CN110840781A

CN110840781A - Preparation method of tremella spore fermentation extract and product thereof

Info

Publication number: CN110840781A
Application number: CN201911116240.7A
Authority: CN
Inventors: 孙劭靖; 郭文逸; 陆震; 毛华; 张天萌; 马良; 石艳丽; 郭学平
Original assignee: Huaxi Biotechnology Co Ltd; Shandong Bloomage Hyinc Biopharm Co Ltd
Current assignee: Huaxi Biotechnology Tianjin Co ltd; Bloomage Biotech Co Ltd
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2020-02-28
Anticipated expiration: 2039-11-15
Also published as: CN110840781B

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 extract 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

Preparation method of tremella spore fermentation extract and product thereof

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 tremella polysaccharide has excellent lubricating property and film forming property, and may be used in cosmetics to form homogeneous film on the surface of skin or hair to moisten, lubricate and eliminate static electricity.

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, CN 105002254 a patent discloses a preparation method and application of tremella fermented extract, in which saccharomyces cerevisiae, streptococcus thermophilus and other bacteria liquid are inoculated into a solution prepared from tremella powder for fermentation, and the tremella extract prepared by the 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 the 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 then filtering to obtain a tremella spore fermentation extract.

The fermentation liquid is obtained by inoculating tremella spores to a fermentation medium for fermentation, and the viscosity of the fermentation liquid is 6000-12000 mpa-s, and is preferably 8000-10000 mpa-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_B110-20 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-.

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 0.5-1 h.

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 0.5-1 h.

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 0.5-1 h.

In the step (4), the filtration pore size is 0.45 μm.

A Tremella spore fermentation extractive solution obtained by the above preparation method is provided. The content of tremella polysaccharide in the tremella spore fermentation extracting solution is 3-5 g/L; the total amino acid content is 70-90 mg/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)_B115 mg/L; pH value of 6.0), shaking the flask at 30 ℃ and 200 r/min for 3 days to obtain seed liquid;

2. three-stage aeration fermentation

(1) Inoculating the seed solution into a 10L fermentation tank at an inoculation amount of 3%, wherein the formula of the culture medium is as in 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: beginning at 10L/min, 15L/min after 24 h, and 20L/min after 48 h till 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 fed-batch manner, and ending the fermentation when the viscosity is 10000mPa & s, so as to obtain a fermentation liquid F1, wherein the content of the tremella polysaccharides is 10.2 g/L;

(2) respectively fermenting according to the fermentation conditions in the step (1) to obtain fermentation liquor F2-F6, wherein the viscosity and the tremella polysaccharide content are shown in 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 value of the supernatant to 6.5, adding 0.5% w/v active carbon (200 meshes) for adsorption at 65 ℃ for 1h, and then filtering to obtain filtrate I;

(3) protein removal: adjusting the pH value of the filtrate I to 9.5, heating at 90 ℃ for 0.75 h, cooling to room temperature, and filtering to obtain a 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 extract 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

And (3) preparing a tremella spore fermentation extract from the fermentation liquid F1 by the method in the reference example 2, and respectively adjusting the pH value to 13.0 or 5.0 in the step (1) to obtain tremella spore fermentation extract S4 and S5.

Example 3 preparation of Tremella spore fermentation extract

Referring to the method of steps (1) to (4) in example 2, fermentation liquids F3, F4 and F5 are respectively prepared into tremella spore fermentation extract liquids, and tremella spore fermentation extract liquids 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 liquors F2 and F6 are respectively prepared into tremella spore fermentation extract liquor, and tremella spore fermentation extract liquor 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 an application method 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 broth with the same viscosity as a raw material, wherein the content of amino acid is increased and then decreased along with the increase of pH (S1-S5) in the step (1), and is stabilized to be more than 75mg/L when the pH range is 7.0-13.0, and is more than 85mg/L when the pH range is 9.0-11.0; 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 100 mg of glucose, adding 20 mL of distilled water for dissolving, transferring to a 250 mL volumetric flask, adding water to the scale, diluting by 10 times before use, respectively sucking 0.4, 0.8, 1.2, 1.6 and 2.0 mL of glucose in a test tube with a plug, and supplementing each volume of glucose to 2.0 mL by using distilled water.

Sample preparation: weighing 250 mg of sample, adding 20 mL of distilled water for dissolving, transferring to a 50 mL volumetric flask, adding water to a constant volume to obtain a scale as a stock solution, weighing 4 mL of the stock solution before use, placing in the 50 mL volumetric flask, adding water to the scale, diluting by 10 times before measurement, and taking 2 mL of the stock solution in a test tube with a plug.

And (3) determination: adding 1.0 mL of 6% redistilled phenol and concentrated H into the standard solution and the sample diluent₂SO₄5.0 mL, shake well 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 broth, as the pH value increases in the cell separation process in step (1) (S1-S5), the tremella polysaccharide in the extract tends to increase first and then decrease. The tremella polysaccharide content in the extract after bacteria removal is high by adopting pH7.0-11.0, 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 fermentation liquors with different viscosities under the same condition, along with the increase of the viscosity, the concentration of tremella polysaccharide tends to increase firstly and then decrease, the viscosity is lower than 6000mPa & S (S6, viscosity =4000mPa & S), the accumulation of 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 percent and uronic acid content 19.3 percent) to prepare 0.5 percent solution as a positive control, and determining the inhibition of different samples on the generation of cell melanin, 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 0.1mol/L by using 10% DMSO solution; monkey head extract solution: preparing 20 mu M of a mao monkey hormone solution by using a cell culture solution; sample solution: preparing the fermented extract into 4% mother liquor with a mao monkey extract solution, and diluting to a 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 cultured at 1X 10⁵The cells were inoculated in 96-well plates at a density of one/mL and cultured at 37 ℃ under 5% CO2 for 24 hours. Adding the sample, reacting for 72 h, cracking the cells with NaOH lysate, heating at 80 ℃ for 30min, and detecting the absorbance by an enzyme-labeling instrument. While inscribing equal bulk densities in 96-well platesThe cells of (3) were used as proliferation controls and the effect of the samples on the level of melanin per cell was examined. The effect of the fermented tremella polysaccharide 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 broth with too high or too low viscosity on melanin is inferior to that of the extract prepared by the fermentation broth with 6000-12000 mPas, and the content of the tremella polysaccharide and the total content of the 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 percent and uronic acid content 19.3 percent) to prepare 0.5 percent solution as a 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 10⁵Inoculating to 96-well plate at 37 deg.C and 5% CO₂Culturing for 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 is carried out by adding sample solution into sample-adding group, adding negative control group andadding equal amount of culture solution into the model group, culturing for 24 h, and collecting the model group and the sample adding group at 7.2J/cm²UVA plus 126 mJ/cm²And 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 hr, adding MTT detection solution, and measuring absorbance with enzyme labeling instrument.

TABLE 5 Tremella polysaccharides 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%, tremella polysaccharide has a certain repair effect and increases with the increase of concentration, and the cell proliferation rate in the sample group at the concentration of 2% can be recovered to the level of 73.22% at the highest. 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 10⁴Inoculating to 24-well plate at 37 deg.C and 5% CO₂Culturing for 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 incubation continued for 48 h before observation by photography.

The result is shown in fig. 2, compared with the control, the tremella polysaccharide fermentation 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 capacity after the S2 treatment is better than that of the tremella polysaccharide S11 solution, which indicates that the tremella polysaccharide fermentation extract has better repair capacity to the damaged cells.

Claims

1. A preparation method of a tremella spore fermentation extract is characterized by comprising the following steps:

2. The method according to claim 1, wherein the viscosity of the fermentation broth is 6000-.

3. The method according to claim 1, wherein in the step (1), the pH is adjusted to 7.0 to 11.0; preferably, the pH is adjusted to 9.0-11.0.

4. The method according to claim 1, wherein in the step (1), the centrifugation speed is 10000-.

5. The method according to claim 1, wherein in the step (2), the pH is 5.5 to 7.0; in the step (3), the pH is 8.5-9.5.

6. 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 0.5-1 h;

7. 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.

8. A Tremella spore fermentation extract obtained by the method of any one of claims 1-7.

9. The tremella spore fermentation extract as claimed in claim 8, wherein the tremella polysaccharide content is 3-5g/L, and the total amino acid content is 70-90 mg/L.

10. A cosmetic comprising the tremella spore fermentation extract of claim 8.