooling to room temperature, and filtering with 0.45 μm polypropylene filter element to obtain Tremella spore fermentation extractive solution S1.
In the same manner, 2 parts of the fermentation broth F1 prepared in example 1 were taken and extracted as described above, except that the pH was adjusted to 9.0 and 11.0 in step (1) to obtain tremella spore fermentation extracts S2 and S3, respectively.
Comparative example 1 preparation of Tremella spore fermentation extract
Referring to the method in example 2, the fermented liquid F1 is used to prepare the fermented extract of tremella spores, and the pH in step (1) is adjusted to 13.0 or 5.0, respectively, to obtain the fermented extract S4 and S5 of tremella spores.
Example 3 preparation of Tremella spore fermentation extract
Referring to the steps (1) to (4) in example 2, the fermentation liquids F3, F4 and F5 are respectively prepared into tremella spore fermentation extract, and tremella spore fermentation extract S7, S8 and S9 are obtained.
Comparative example 2 preparation of Tremella spore fermentation extract
Referring to the method of steps (1) to (4) in example 2, the fermentation liquids F2 and F6 are respectively prepared into tremella spore fermentation extract liquids, and tremella spore fermentation extract liquids S6 and S10 are obtained.
The samples obtained in each example and comparative example were as follows:
comparative example 3 preparation of Tremella polysaccharide
Referring to CN 107446825A a tremella strain and the method of application thereof, tremella polysaccharide is prepared from fermentation broth F6, and tremella polysaccharide S11 is obtained.
Example 4 amino acid content in Tremella spore fermentation extract
The content and type of amino acids contained in the extract liquid samples prepared in each example and comparative example were analyzed by an automatic amino acid analyzer using the liquid medium of example 1 as a control, and the results are shown in Table 2.
TABLE 2 amino acid content in different samples of Tremella spore fermentation extractive solution
As can be seen from the data in table 2, 8 new amino acids such as hydroxyproline, serine, proline, glycine, valine, leucine, ornithine, lysine and the like were detected in the fermentation broth by fermentation; the content of threonine and alanine is also increased; the total amino acid content is increased by 7-8 times. This indicates an increase in active substance after fermentation. Taking tremella spore fermentation liquor with the same viscosity as a raw material, wherein along with the rise of pH (S1-S5) in the step (1), the content of amino acid shows a trend of rising firstly and then falling, when the pH range is 7.0-13.0, the content of amino acid is stabilized to be more than 75mg/L, and when the pH is 9.0-11.0, the content of amino acid is more than 85 mg/L; by adopting the same extraction process, along with the increase of the viscosity of the tremella spore fermentation liquor (S2, S6-S10), the content of amino acid tends to increase firstly and then decrease, and the content of amino acid in the obtained extracting solution is not further increased by the fermentation liquor with overhigh viscosity (S10, viscosity =14000mPa · S).
Example 5 Tremella polysaccharide content in Tremella spore fermentation extract
The content and the type of the amino acid contained in the extract samples prepared in the examples and the comparative examples are analyzed, and the content of the high molecular weight tremella polysaccharide in the product is determined by adopting a phenol-sulfuric acid method, and the method comprises the following specific steps:
preparing standard yeast: accurately weighing glucose 100 mg, adding 20 mL distilled water for dissolving, transferring to a 250 mL volumetric flask, adding water to the scale, diluting by 10 times before use, sucking 0.4,0.8,1.2,1.6 and 2.0 mL respectively into a test tube with a plug, and supplementing to 2.0 mL with distilled water respectively.
Sample preparation: weighing a 250 mg sample, adding 20 mL distilled water for dissolving, transferring to a 50 mL volumetric flask, adding water to a constant volume to be marked as a stock solution, measuring stock solution 4 mL before use, placing in a 50 mL volumetric flask, adding water to be marked, measuring and diluting by 10 times before use, and taking 2 mL to be placed in a test tube with a plug.
And (3) determination: adding 6% redistilled phenol 1.0 mL and concentrated H into the above standard solution and sample diluent 2 SO 4 5.0 mL, shake and cool. After reacting for 20 min at room temperature, measuring OD at 490 nm, using 2.0 mL water as blank according to the same color development operation, using sugar concentration as ordinate and absorbance value as abscissa, obtaining a standard curve and a regression equation: y = 0.0122x-0.0384 (R = 0.9994) is shown in fig. 1. The polysaccharide concentration was obtained from the regression equation of the standard curve, and the high molecular weight tremella polysaccharide content in the product was calculated, with the results shown in table 3:
TABLE 3 Tremella polysaccharide content in different Tremella spore fermentation extractive solution samples
S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | |
Content (g/L) | 4.46 | 4.67 | 4.57 | 4.02 | 3.99 | 3.24 | 4.29 | 4.51 | 4.11 | 3.52 |
As can be seen from the data in Table 3, with the same tremella spore fermentation liquid, as the pH value increases (S1-S5) in the process of separating the mycelia in step (1), the tremella polysaccharide in the extract tends to increase first and then decrease. The tremella polysaccharide content in the extract after bacteria removal by adopting pH7.0-11.0 is high, when the pH is lower than 7 (S5, pH = 5) during the bacteria separation, the bacteria and the polysaccharide can not be effectively separated, and when the pH is higher than 11.0 (S4, pH = 13) during the bacteria separation, some polysaccharide conformation is changed and separated out, so that serious loss is caused. In the extracting solutions (S2, S6-S10) prepared by adopting the same conditions and using the fermentation liquors with different viscosities, along with the increase of the viscosity, the concentration of the tremella polysaccharide is increased firstly and then decreased, the viscosity is lower than 6000mPa & S (S6, viscosity =4000mPa & S), the accumulation of the sugar content in the fermentation liquor is not high, and after the viscosity exceeds 12000mPa & S (S10, viscosity =14000mPa & S), although the polysaccharide content in the fermentation liquor is further increased, the polysaccharide loss is caused due to the difficulty in separation caused by the excessive viscosity, and finally the polysaccharide content in the extracting solution is not further increased.
Example 6 skin-improving action of Tremella spore fermentation extract
1. Whitening effect
Taking tremella spore fermentation extract S2, S6, S7, S8, S9 and S10 as test samples, taking tremella polysaccharide S11 (total sugar content 85.2%, uronic acid content 19.3%) to prepare 0.5% solution as positive control, and determining the inhibition of different samples on cell melanin generation, wherein the method comprises the following steps:
cell culture solution: 1640 culture medium containing 10% FBS; naOH lysate: preparing lysis solution with the concentration of 0.1 mol/L by using 10% DMSO solution; mao Housu solution: preparing 20 mu M Mao Housu solution by using the cell culture solution; sample solution: preparing the fermentation extract into 4% mother liquor by using Mao Housu solution, and diluting to the target concentration before use; MTT: and (3) preparing an MTT (methyl thiazolyl tetrazolium) acting solution by using PBS (phosphate buffer solution), filtering and sterilizing by using a filter membrane of 0.22 mu m, and storing for later use in a refrigerator at 4 ℃.
B16 cells were plated at 1X 10 5 The cells were inoculated in 96-well plates at a density of 24/mL and cultured at 37 ℃ under 5% CO2 conditions at h. Adding a sample to react for 72 h, cracking cells by using NaOH lysate, heating at 80 ℃ for 30min, and detecting absorbance by using an enzyme-labeling instrument. Meanwhile, cells with the same volume density are inoculated in a 96-well plate to serve as proliferation control, and the influence of the sample on the melanin level in a unit cell is examined. The effect of the tremella polysaccharide fermentation extract on the amount of melanin produced by the cells is shown in table 4 (the melanin inhibition rate of the negative control is 0%, and the tremella polysaccharide S11 solution of the positive control is 0.5%).
TABLE 4 influence of Tremella spore fermentation extract on melanin level per cell
The result shows that when the concentration of the tremella polysaccharide and the tremella spore fermentation extract is 1% -5%, both the tremella polysaccharide and the tremella spore fermentation extract have a certain inhibition effect on melanin, the effect of the fermentation extract is particularly remarkable, and the inhibition rate of the fermentation extract with the concentration of 5% on the melanin level of a unit cell is 5.40% -5.92% and is higher than 4.33% of the tremella polysaccharide. However, the effect of the extract prepared by the fermentation liquor with the excessively high or excessively low viscosity on inhibiting melanin is not as good as that of the extract prepared by the fermentation liquor with the viscosity of 6000-12000mPa & s, and the content of the tremella polysaccharide and the total content of amino acid in the extract show the same trend.
2. Repair effect on combined irradiation damage of UVA and UVB
Taking tremella spore fermentation extract S2, S6, S7, S8, S9 and S10 as test samples, taking tremella polysaccharide S11 (total sugar content 85.2%, uronic acid content 19.3%) to prepare 0.5% solution as positive control, and determining the repair effect of different samples on HaCaT cells damaged by ultraviolet irradiation, wherein the method comprises the following steps:
cell culture solution: DMEM medium containing 10% FBS; sample solution: preparing a sample into a mother solution by using a cell culture solution, and diluting the mother solution to a target concentration before use; MTT: and (3) preparing an MTT (methanol to transfer) acting solution with the concentration of 5mg/mL by using PBS (phosphate buffer solution), filtering and sterilizing by using a filter membrane of 0.22 mu m, and storing for later use in a refrigerator at 4 ℃.
Cell damage and sample application treatment: haCaT cells were cultured at 1X 10 5 Inoculating to 96-well plate at 37 deg.C and 5% CO 2 Culturing 24 h under the condition. Cells in a 96-well plate were divided into a negative control group, a model group, and a sample addition group. The protection experiment comprises adding sample solution into the sample adding group, adding equal amount of culture solution into the negative control group and the model group, culturing for 24 h, and adding 7.2J/cm into the model group and the sample adding group 2 UVA plus 126 mJ/cm 2 And carrying out combined irradiation on UVB, adding MTT detection solution, and measuring absorbance by using an enzyme-labeling instrument. The damage repairing experiment includes irradiating the model group and the sample adding group, adding sample solution into the sample adding group, adding equal amount of culture solution into the negative control group and the model group, culturing for 24 h, adding MTT detection solution, and measuring absorbance with enzyme labeling instrument.
TABLE 5 Tremella polysaccharide repairing effect on HaCaT cell ultraviolet injury
The results of the repair effect of tremella polysaccharide on ultraviolet injury are shown in table 5 (the proliferation rate of the negative control group is 100%, the positive control is 0.5% tremella polysaccharide S11 solution), after UVA/UVB combined irradiation injury, the cell proliferation rate of the model group is reduced to 52.4%, the tremella polysaccharide has a certain repair effect and is increased along with the increase of the concentration, and the cell proliferation rate in the sample group with the concentration of 2% can be recovered to the level of 73.22% to the maximum. The cell proliferation rate and the content of tremella polysaccharide and the total content of amino acid in the extracting solution show the same trend.
3. Scratch damage repair experiment
The repair of damaged cells by different samples is determined by taking tremella spore fermentation extract S2 and tremella polysaccharide S11 (total sugar content 85.2%, uronic acid content 19.3%) 0.5% aqueous solution as test samples, and the method comprises the following steps:
cell culture solution: DMEM medium containing 10% FBS; sample solution: the samples were made into stock solutions using cell culture medium containing 2.5% serum.
Cell damage and sample application treatment: haCaT cells were cultured at 5X 10 4 Inoculating to 24-well plate at 37 deg.C and 5% CO 2 Culturing 24 h under the condition. On the near-confluent monolayer cells, a 200 μ L gun head was used to vertically scribe lines in each well of the 24-well plate. The wells were discarded, the sample solution (final serum content 2.5%) was added, and the cells were incubated for 48 h and visualized by photography.
The result is shown in fig. 2, compared with the control, the tremella polysaccharide fermented extract S2 and the 0.5% tremella polysaccharide S11 solution both have the effect of promoting the migration of cells to the wound, and it can be seen from the figure that the cell migration capability after the S2 treatment is better than that of the tremella polysaccharide S11 solution, which indicates that the tremella polysaccharide fermented extract has better capability of repairing damaged cells.
Claims (8)
1. A preparation method of a tremella spore fermentation extract is characterized by comprising the following steps:
(1) And (3) removing thalli: adjusting the pH value of the fermentation liquid, and centrifuging to obtain a supernatant;
(2) Decoloring and removing impurities: adjusting the pH value of the supernatant, adding activated carbon for adsorption, and filtering to obtain a filtrate I;
(3) Protein removal: adjusting the pH value of the filtrate I, heating, cooling to room temperature, and filtering to obtain a filtrate II;
(4) Adsorbing and removing impurities: adding activated carbon into the filtrate II for adsorption, and filtering to obtain a tremella spore fermentation extract;
the viscosity of the fermentation liquor is 6000 to 12000mPa & s;
in the step (1), the pH is adjusted to 7.0-11.0;
in the step (2), the pH value is 5.5-7.0; in the step (3), the pH value is 8.5-9.5;
in the step (3), the heating temperature is 80-90 ℃, and the heating time is 0.5-1h.
2. The method according to claim 1, wherein the viscosity of the fermentation broth is 8000 to 10000 mPa-s.
3. The method according to claim 1, wherein in the step (1), the pH is adjusted to 9.0 to 11.0.
4. The method according to claim 1, wherein in the step (1), the centrifugation speed is 10000 to 15000rpm, and the centrifugation time is 15 to 30min.
5. The preparation method according to claim 1, wherein in the step (2) and the step (4), the addition amount of the activated carbon is 0.2-1%w/v, the adsorption temperature is 60-70 ℃, and the adsorption time is 0.5-1h.
6. The production method according to claim 1, wherein in the step (2), the filtration pore size is 1.2 μm; in the step (3), the filtering pore diameter is 0.65 μm; in the step (4), the filtration pore size is 0.45 μm.
7. The tremella spore fermentation extract obtained by the preparation method according to any one of claims 1 to 6, wherein the tremella polysaccharide content is 3-5g/L, and the total amino acid content is 70-90mg/L.
8. A cosmetic comprising the tremella spore fermentation extract of claim 7.
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