CN111808901A - Preparation method of cordyceps militaris fermentation extract, product obtained by preparation method and application of product - Google Patents
Preparation method of cordyceps militaris fermentation extract, product obtained by preparation method and application of product Download PDFInfo
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- CN111808901A CN111808901A CN202010646362.3A CN202010646362A CN111808901A CN 111808901 A CN111808901 A CN 111808901A CN 202010646362 A CN202010646362 A CN 202010646362A CN 111808901 A CN111808901 A CN 111808901A
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- cordyceps militaris
- fermentation
- fermentation extract
- extract
- cordyceps
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
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Abstract
The invention discloses a preparation method of cordyceps militaris fermentation extract, an obtained product and application. The invention makes the microorganism utilize and decompose the cordyceps militaris powder to the maximum extent to release the effective components by selecting proper strains and culture media; the cordycepin content after microbial decomposition is stable, and the cordycepic acid content is improved by 6-8 times compared with that of an extraction method; the extract not only contains the functional components of the cordyceps militaris, but also contains some active substances generated by lactobacillus fermentation, and the two substances are combined for synergistic interaction, so that the nutrient substances, the nutrient density and the physiological activity of the cordyceps militaris fermentation extract are greatly improved, the value of the cordyceps militaris fermentation extract is far higher than that of the cordyceps militaris or lactobacillus fermentation extract, and the cordyceps militaris fermentation extract is a natural high-quality skin care product, food or health care product raw material.
Description
Technical Field
The invention relates to a preparation method of a cordyceps militaris fermentation extract, in particular to a method for preparing a cordyceps militaris fermentation extract by fermenting lactic acid bacteria serving as fermentation strains in a fermentation medium taking cordyceps militaris as a unique nitrogen source, and also relates to the cordyceps militaris fermentation extract and application of the cordyceps militaris fermentation extract.
Background
Cordyceps militaris is a model species of Ascomycota, Hypocreales, Clavicipitaceae, Cordyceps. The scientific name is Cordyceppsmilitaris, also called cordyceps militaris and cordyceps militaris, called cordyceps militaris for short, and the cordyceps militaris cultured by living pupa is generally called cordyceps militaris. Cordyceps militaris is distributed worldwide, and the quantity of natural resources is small. The main active ingredients in Cordyceps militaris comprise cordycepin, cordycepic acid and Cordyceps polysaccharide. Cordycepin (cordycepin), also known as cordycepin, 3-deoxyadenosine, is the first nucleoside antibiotic isolated from fungi. The cordycepin contained in the cordyceps militaris can obviously inhibit various inflammatory factors due to the specific antibacterial and antiviral effects of the cordycepin. Cordycepic acid is another important quality index of cordyceps militaris, and has the effects of resisting free radicals, resisting oxidation, promoting urination and dehydration, relieving cough, eliminating phlegm and relieving asthma. Cordyceps polysaccharide has anti-inflammatory and antioxidant effects, and can improve immunity, reduce blood sugar activity, resist tumor, and resist radiation.
Cordyceps militaris has been widely added into cosmetics and health foods, and nowadays, the application method of cordyceps militaris mostly takes cordyceps militaris as a raw material to extract effective components thereof, or takes cordyceps militaris living bodies as strains to obtain cordyceps militaris extracts or single effective components thereof by a fermentation method. For example, patent CN 107602660 a discloses a cordyceps militaris polypeptide product, and a preparation method and application thereof, in the patent, cordyceps militaris raw materials are added with an extracting solution and ground to obtain homogenate, and the homogenate is centrifuged and filtered, and then hydrolyzed by an enzyme preparation to obtain the polypeptide product. The cordyceps militaris polypeptide product prepared by the method is mostly applied to health-care food or beauty and skin care products. However, this method requires a large amount of enzyme. For example, patents CN 109136112A and CN 109294927 a disclose methods for improving cordycepin and cordyceps polysaccharide in cordyceps militaris, and these patents only research methods for improving a certain component and applications thereof.
At present, in the prior art, the utilization of active ingredients in cordyceps militaris is single, the utilization rate is low, and the effect of cordyceps militaris cannot be fully exerted.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a preparation method of cordyceps militaris fermentation extract, which can also be called as a utilization method of cordyceps militaris. By the method, the whole Cordyceps militaris is fully utilized, and the fermentation extract is rich in nutrient components.
At present, no relevant report that lactic acid bacteria are used for fermentation culture in a fermentation culture medium with cordyceps militaris as a unique nitrogen source is seen. According to the invention, by selecting proper strains and culture media, the production period is shortened, the utilization rate of cordyceps militaris is improved, and the obtained fermentation extract has higher content of active substances such as cordycepin, cordycepic acid and amino acid; and the product is stable, and is beneficial to later purification.
The specific technical scheme of the invention is as follows:
a preparation method of cordyceps militaris fermentation extract comprises the following steps: carrying out fermentation culture on lactic acid bacteria in a fermentation culture medium taking cordyceps militaris as a nitrogen source to obtain cordyceps militaris fermentation extract, wherein the fermentation culture medium taking cordyceps militaris as the nitrogen source comprises the following components: 5-10 g/L of cordyceps militaris powder, 5g/L of glucose, 15-20g/L of fructose, 3-8 g/L of sodium acetate, 1-3g/L of magnesium sulfate and the balance of water.
Furthermore, in the fermentation medium, the cordyceps militaris powder is 8-10 g/L and the fructose is 18g/L, so that the effect is best. When the content of sodium acetate is 3 to 8g/L, the content of the effective component in the extract tends to increase with the increase of the content of sodium acetate, but the effect is equivalent when the content of sodium acetate is 5 to 8g/L, and therefore, 5g/L of sodium acetate is preferable. When magnesium sulfate is 1 to 3g/L, the content of the effective component in the extract tends to increase with the increase of the content of magnesium sulfate, but the effect is equivalent when the content of magnesium sulfate is 2 to 3g/L, and therefore, sodium acetate is preferably 2 g/L.
Preferably, when the following contents are selected from the fermentation medium taking cordyceps militaris as a nitrogen source, the effect is better: 8-10 g/L of cordyceps militaris powder, 5g/L of glucose, 15-20g/L of fructose, 5-8g/L of sodium acetate, 2-3g/L of magnesium sulfate and the balance of water.
More preferably, when the following contents are selected from the fermentation medium taking cordyceps militaris as a nitrogen source, the effect is best: 8g/L of cordyceps militaris powder, 5g/L of glucose, 18g/L of fructose, 5g/L of sodium acetate, 2g/L of magnesium sulfate and the balance of water.
The preparation method specifically comprises the following steps:
(1) carrying out seed culture on lactobacillus to obtain a seed solution;
(2) inoculating the seed solution into a fermentation medium using cordyceps militaris as a nitrogen source, ventilating, stirring and culturing, and ending fermentation until no residual sugar exists to obtain a fermentation liquid;
(3) and treating the fermentation liquor to obtain cordyceps militaris fermentation extract.
Further, the cordyceps militaris powder is powder formed by drying and crushing natural cordyceps militaris sporocarp, and the particle size of the powder has no special requirement and is generally 40 meshes or below. The Cordyceps militaris raw material contains various active ingredients such as cordycepin, cordycepic acid, Cordyceps polysaccharide and the like.
Further, various lactic acid bacteria species disclosed in the prior art of lactic acid bacteria, such as lactobacillus, leuconostoc, bifidobacterium, and the like, preferably lactobacillus. Each of the lactic acid bacteria can be purchased from the market.
Further, in step (1), the lactic acid bacteria can select a suitable seed culture medium and seed culture conditions from the disclosed prior art to obtain a seed solution required for fermentation culture. The conditions for seed culture, for example, temperature, pH, etc., may be selected to be optimum for each species.
Further, in the step (2), inoculating the seed solution into a fermentation culture medium taking cordyceps militaris as a nitrogen source according to the inoculation amount of 5-10%, performing fermentation culture, and stopping fermentation when residual sugar is exhausted without controlling the pH value in the fermentation process. The fermentation temperature and pH are optimum for each strain, for example, the fermentation temperature of lactobacillus is maintained at about 37 deg.C, pH is maintained at about 6.2-6.6, and aeration stirring is carried out during the culture process, with aeration amount of 0.3-0.6 vvm.
Further, in the step (3), after the fermentation is finished, the fermentation liquid is sequentially subjected to operations of heating sterilization, pH adjustment and filtration to obtain the cordyceps militaris fermentation extract. The sterilization is carried out at 60-70 deg.C and the heating time is about 1-2 h. Adjusting pH to 5.0-6.0. Filtration is generally carried out using a filter membrane having a pore size of 0.45. mu.m.
The invention also protects the cordyceps militaris fermentation extract obtained by the method. The Cordyceps militaris fermentation extract is rich in nutrition, high in activity, and rich in cordycepin and cordycepic acid, and is a good raw material for food, health product and cosmetic.
According to the invention, microbial lactic acid bacteria are adopted as strains, cordyceps militaris powder is utilized for fermentation to prepare cordyceps militaris fermentation extract, and the cordyceps militaris powder is used as a nitrogen source, so that the cordyceps militaris powder is decomposed to the maximum extent by utilizing microorganisms, various active ingredients in the cordyceps militaris powder are released, various active ingredients in the cordyceps militaris are all introduced into the fermentation extract, and the utilization rate of the cordyceps militaris is improved. In addition, after being decomposed by microorganisms, other active products such as various small molecule essential amino acids, lactic acid, sterol, phytic acid, vitamins and the like can be metabolized. The nutrient components in the fermented extract of cordyceps militaris obtained after fermentation are very rich, and the combination of microbial fermentation and cordyceps militaris has a good synergistic interaction effect, so that the nutrient substances, the nutrient density and the physiological activity of the fermented extract of cordyceps militaris are greatly improved, and the value of the fermented extract of cordyceps militaris far exceeds that of cordyceps militaris or microbial fermentation.
Further, the cordyceps militaris fermentation extract obtained by the invention has the following effects: 1. has the function of softening cuticle, can well play the effect of smoothing and tendering skin, and is particularly suitable for removing dandruff of scalp and oily skin; 2. the moisturizing and skin-moistening cream has good effects of moisturizing and moistening skin, can prevent the loss of moisture, and forms a protective film on the surface layer of the skin; 3. has the whitening effect: can inhibit melanin precipitation, effectively improve freckle, mottle, etc., whiten skin more, promote cell metabolism, and rapidly renew skin cells; 4. has antibacterial and antiseptic effects, and can prevent bacteria from invading skin and regulate skin pH. Compared with the cordyceps militaris products reported in the prior art, the cordyceps militaris fermentation extract disclosed by the invention is short in preparation period, low in cost, rich in components, diversified in effect and better in application value.
Furthermore, in the cordyceps militaris fermentation extract, the cordycepin content is 40-90mg/L, the cordycepic acid content is 14-17g/L, and the total amino acid content is 1-2.5 g/L.
Further, the invention also provides application of the cordyceps militaris fermentation extract in health care products, foods or cosmetics.
Furthermore, the invention also provides a health-care product, food or cosmetic, and the health-care product, food or cosmetic contains the cordyceps militaris fermentation extract.
The invention has the following advantages:
1. the invention takes the cordyceps militaris powder as a unique nitrogen source, and makes microorganisms utilize and decompose the cordyceps militaris powder to the maximum extent by selecting a proper strain and a proper fermentation medium formula, so that the effective ingredients in the cordyceps militaris are fully released.
2. The cordycepin content after microbial decomposition is stable and basically consistent with that obtained by an extraction method, and the cordycepic acid content is improved by 6-8 times compared with that obtained by the extraction method.
3. The extract not only contains the functional components of the cordyceps militaris, but also contains some active substances generated by microbial fermentation, and the two are combined for synergistic interaction, so that the nutrient substances, the nutrient density and the physiological activity of the cordyceps militaris fermentation extract are greatly improved, and the value of the cordyceps militaris fermentation extract is far higher than that of the cordyceps militaris or the microbial fermentation extract.
4. The invention has simple process, short production period and convenient operation, can prepare the cordyceps militaris fermentation extract with higher active matter content by simple microbial catabolism and purification process, has stable product quality, and is a high-quality food, health-care product or cosmetic raw material.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.
In the following examples, the lactic acid bacteria used were commercially available products. The cordyceps militaris powder is obtained by drying and crushing commercially available cordyceps militaris sporocarp, and the particle size is about 40 meshes.
Example 1
(1) Seed liquid preparation
Inoculating lactobacillus into liquid MRS culture medium, adjusting pH to 6.2-6.6, standing at 37 deg.C, shake-flask culturing for 2 days to obtain seed solution;
(2) fermentation culture
Inoculating the seed solution into a 5L fermentation tank according to the inoculation amount of 5wt%, wherein the formula of a fermentation medium is as follows: 8g/L of cordyceps militaris powder, 5g/L of glucose, 18g/L of fructose, 5g/L of sodium acetate, 2g/L of magnesium sulfate and the balance of water, wherein the pH value is 6.2-6.6. After inoculation, ventilating, stirring and culturing at 37 ℃, wherein the rotating speed is 200 r/min, the ventilation quantity is 0.5vvm, the pH value is not controlled in the fermentation process, and the fermentation is finished when no residual sugar exists, so that fermentation liquor is obtained;
(3) working up of the fermentation liquor
Heating the fermentation liquid in 65 deg.C water bath for 1h, adjusting pH to 5.0-6.0, and filtering with 0.45 um filter membrane to obtain Cordyceps militaris fermentation extractive solution S1.
Example 2
Preparing cordyceps militaris fermentation extract according to the method of the embodiment 1, except that: 5g/L of glucose and 15g/L of fructose in the fermentation medium. The obtained cordyceps militaris fermentation extract is recorded as cordyceps militaris fermentation extract S2.
Example 3
Preparing cordyceps militaris fermentation extract according to the method of the embodiment 1, except that: 5g/L of glucose and 20g/L of fructose in the fermentation medium. The obtained cordyceps militaris fermentation extract is recorded as cordyceps militaris fermentation extract S3.
Example 4
Preparing cordyceps militaris fermentation extract according to the method of the embodiment 1, except that: the content of the cordyceps militaris powder in the fermentation medium is 5 g/L. The obtained cordyceps militaris fermentation extract is recorded as cordyceps militaris fermentation extract S4.
Example 5
Preparing cordyceps militaris fermentation extract according to the method of the embodiment 1, except that: the content of the cordyceps militaris powder in the fermentation medium is 10 g/L. The obtained cordyceps militaris fermentation extract is recorded as cordyceps militaris fermentation extract S5.
Comparative example 1
Preparing cordyceps militaris fermentation extract according to the method of the embodiment 1, except that: 5g/L of glucose and 10g/L of fructose in the fermentation medium. The obtained cordyceps militaris fermentation extract is recorded as cordyceps militaris fermentation extract D1.
Comparative example 2
Preparing cordyceps militaris fermentation extract according to the method of the embodiment 1, except that: 5g/L of glucose and 25g/L of fructose in the fermentation medium. The obtained cordyceps militaris fermentation extract is recorded as cordyceps militaris fermentation extract D2.
Comparative example 3
Preparing cordyceps militaris fermentation extract according to the method of the embodiment 1, except that: the content of the cordyceps militaris powder in the fermentation medium is 3 g/L. The obtained cordyceps militaris fermentation extract is recorded as cordyceps militaris fermentation extract D3.
Comparative example 4
Preparing cordyceps militaris fermentation extract according to the method of the embodiment 1, except that: the content of the cordyceps militaris powder in the fermentation medium is 15 g/L. The obtained cordyceps militaris fermentation extract is recorded as cordyceps militaris fermentation extract D4.
Comparative example 5
Preparing a cordyceps militaris extracting solution by an extraction method: taking 8g of cordyceps militaris powder, adding 50-70wt% of ethanol solution, wherein the material-liquid ratio is 1 g: 20-30ml, then oscillating and extracting in a water bath at 20-30 ℃ for 2-3h, and filtering after extraction is finished to obtain filtrate; removing alcohol from the filtrate, concentrating, and centrifuging to obtain supernatant; loading the supernatant into macroporous adsorbent resin column at flow rate of 1.5-2.5BV/h, desorbing with 25-35wt% ethanol at flow rate of 0.5-1.5BV/h, and collecting desorption solution to obtain Cordyceps militaris extractive solution D5.
Comparative example 6
A fermentation extract was prepared according to the method of example 1, except that: the fermentation medium comprises the following components: 8g/L of peptone, 5g/L of glucose, 18g/L of fructose, 5g/L of sodium acetate, 2g/L of magnesium sulfate and the balance of water, wherein the pH value is 6.2-6.6. The resulting sample was designated as D6.
Comparative example 7
A fermentation extract was prepared according to the method of example 1, except that: the fermentation medium comprises the following components: 10g/L of yeast powder, 8g/L of cordyceps powder, 5g/L of glucose, 18g/L of fructose and the balance of water, wherein the pH value is about 7.0. Yeast (purchased from the market) is used as a fermentation strain, and the obtained cordyceps militaris fermentation extract is recorded as cordyceps militaris fermentation extract D7.
Test example 1 content of amino acids in fermented extract of Cordyceps militaris
The content and kind of amino acids contained in the fermentation extract samples prepared in each example and comparative example were analyzed using an amino acid autoanalyzer using the liquid MRS medium of example 1 as a control, and the results are shown in table 1.
As is clear from the data in Table 1 above, the amino acid content in the samples according to the examples of the present invention was stabilized at 1g/L or more, and 4 new amino acids, hydroxyproline, proline, glycine and cysteine, were detected. In contrast, the amino acid content in each of the samples of comparative examples D3, D5 and D6 was significantly lower than that of the samples of examples, and the kind of amino acid was also lower than that of the samples of examples. The amino acid content differences of S1, S5 and D4 are not large, which shows that the amino acid components in the stock solution mainly come from cordyceps sinensis powder, meanwhile, the metabolic decomposition capacity of microorganisms is limited, and the amino acid content is influenced by too high and too low addition of cordyceps sinensis, so that the amino acid content in the fermentation extract is greatly increased, and the total amino acid content is increased by 3-6 times compared with that of the extraction method and the single lactobacillus fermentation extract. This indicates an increase in active substance after fermentation.
Test example 2 content of Cordycepin in Cordyceps militaris fermentation extract
The method for measuring the cordycepin content in the fermentation extract samples prepared in the examples and the comparative examples comprises the following specific steps:
sample preparation: the cordyceps militaris fermentation extract of each example and comparative example is diluted by 10 times.
Preparation of a standard sample: accurately weighing 1mg of standard cordycepin, adding 50 mL of distilled water for dissolving, transferring to a 100mL volumetric flask, adding water to the scale, and diluting by 10 times before use.
Chromatographic conditions are as follows: detection was performed on a liquid chromatograph, column: CAPCELL PAK-C18 column, 0.04M potassium dihydrogen phosphate and 5% acetonitrile as mobile phase, 1.0ml/min flow rate, and 35 deg.C of column temperature. The detection wavelength is 260nm, and the sample injection amount is 10 ul.
And (3) determination: the cordycepin content was calculated from the peak area and the results are shown in table 2 below.
As can be seen from the above table, the content of cordycepin in each example is determined by the addition amount of cordyceps powder, and as can be seen from comparison of examples 1, 4, and 5 with comparative examples 3 and 4, cordycepin increases with the increase of the addition amount of cordyceps powder, but when the addition amount of cordyceps is too high, the release of cordycepin decreases because the metabolic decomposition capability of microorganisms is limited. As can be seen from the comparison between the example 1 and the comparative example 5, the extraction method has lower cordycepin content than the cordyceps militaris fermentation extracting solution prepared by the method, and the cordycepin can be lost due to complicated steps of the extraction method. As can be seen by comparing example 1 with comparative examples 6 and 7, cordycepin is mainly derived from cordyceps powder, and has a certain correlation with the decomposition capability of the strain but has no correlation with the metabolic production capability.
According to the literature report, the cordycepin is dissolved in water and is easy to separate from the cordyceps, and the microbial decomposition method can separate the cordycepin from the cordyceps powder to the maximum extent, does not consume the cordycepin and ensures that the cordycepin is accumulated to the maximum extent.
Experimental example 3 content of Cordycepin in Cordyceps militaris fermentation extract
1. Preparing a reagent:
and (3) Nash reagent: fresh configuration is required. 150g of ammonium acetate is accurately weighed, dissolved in distilled water, added with 2mL of glacial acetic acid and 2mL of acetylacetone, and subjected to constant volume to 1000 mL.
0.015mol/L sodium periodate solution: 3.2g of sodium periodate are accurately weighed and dissolved in 0.12mol/L hydrochloric acid.
0.1% of L-rhamnose: accurately weighing 0.1g of L-rhamnose, dissolving in distilled water, and diluting to 100 ml.
Preparation (storage) of cordycepic acid standard solution: weighing 100mg of cordycepic acid standard substance, dissolving in distilled water, and diluting to 100ml to obtain 1mg/ml cordycepic acid standard solution. When used, the solution is diluted ten times.
Test solution: the cordyceps militaris fermentation extract of each example and comparative example is diluted by 200 times.
2. The determination method comprises the following steps:
(1) cordycepic acid standard curve sample system
Mixing the standard cordycepic acid solution with water according to the table above, then respectively adding 1ml of sodium periodate solution, uniformly mixing, standing at room temperature for 10min, adding 2ml of 0.1% rhamnose into each test tube to remove excessive sodium periodate, shaking, uniformly mixing, adding 4ml of freshly prepared Nash reagent, carrying out heat preservation in a water bath at 53 ℃ for 15min, and then rapidly cooling to room temperature. Measuring absorbance at 412nm, and plotting cordycepic acid content (ug/ml) and A412The standard curve of (2).
(2) Sample detection
Taking 50ul of each sample solution, fixing the volume to a 100ml volumetric flask, then taking 1ml, putting the 1ml into a test tube with a plug, adding 1ml of water into a blank control, then adding 1ml of sodium periodate solution, uniformly mixing, standing at room temperature for 10min, adding 2ml of 0.1% rhamnose into each test tube to remove excessive sodium periodate, shaking, uniformly mixing, adding 4ml of freshly prepared Nash reagent, keeping the temperature in a water bath at 53 ℃ for 15min, and then rapidly cooling to room temperature. Absorbance at 412 nm. The absorbance was brought into the standard curve and multiplied by the dilution factor to obtain the cordycepic acid content, the results of which are shown in table 4 below.
As can be seen from the above table, in each example, cordycepic acid is generated by carbon source metabolism of lactobacillus, and as can be seen from comparison of examples 1, 2 and 3 with comparative examples 1 and 2, the content of cordycepic acid increases with the addition amount of fructose, but when the addition amount of fructose is too high, the content of cordycepic acid does not increase significantly. As can be seen from the comparison between the example 1 and the comparative example 5, the content of cordycepic acid in the cordyceps militaris fermentation extract prepared by the extraction method is remarkably reduced compared with the cordyceps militaris fermentation extract prepared by the method, because the content of cordycepic acid in the extract obtained by the extraction method is very low. As can be seen by comparing the example 1, the comparative example 6 and the comparative example 7, the cordycepic acid is mainly derived from the metabolic synthesis of microorganisms and exists in the cordyceps sinensis powder in a small amount, the lactic acid bacteria can utilize a carbon source to the maximum extent to generate the cordycepic acid, and the content of the cordycepic acid in the yeast fermentation extract is not much different from that in the comparative example D5, which indicates that the cordycepic acid is also from the cordyceps sinensis powder. In conclusion, the cordyceps acid fermentation extract decomposed by the lactic acid bacteria not only contains the cordycepic acid in the raw material cordyceps militaris, but also contains the cordycepic acid generated by metabolism of microorganisms by utilizing nutrient components, so that the content of the cordycepic acid in the cordyceps fermentation extract prepared by fermentation is improved by 6-8 times compared with the extract prepared by the extraction method.
Test example 4 improving effect of fermented extract of Cordyceps militaris on skin
1. Whitening efficacy test
The method for determining the inhibition of different samples on the generation of the melanin of the cells by taking cordyceps militaris fermentation extracting solutions S1, S2 and S3 as test samples and cordyceps militaris extracting solution D5 and sample D6 as controls comprises the following steps:
(1) effect on proliferation of melanoma cells in B16 mice
B16 mouse melanoma cells were collected at 2X 10 in logarithmic growth phase4The cells were seeded at a density of 100. mu.L/well in 96-well cell culture plates and routinely cultured overnight in a carbon dioxide incubator at 37 ℃ in 5% CO 2. The samples were prepared to a concentration of 1.5% (v/v) using serum-containing complete medium and filter sterilized with a 0.22 μm filter. Discarding the old culture solution, adding 1.5% (v/v) of sample into the experimental group, adding the same amount of serum-containing cell culture solution into the normal control group, continuously culturing for 24h, and detecting the relative proliferation rate of cells by adopting a WST-1 method. The relative proliferation rate (RGR) is the ratio of the absorbance of the sample group to the absorbance of the normal control group.
(2) Melanin content detection
Taking B16 cells in logarithmic growth phase at 2X 104Inoculating to 6-well culture plate at density of 3mL per well, culturing in carbon dioxide incubator at 37 deg.C with 5% CO2 for 24 hr, discarding old culture solution, adding 3mL serum-containing complete culture medium into normal control group, adding 3mL sample solution into experimental group, and adding into each well of control group and experimental group60 μ L of forskolin solution (2 mM) was added to stimulate melanin production, and the culture was continued for 72 h.
The method for measuring the melanin content in the cells comprises the following steps: pancreatin digestion, centrifuging to remove supernatant, adding 500uL of NaOH solution with the concentration of 1mol/L (containing 10% DMSO) to crack cells, heating at 80 ℃ for 30min, centrifuging at 3000rpm for 10min, taking supernatant, adding the supernatant into a 96-well plate, measuring the absorbance at 450nm to obtain the total melanin, taking the melanin content of a control group as 100%, and obtaining the relative value and the inhibition rate of the melanin content of an experimental group, wherein if a sample has obvious influence on the proliferation of B16 cells, the ratio of the relative value of the melanin content to the corresponding proliferation rate of B16 cells is the change rate of the melanin production of a unit cell.
(3) Results of the experiment
The relative proliferation rate of cells and the effect of the fermented extract of cordyceps militaris on melanin content are shown in table 5 below.
As shown by the results, under the concentration of 1.5% (v/v), all samples have certain inhibition effect on the proliferation of mouse melanoma cells B16, the inhibition rates of S1, S2 and S3 on the total melanin are equivalent (45% -55%), but compared with D5 and D6, S1, S2 and S3 have more significant inhibition effect on the melanin secretion level of cells, D5 and D6 have inhibition effect on the proliferation of melanoma cells, which shows that lactic acid fermentation extract and cordyceps militaris extract have certain whitening effect, but the effect is reduced compared with the embodiment, the fermentation extract prepared by decomposing and metabolizing cordyceps militaris by lactic acid bacteria has more significant inhibition effect on the proliferation of melanoma cells, so that the conclusion can be concluded that the lactic acid bacteria ferment by using cordyceps militaris, and the obtained fermentation liquid not only enables nutrients in cordyceps militaris powder to be released to the maximum degree, in the fermentation process, some substances which are not contained in other nitrogen sources can be metabolized by the lactic acid bacteria, or the combination of the cordyceps militaris ingredient and the lactic acid bacteria fermentation ingredient achieves the synergistic effect in the aspect of skin whitening.
Anti-inflammatory efficacy test
The inhibition effect of different samples on proinflammatory factors is determined by taking cordyceps militaris fermentation extracting solutions S1, S2 and S3 as test samples and D5 and D6 as controls under the concentration of 1.5% (v/v).
Using mouse macrophage model, LPS was used to stimulate the production of inflammatory factors, and samples were examined for their ability to inhibit the secretion of inflammatory factors. Mother liquor with the concentration of 50 ten thousand units/mL is prepared by LPS (lipopolysaccharide) by serum-free 1640 culture solution, and is filtered and sterilized by a 0.22 mu m filter membrane, and is stored in a refrigerator at the temperature of-20 ℃ and diluted into action solution with the concentration of 1 ten thousand units/mL before use. The sample contacted with the cells was prepared with LPS working solution.
Raw264.7 cells in logarithmic growth phase were taken at 1X 105Perml inoculated in 24-well plates at 37 ℃ with 5% CO2Culturing for 24h under the condition, adding a sample with each hole being 1mL, adding LPS action liquid without the sample into the model group, adding serum-free 1640 culture liquid without LPS into the negative control group, continuously culturing for 24h, and determining the content of inflammatory factors in culture supernatant according to the specifications of ELISA detection kits of IL-6, TNF-alpha and IL-1 beta of mice. The inhibition of inflammatory factors was calculated for each sample using the following formula:
inhibition (%) =100% - (sample group inflammatory factor content-negative control group inflammatory factor content)/(model group inflammatory factor content-negative control group inflammatory factor content) × 100%.
The results are shown in Table 6, and the mouse macrophage secretion of IL-6 and TNF-alpha proinflammatory factors is significantly increased under the stimulation of LPS, while the IL-1 beta is increased only in a small amount. All samples have certain inhibition effects on three inflammatory factors of IL-6, TNF-alpha and IL-1 beta of mice by taking the three inflammatory factors as research indexes, the anti-inflammatory effect of D6 is least remarkable, the inhibition rates of S1, S2 and S3 on the three inflammatory factors respectively reach 69-71%, 86-91% and 50-55%, and the inhibition effects of S1, S2 and S3 on the proinflammatory factors are more remarkable compared with those of D5 and D6.
Radical scavenging efficacy experiment
The method for determining the scavenging capacity of different samples on free radicals by taking cordyceps militaris fermentation extracting solutions S1, S2 and S3 as test samples and taking D5 and D6 as controls comprises the following steps:
(1) preparing a solution:
8.8mM hydrogen peroxide: 0.0997g of hydrogen peroxide were weighed out accurately and dissolved in a 100mL brown volumetric flask with purified water.
9mM ferrous sulfate: 0.2502g of ferrous sulfate were weighed out accurately and dissolved in a 100mL brown flask with purified water.
9mM salicylic acid-ethanol: 0.1243g of salicylic acid were weighed out accurately and dissolved in a 100mL brown flask using absolute ethanol.
Preparation of sample solution: accurately sucking 5mL of each prepared sample, and adding purified water to reach a constant volume of 100 mL.
(2) Test method
Several tubes were taken, and the reaction was started by pipetting 0.2mL of 9mM ferrous sulfate, 0.2mL of 9mM salicylic acid-ethanol solution, 3.0mL of each sample solution, and finally 0.25mL of 8.8mM hydrogen peroxide solution. The reaction was carried out at 37 ℃ for 30 minutes, and the light absorption value was measured at 510nm with purified water as a reference. The OH radical clearance was calculated as follows.
(3) Calculation of results
As can be seen from the above table, S1, S2 and S3 can remove OH free radicals, and the removal rate of OH free radicals can be maintained at about 55-65%, but D5 and lactobacillus fermentation extract D6 prepared by the extraction method can remove OH free radicals, but the removal rate is only 37.16% and 6.44%, and the effect is poor. Therefore, the microorganism utilizes the cordyceps militaris for fermentation, the obtained fermentation liquor not only enables nutrient substances in the cordyceps militaris powder to be released to the maximum extent, but also can metabolize substances which are not available by adopting other nitrogen sources in the fermentation process of the lactobacillus, or the combination of the cordyceps militaris component and the lactobacillus fermentation component realizes the synergistic effect on the removal of free radicals.
Claims (10)
1. A preparation method of cordyceps militaris fermentation extract is characterized by comprising the following steps: carrying out fermentation culture on lactic acid bacteria in a fermentation culture medium taking cordyceps militaris as a nitrogen source to obtain cordyceps militaris fermentation extract, wherein the fermentation culture medium taking cordyceps militaris as the nitrogen source comprises the following components: 5-10 g/L of cordyceps militaris powder, 5g/L of glucose, 15-20g/L of fructose, 3-8 g/L of sodium acetate and 1-3g/L of magnesium sulfate.
2. The method of claim 1, wherein: the fermentation medium taking cordyceps militaris as a nitrogen source comprises the following components: 8g/L of cordyceps militaris powder, 5g/L of glucose, 18g/L of fructose, 5g/L of sodium acetate and 2g/L of magnesium sulfate.
3. The method according to claim 1 or 2, characterized in that: the method comprises the following steps:
(1) carrying out seed culture on lactobacillus to obtain a seed solution;
(2) inoculating the seed solution into a fermentation medium using cordyceps militaris as a nitrogen source, ventilating, stirring and culturing, and ending fermentation until no residual sugar exists to obtain a fermentation liquid;
(3) and treating the fermentation liquor to obtain cordyceps militaris fermentation extract.
4. The method of claim 1, 2 or 3, wherein: the lactic acid bacteria include lactobacillus, leuconostoc or bifidobacterium, preferably lactobacillus.
5. The method of claim 3, wherein: inoculating the seed liquid into a fermentation culture medium taking cordyceps militaris as a nitrogen source according to the inoculation amount of 5-10%.
6. The method of claim 1, 2 or 3, wherein: in the step (2), the ventilation amount is kept at 0.3-0.6vvm during fermentation culture.
7. The method of claim 3, wherein: in the step (3), sequentially carrying out operations of heating sterilization, pH adjustment and filtration on the fermentation liquor to obtain cordyceps militaris fermentation extract; preferably, the temperature for heat sterilization is 60-70 deg.C, and pH is adjusted to 5.0-6.0.
8. The cordyceps militaris fermentation extract prepared by the preparation method of the cordyceps militaris fermentation extract according to any one of claims 1 to 7; preferably, the cordycepin content is 40-90mg/L, the cordycepic acid content is 14-17g/L, and the total amino acid content is 1-2.5 g/L.
9. The application of the cordyceps militaris fermentation extract liquid as claimed in claim 8 in health care products, foods or cosmetics.
10. A health product, food or cosmetic is characterized in that: comprises the cordyceps militaris fermentation extract liquid as claimed in claim 8 or 9.
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