CN110801413A - Chlorella fermented extract and preparation method thereof - Google Patents
Chlorella fermented extract and preparation method thereof Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9706—Algae
- A61K8/9722—Chlorophycota or Chlorophyta [green algae], e.g. Chlorella
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- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
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- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
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Abstract
The invention discloses a chlorella fermented extract and a preparation method thereof. The invention utilizes the culture solution of strains such as lactobacillus plantarum and the like to carry out liquid fermentation culture on chlorella powder to obtain a fermentation product, and the fermentation product is centrifuged, filtered and sterilized to obtain the chlorella powder fermentation extract. The method has the advantages of mild reaction conditions, simple preparation process and no addition of any chemical component. Experiments prove that the chlorella powder fermentation extract prepared by the method is rich in functional components such as protein, crude polysaccharide, total amino acid, lutein and the like, has good effects of resisting free radicals, improving skin irritation, resisting aging and the like, has high biological safety, and can be widely applied to various cosmetics.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a chlorella fermented extract and a preparation method thereof.
Background
Chlorella (Chlorella vulgaris) Chlorella, Chlorophyceae, Chlorella family. Unicellular algae, which are often single-grown, also have multicellular aggregates. One of the earliest lives on the earth is a high-efficiency photosynthetic plant which grows and breeds by photoautotrophy and has extremely wide distribution. The chlorella has high application value, and forms a microalgae biotechnology together with development and utilization of microalgae such as spirulina, scenedesmus, dunaliella and the like, so the chlorella has wide development prospect.
The chlorella contains abundant proteins with the content of 50-60%, can be used as an important source of single-cell proteins, and also contains various amino acids, dietary fibers, vitamins and other nutritional ingredients necessary for human and animals. The chlorella also has the advantages of low fat, low sugar, low calorie and the like, is a high-quality green nutrient source food, and has great economic value in the industries of food, health-care food and the like. The chlorella pyrenoidosa is a natural resource with great economic value and is determined as a green nutritional source health food in twenty-first century by the Food and Agriculture Organization (FAO) of the United nations. Chlorella contains various bioactive substances such as Chlorella growth factor COF, polysaccharide and glycoprotein, and has significant effects in resisting tumor, enhancing immunity, resisting virus infection, etc. Chlorella contains many pigments such as chlorophyll, lutein, and astaxanthin, and it is found that the Chlorella can accumulate astaxanthin, and there is a possibility that it becomes a high-quality natural astaxanthin alga source following Haematococcus pluvialis.
At present, research on the application of chlorella mainly focuses on the fields of food, medicine, energy, aquaculture and the like, and few researches and reports on the application of chlorella in the field of cosmetics are available.
Disclosure of Invention
In order to solve the problems, the invention provides a chlorella fermented extract and a preparation method thereof. The invention utilizes the culture solution of strains such as lactobacillus plantarum and the like to carry out liquid fermentation culture on chlorella powder to obtain a fermentation product, and the fermentation product is centrifuged, filtered and sterilized to obtain the chlorella powder fermentation extract. The chlorella powder fermentation extract prepared by the method is rich in functional components such as protein, crude polysaccharide, total amino acid and lutein, has good effects of resisting free radicals, improving skin irritation and resisting aging, and develops a new application of chlorella in the field of skin care.
In order to achieve the purpose, the invention adopts the following technical scheme: a preparation method of a chlorella fermented extract is characterized by comprising the following steps:
(1) preparing seeds: inoculating the preserved strains into a seed culture medium, and performing expanded culture to obtain a strain suspension; the strain is at least one of lactobacillus plantarum, lactobacillus casei, lactobacillus paracasei and lactococcus lactis, and lactobacillus plantarum is preferred;
(2) liquid fermentation culture: inoculating the bacterial suspension cultured in the step (1) into a fermentation medium containing chlorella powder and glucose, and performing static culture at 28-32 ℃ for 24-48 h (until the content of glucose is 0) to obtain a fermentation liquid;
(3) adjusting the pH value: adjusting the pH value of the fermentation liquor to 5.5-7.5;
(4) centrifuging, filtering and sterilizing: and (4) centrifuging the fermentation liquor obtained in the step (3) to remove residues, collecting supernatant, and then filtering and sterilizing to obtain the chlorella fermentation extract.
Further, the seed culture medium in the step (1) comprises the following components in percentage by weight: sterilizing an M17 culture medium and 5-20 g/l of glucose for 30min at 115 ℃; standing and culturing at the temperature of 28-32 ℃ for 12-36 h to obtain the bacterial suspension.
Further, the bacterial suspension inoculation amount (volume ratio) in the step (2) is as follows: 10 to 20 percent.
Further, the fermentation medium in the step (2) comprises the following components in percentage by weight: 5-15 g/l of chlorella powder, 10-20 g/l of glucose and the balance of distilled water, and sterilizing at 115 ℃ for 30 min.
Further, in the step (3), the pH value of the fermentation liquor is adjusted to 5.5-7.5 by using 1mol/L NaOH solution.
Further, in the step (4), the centrifugal power is 4000-8000 r/min, and the centrifugation is 10-30 min.
Further, the filtration sterilization of the step (4) is as follows: filtering and sterilizing by a 1.2 mu m fine filtering paperboard and a 0.22 mu m polyethersulfone filter element.
The chlorella fermented extract prepared by the method comprises the following main effective components and contents (the dosage of the chlorella powder is about 10g/1.1L of fermentation liquor): the protein is more than or equal to 500mg/100 ml; the crude polysaccharide is more than or equal to 440mg/100 ml; the total amino acid is more than or equal to 190mg/100 ml; the lutein content is more than or equal to 240 mu g/100 ml.
The chlorella fermented extract prepared by the method has good effects of resisting free radicals, improving skin irritation and resisting aging, and can be used as effective component for preparing cosmetics.
The invention has the beneficial effects that:
1. the chlorella powder is used as a raw material, active ingredients of the chlorella powder are fully released by utilizing a microbial fermentation technology, and the prepared chlorella fermentation extract is rich in protein, crude polysaccharide, total amino acids and lutein. The active components act together to ensure that the chlorella fermented extract has good effects of resisting free radicals, improving skin irritation, resisting aging and the like, and develops new application of the chlorella in the field of skin care.
2. The invention is proved by tests that:
1) the chlorella powder fermentation extract can improve the clearance rate of ABTS free radicals under the concentration of 4.5-40.5% (V/V), is in a dose-dependent model, and has statistical difference compared with a negative control group when the concentration of a sample is more than or equal to 4.5% (V/V);
2) the chlorella fermentation product of the invention acts for 48 hours at the concentration of 0.16-10.00% (V/V), can improve the survival rate of NHDFs cells, is in a dose-dependent model, and has statistical difference compared with a negative control group when the concentration of a sample is more than or equal to 0.63% (V/V);
3) the chlorella fermentation product of the invention acts for 48 hours under the concentration of 10% (V/V), can improve the type I collagen synthesis capacity of NHDFs cells, is higher than a positive control (50 mug/ml ascorbic acid), and has statistical difference compared with the negative control. The product has good effects of resisting free radicals, improving skin irritation, resisting aging and the like.
3. The method has the advantages of mild reaction conditions, simple and efficient preparation process, no addition of any chemical components and capability of realizing industrial large-scale production.
Drawings
FIG. 1 shows the scavenging effect of chlorella fermented extract on ABTS free radicals; wherein: p value < 0.01;
FIG. 2 is a graph showing the effect of chlorella fermented extract on the relative viability of NHDFs cells; wherein: p value <0.05, x: p value < 0.01;
FIG. 3 is a graph showing the effect of chlorella fermentation extract on the relative amount of type I collagen; wherein: p value < 0.01.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to illustrate the present invention and are not intended to limit the scope thereof. The experimental methods used in the following examples are not specifically described, and are all conventional methods. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The strains used in the following examples were purchased from the China center for Industrial culture Collection of microorganisms (CICC).
Example 1: preparation of chlorella fermented extract
The chlorella fermented extract is prepared by the following method:
(1) preparing seeds: inoculating the preserved lactobacillus plantarum into a seed culture medium, and performing static culture at 30 ℃ for 24 hours to obtain a bacterial suspension;
the seed culture medium comprises the following components in percentage by weight: m17 medium, glucose 20g/l, 115 ℃ sterilization for 30 min.
(2) Liquid fermentation culture: inoculating the bacterial suspension cultured in the step (1) into a fermentation medium according to the inoculation amount of 10% of the volume ratio, standing and culturing for 36h at 30 ℃ with the glucose content of 0, and ending fermentation to obtain fermentation liquor;
the fermentation medium comprises the following components in percentage by weight: 10g/l of chlorella powder, 15g/l of glucose and the balance of distilled water, and sterilizing at 115 ℃ for 30 min.
(3) Adjusting the pH value: the pH of the fermentation liquor is adjusted to about 6.0 by using 1mol/LNaOH solution.
(4) Centrifuging, filtering and sterilizing: firstly, centrifuging the fermentation liquor obtained in the step (3) at 8000r/min for 10min, removing residues and collecting supernatant; then filtering and sterilizing by a fine filtering paper board with the diameter of 1.2 mu m and a polyether sulfone filter element with the diameter of 0.22 mu m to obtain the chlorella fermented extract.
Detecting main functional components and contents in the chlorella fermented extract. The results are as follows:
TABLE 1 main effective components and contents
Example 2: preparation of chlorella fermented extract
The chlorella fermented extract is prepared by the following method:
(1) preparing seeds: inoculating the preserved lactobacillus plantarum into a seed culture medium, and performing static culture at 30 ℃ for 28 hours to obtain a bacterial suspension;
the seed culture medium comprises the following components in percentage by weight: m17 medium, glucose 15g/l, 115 ℃ sterilization for 30 min.
(2) Liquid fermentation culture: inoculating the bacterial suspension cultured in the step (1) into a fermentation medium according to the inoculum size of 15% by volume, standing and culturing for 30h at 30 ℃ with the glucose content of 0, and finishing fermentation to obtain fermentation liquor;
the fermentation medium comprises the following components in percentage by weight: 10g/l of chlorella powder, 12g/l of glucose and the balance of distilled water, and sterilizing at 115 ℃ for 30 min.
(3) Adjusting the pH value: the pH of the fermentation liquor is adjusted to about 6.0 by using 1mol/LNaOH solution.
(4) Centrifuging, filtering and sterilizing: firstly, centrifuging the fermentation liquor obtained in the step (3) at 6000r/min for 15min, removing residues and collecting supernatant; then filtering and sterilizing by a fine filtering paper board with the diameter of 1.2 mu m and a polyether sulfone filter element with the diameter of 0.22 mu m to obtain the chlorella fermented extract.
Example 3: preparation of chlorella fermented extract
The chlorella fermented extract is prepared by the following method:
(1) preparing seeds: inoculating the preserved lactobacillus plantarum into a seed culture medium, and performing static culture at 30 ℃ for 20 hours to obtain a bacterial suspension;
the seed culture medium comprises the following components in percentage by weight: m17 medium, glucose 10g/l, 115 ℃ sterilization for 30 min.
(2) Liquid fermentation culture: inoculating the bacterial suspension cultured in the step (1) into a fermentation medium according to the inoculation amount of 20% of the volume ratio, standing and culturing for 38h at 30 ℃ with the glucose content of 0, and finishing fermentation to obtain fermentation liquor;
the fermentation medium comprises the following components in percentage by weight: 10g/l of chlorella powder, 20g/l of glucose and the balance of distilled water, and sterilizing at 115 ℃ for 30 min.
(3) Adjusting the pH value: the pH of the fermentation liquor is adjusted to about 6.0 by using 1mol/LNaOH solution.
(4) Centrifuging, filtering and sterilizing: firstly, centrifuging the fermentation liquor obtained in the step (3) for 20min at 5000r/min, removing residues and collecting supernatant; then filtering and sterilizing by a fine filtering paper board with the diameter of 1.2 mu m and a polyether sulfone filter element with the diameter of 0.22 mu m to obtain the chlorella fermented extract.
Test example 1: detection of ABTS free radical scavenging capability of chlorella fermentation extract
This example is intended to evaluate the anti-radical efficacy of the chlorella fermented extract prepared in example 1
The samples were prepared into solutions to be tested with corresponding concentrations (0.5, 1.5, 4.5, 13.5, 40.5% (v/v)) with distilled water, reaction systems were prepared in 96-well plates according to the amounts of reagents added in table 2, and mixed well, with 3 multiple wells per concentration and 1 background control well.
TABLE 2ABTS radical scavenging test reaction System
After incubation for 2-6min at room temperature, reading the absorbance OD value at 734nm, calculating the clearance rate of the sample to ABTS free radicals, and obtaining the SD value according to the standard deviation calculation formula.
The clearance rate calculation method comprises the following steps:
in the formula: c1-blank ABTS system absorbance value;
c2-blank absorbance value without ABTS system;
t1-sample set has ABTS system absorbance value;
t2-sample set Absorbance value without ABTS system.
As shown in Table 3, the sample (Chlorella fermented extract) was able to increase the scavenging rate of ABTS free radicals at a concentration of 4.5-40.5% (V/V), and was in a dose-dependent model, and when the sample concentration was 4.5% (V/V) or more, it was statistically different from the negative control group (see FIG. 1).
TABLE 3ABTS free radical scavenging test results analysis (Mean + -SD)
Test example 2: evaluation of skin irritation improving and anti-aging effects of chlorella extract
This example is intended to evaluate the anti-aging efficacy of the chlorella extract prepared in example 1.
1. In vitro human fibroblast proliferation assay
A sample (chlorella fermentation product) is prepared into 10% (V/V) solution by using a DMEM medium, and after the solution is filtered by a 0.22 mu m filter membrane, the sample solution is continuously diluted to 0.08% (V/V) in pairs for 8 gradient concentrations.
Table 4 design table of concentration of each test group
Cells in logarithmic growth phase were collected at a cell density of 8X 103One well was inoculated to a 96-well plate containing 100. mu.l of culture medium per well. In an incubator (37 ℃, 5% CO)2) After 24h incubation, the medium was aspirated, the cells were washed twice with PBS buffer, then the drug was added according to table 4, 100 μ l per well, with untreated cells as negative control, 3 replicate wells per group.
Adding the drug into an incubator (37 ℃, 5% CO)2) Then, the culture is continued for 48h, and then a CCK-8 cytotoxicity test is carried out, and the OD value of absorbance is read at 450 nm. The cell viability was calculated according to the following formula, the SD value was calculated according to the standard deviation, and only the cell culture medium was added to the blank wells.
As shown in Table 5, the survival rate of NHDFs cells was improved by exposing the sample (chlorella fermentation product) to 0.16-10.00% (V/V) for 48h, and the sample was statistically different from the negative control group when the sample concentration was 0.63% (V/V) or higher in a dose-dependent manner (see FIG. 2).
TABLE 5 analysis of cell viability assay results (action 48h)
2. In vitro firming anti-wrinkle efficacy assessment (collagen I content)
And detecting the content of the type I collagen by an ELISA method.
Human skin fibroblasts (NHDFs) in logarithmic growth phase were collected at a cell density of 6X 104One/well was inoculated into 12-well plates. In an incubator (37 ℃, 5% CO)2) After 24h of medium culture, according to the cytotoxicity result, a sample (chlorella fermentation product) is added according to the concentration of 10% (v/v), 50 mu g/ml ascorbic acid (VC) is used as a positive control, untreated cells are used as a negative control, a reaction system reagent and a solvent are used as a solvent control, and 4 repeat wells are arranged in each group.
Adding the drug into an incubator (37 ℃, 5% CO)2) Culturing for 48h, and collecting the culture solution. And (3) detecting the content of the type I collagen in the culture solution by adopting a Col I ELISA kit, calculating the relative content of the type I collagen relative to a solvent control, and obtaining an SD value according to a standard deviation calculation formula.
As shown in Table 6, the sample (chlorella fermentation product) acted at 10% (V/V) concentration for 48h can increase the type I collagen synthesis capacity of NHDFs cells to 4.43 + -0.34 times of that of the negative control, which is higher than that of the positive control, and has statistical difference compared with the negative control. It is shown that the sample has certain firming and anti-wrinkle effects at this concentration (see figure 3).
TABLE 6 analysis of collagen type I relative content results
| Concentration (%, V/V) | Relative content of type I collagen | SD |
| Negative control | 1.00 | 0.19 |
| Positive control | 2.90 | 0.05 |
| 10 | 4.43 | 0.34 |
Claims (10)
1. A preparation method of a chlorella fermented extract is characterized by comprising the following steps:
(1) preparing seeds: inoculating the preserved strains into a seed culture medium, and performing expanded culture to obtain a strain suspension; the strain is at least one of lactobacillus plantarum, lactobacillus casei, lactobacillus paracasei and lactococcus lactis;
(2) liquid fermentation culture: inoculating the bacterial suspension cultured in the step (1) into a fermentation medium containing chlorella powder and glucose, and performing static culture at 28-32 ℃ for 24-48 h to obtain a fermentation liquid;
(3) adjusting the pH value: adjusting the pH value of the fermentation liquor to 5.5-7.5;
(4) centrifuging, filtering and sterilizing: and (4) centrifuging the fermentation liquor obtained in the step (3) to remove residues, collecting supernatant, and then filtering and sterilizing to obtain the chlorella fermentation extract.
2. The method for preparing fermented extract of chlorella according to claim 1, wherein the fermentation medium in the step (2) comprises the following components by weight: 5-15 g/l of chlorella powder, 10-20 g/l of glucose and the balance of distilled water.
3. The method for preparing fermented extract of chlorella according to claim 1, wherein the seed culture medium in step (1) comprises the following components in percentage by weight: m17 culture medium and 5-20 g/l glucose.
4. The method for preparing a fermented chlorella extract according to claim 1, wherein the step (1) comprises performing static culture at 28-32 ℃ for 12-36 hours to obtain a bacterial suspension.
5. The method for preparing fermented extract of chlorella according to claim 1, wherein the inoculation amount of the bacterial suspension in the step (2) is as follows according to the volume ratio: 10 to 20 percent.
6. The method for preparing fermented extract of chlorella according to claim 1, wherein in the step (3), the pH of the fermentation broth is adjusted to 5.5 to 7.5 with 1mol/L NaOH solution.
7. The method for preparing fermented extract of chlorella according to claim 1, wherein in the step (4), the centrifugal power is 4000 to 8000r/min and the centrifugation is 10 to 30 min.
8. The method for preparing fermented extract of chlorella according to claim 1, wherein the filtration and sterilization in step (4) are as follows: filtering and sterilizing by a 1.2 mu m fine filtering paperboard and a 0.22 mu m polyethersulfone filter element.
9. A fermented extract of chlorella produced by the method of any one of claims 1 to 8.
10. Use of the fermented extract of chlorella as claimed in claim 9 for preparing cosmetics for scavenging free radicals, improving skin irritation and resisting aging.
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