CN111973498A

CN111973498A - Ganoderma lucidum fermentation product and preparation method and application thereof

Info

Publication number: CN111973498A
Application number: CN202011025781.1A
Authority: CN
Inventors: 何敬愉; 胡根华; 吴知情; 龚盛昭
Original assignee: Guangzhou Huanya Cosmetic Science and Technology Co Ltd
Current assignee: Guangzhou Huanya Cosmetic Science and Technology Co Ltd
Priority date: 2020-09-25
Filing date: 2020-09-25
Publication date: 2020-11-24

Abstract

The invention provides a lucid ganoderma fermentation product and a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) enzymolysis: adding protease into the ganoderma lucidum extract for enzymolysis reaction, and then inactivating the protease to obtain ganoderma lucidum hydrolysate; (2) fermentation: inoculating microorganism into the Ganoderma hydrolysate, adding culture medium, performing fermentation reaction, sterilizing after the fermentation reaction is finished, and collecting clear liquid to obtain Ganoderma fermented product. The preparation method of the ganoderma lucidum fermented product fully utilizes the advantages of enzymolysis reaction and microbial transformation, can prepare the ganoderma lucidum fermented product which has strong inoxidizability and good whitening effect and can inhibit the generation of inflammatory factors, and has wide application prospect in the field of cosmetics.

Description

Ganoderma lucidum fermentation product and preparation method and application thereof

Technical Field

The invention belongs to the technical field of fine chemical engineering, and relates to a ganoderma lucidum fermentation product, and a preparation method and application thereof.

Background

With the continuous improvement of living standard, people pay more and more attention to beauty and skin care, and pay more and more attention to the efficacy and components of beauty and skin care cosmetics. In recent years, extracts or active substances from Chinese herbal medicines have the advantages of stability, lasting efficacy and low toxic and side effects, and are popular among people as additives of beauty and skin care cosmetics.

Ganoderma lucidum is called Shen Zhi from Shen nong Ben Cao and listed as the top grade from Ben Cao gang mu, and contains various active ingredients including polysaccharide, triterpene, polypeptide, sterol, polyacetylene, alkaloid, amino acid, etc. Modern dermatology researches show that the ganoderma lucidum extract can not only induce the secretion of cell factors, further improve the mitosis and growth capacity of cells, accelerate the shedding of aging cells and promote metabolism, but also increase the thickness of an epidermal layer and a dermal layer, help to improve the tissue structure of facial skin and delay aging from the deep layer of the skin.

The traditional Chinese medicine fermentation technology mainly applies modern biotechnology such as fermentation engineering and the like to the research of traditional Chinese medicines, a plurality of beneficial strains are added in the traditional Chinese medicine fermentation process, and the obtained fermentation product not only contains active substances of medicinal materials, but also contains secondary metabolites of microorganisms, so that the efficacy of the fermentation product is enhanced.

CN105147587A discloses a preparation method of ganoderma lucidum fermentation raw stock, which comprises the following steps: mixing dry ganoderma powder, water and wine yeast to obtain an initial system, and performing fermentation culture on the initial system to obtain ganoderma fermentation raw stock, wherein the ganoderma fermentation raw stock has the functions of whitening, resisting aging, removing free radicals and inhibiting tyrosinase, but does not have the function of inhibiting the generation of inflammatory factors.

CN108004282A discloses a method for preparing a fermented product of ganoderma lucidum fruiting body, which comprises: mixing Ganoderma fruiting body with water, and making into Ganoderma serous fluid; inoculating saccharomyces cerevisiae into the ganoderma lucidum serous fluid, and fermenting to obtain ganoderma lucidum fermentation liquor; inoculating lactobacillus bulgaricus into the ganoderma lucidum fermentation liquor, and performing secondary fermentation to obtain a ganoderma lucidum fruiting body fermentation product. The ganoderma lucidum fruiting body fermentation product has high nutritive value, has oxidation resistance and whitening effect, but has low inhibition rate on tyrosinase activity.

CN105935143A discloses a method for preparing ganoderma lucidum ferment, which comprises the steps of ganoderma lucidum extraction, liquid medium preparation, monomer fermentation, mixed fermentation and enzymolysis, so as to increase the content of ganoderma lucidum polysaccharide and triterpenes effective active ingredients in ganoderma lucidum ferment.

In conclusion, the lucid ganoderma fermentation product which is strong in oxidation resistance, good in whitening effect and capable of inhibiting the generation of inflammatory factors and the preparation method thereof are provided, and the lucid ganoderma fermentation product has important significance in the fields of cosmetics preparation and lucid ganoderma fermentation.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides the lucid ganoderma fermentation product, and the preparation method and the application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a method for preparing a ganoderma lucidum fermented product, comprising the following steps:

(1) enzymolysis: adding protease into the ganoderma lucidum extract for enzymolysis reaction, and then inactivating the protease to obtain ganoderma lucidum hydrolysate;

(2) fermentation: inoculating microorganism into the Ganoderma hydrolysate, adding culture medium, performing fermentation reaction, sterilizing after the fermentation reaction is finished, and collecting clear liquid to obtain Ganoderma fermented product.

According to the preparation method, the enzymolysis reaction and the fermentation reaction are combined, the ganoderma lucidum extracting solution is subjected to enzymolysis, the ganoderma lucidum hydrolysate rich in various active substances is obtained, microorganisms in the subsequent fermentation process can synthesize various secondary metabolites by taking the active substances as substrates, and the types of the active substances are increased, so that the ganoderma lucidum leavening which is high in oxidation resistance, good in whitening effect and capable of inhibiting the generation of inflammatory factors is prepared.

Preferably, the ganoderma lucidum extract in the step (1) is obtained by hot water extraction of ganoderma lucidum.

Preferably, the protease of step (1) comprises an amylase.

Preferably, the amylase comprises an alpha-amylase and/or a beta-amylase.

Preferably, the mass ratio of the alpha-amylase to the ganoderma lucidum extracting solution is (0.05-2): 100, including but not limited to 0.1:100, 0.5:100, 1:100, 1.5:100 or 1.7: 100.

Preferably, the mass ratio of the beta-amylase to the lucid ganoderma extracting solution is (0.05-2): 100, including but not limited to 0.1:100, 0.5:100, 1:100, 1.5:100 or 1.7: 100.

Preferably, the pH of the enzymatic hydrolysis reaction in the step (1) is 4.0-6.5, including but not limited to 4.2, 4.4, 4.6, 4.8, 5.0, 5.5, 5.8, 6.0, 6.2 or 6.4, preferably 5.0-6.5.

Preferably, the temperature of the enzymolysis reaction in the step (1) is 28-40 ℃, including but not limited to 30 ℃, 32 ℃, 34 ℃, 36 ℃ or 38 ℃, preferably 28-35 ℃.

Preferably, the time of the enzymolysis reaction in the step (1) is 2-8 h, including but not limited to 3h, 4h, 5h, 6h or 7h, preferably 2-5 h.

Preferably, the stirring speed of the enzymolysis reaction in the step (1) is 100-250 rpm, including but not limited to 120rpm, 140rpm, 160rpm, 200rpm, 220rpm or 240rpm, preferably 150-250 rpm.

Preferably, the inactivation temperature in the step (1) is 80-120 ℃, including but not limited to 85 ℃, 90 ℃, 95 ℃, 100 ℃, 105 ℃, 110 ℃ or 115 ℃, preferably 80-100 ℃.

Preferably, the inactivation time in step (1) is 20-35 min, including but not limited to 22min, 24min, 26min, 28min, 30min, 32min or 34min, preferably 20-30 min.

Preferably, the microorganism of step (2) comprises lactic acid bacteria.

Preferably, the lactic acid bacteria comprise Lactococcus lactis (Lactococcus lactis) and/or Lactobacillus plantarum (Lactobacillus plantarum).

Preferably, the Lactococcus lactis comprises Lactococcus lactis ATCC 13675.

Preferably, the Lactobacillus plantarum comprises Lactobacillus plantarum ATCC 8014.

Preferably, the concentration of the bacterial liquid of the lactic acid bacteria is 10⁵～10⁸CFU/mL, including but not limited to 10⁶CFU/mL or 10⁷CFU/mL, preferably 10⁵～10⁷CFU/mL。

Preferably, the culture medium of step (2) comprises MRS broth.

Preferably, the adding amount ratio of the lucid ganoderma hydrolysate to the lactobacillus liquid to the MRS broth culture medium is (1-100) mL (1-5) g, including but not limited to 20mL:1mL:2g, 30mL:3mL:3g or 80mL:4mL:4g, preferably (1-30) mL (1-5) g.

Preferably, the microorganism of step (2) comprises yeast.

Preferably, the yeast comprises Saccharomyces cerevisiae and/or Kluyveromyces lactis.

Preferably, the Saccharomyces cerevisiae comprises Saccharomyces cerevisiae ATCC 9763.

Preferably, the Kluyveromyces lactis comprises Kluyveromyces lactis ATCC 10689.

Preferably, the concentration of the bacterial liquid of the yeast is 10⁵～10⁸CFU/mL, including but not limited to 10⁶CFU/mL or 10⁷CFU/mL, preferably 10⁵～10⁷CFU/mL。

Preferably, the medium of step (2) comprises a YPD medium.

Preferably, the adding amount ratio of the ganoderma lucidum hydrolysate to the yeast strain liquid to the YPD culture medium is (1-100) mL and (1-5) g, including but not limited to (20) mL:1mL:2g, 30mL:3mL:3g or 80mL:4mL:4g, preferably (1-30) mL, (1-5) mL and (1-5) g.

Preferably, deionized water is added for the fermentation reaction.

Preferably, the volume of the deionized water is 3-5 times of the volume of the ganoderma lucidum hydrolysate.

Preferably, the temperature of the fermentation reaction in the step (2) is 25-40 ℃, including but not limited to 28 ℃, 30 ℃, 32 ℃, 34 ℃, 36 ℃ or 38 ℃, preferably 28-35 ℃.

Preferably, the fermentation reaction time in the step (2) is 36-120 h, including but not limited to 42h, 48h, 64h, 70h, 96h, 102h, 108h or 114h, preferably 36-96 h.

Preferably, the temperature for sterilization in the step (2) is 100-121 ℃, including but not limited to 105 ℃, 110 ℃, 115 ℃ or 120 ℃, preferably 115-121 ℃.

Preferably, the time for sterilization in the step (2) is 20-30 min, including but not limited to 22min, 24min, 26min or 28 min.

Preferably, the supernatant obtained in step (2) is obtained by centrifugation.

Preferably, the centrifugation radius of the centrifugation is 6-15 cm, including but not limited to 7cm, 8cm, 9cm, 12cm, 13cm or 14cm, preferably 8-12 cm, and further preferably 8-11 cm.

Preferably, the centrifugation time is 10-30 min, including but not limited to 12min, 14min, 16min, 18min, 24min, 26min or 28min, preferably 10-20 min.

Preferably, the rotation speed of the centrifugation is 3000-4700 rpm, including but not limited to 3200rpm, 3400rpm, 3600rpm, 3800rpm, 4200rpm, 4400rpm or 4600rpm, preferably 3500-4700 rpm.

In the present invention, as a preferred preparation method, the preparation method comprises the steps of:

(1) adding alpha-amylase and beta-amylase into a lucid ganoderma extracting solution, wherein the mass ratio of the alpha-amylase to the beta-amylase to the lucid ganoderma extracting solution is (0.05-2): 100), adjusting the pH to 4.0-6.5, heating to 28-40 ℃, stirring to 100-250 rpm, carrying out enzymolysis reaction for 2-8 h, and then inactivating the amylase at 80-120 ℃ for 20-35 min to obtain lucid ganoderma hydrolysate;

(2) inoculating mixed bacteria of saccharomyces cerevisiae and kluyveromyces lactis into the ganoderma lucidum hydrolysate, and adding an YPD culture medium and deionized water with the volume of 3-5 times that of the ganoderma lucidum hydrolysate, wherein the bacteria liquid concentration of the mixed bacteria is 10⁵～10⁸CFU/mL, the adding amount ratio of the lucid ganoderma hydrolysate, the mixed bacteria liquid and the YPD culture medium is (1-100) mL, (1-5) mL, (0.1-5) g, fermentation is carried out at 25-40 ℃ for 36-120 h, sterilization is carried out at 100-121 ℃ for 20-30 min, and the centrifugal radius is 6 toCentrifuging at 3000-4700 rpm for 10-30 min at 15cm, and collecting supernatant to obtain yeast fermentation liquid;

inoculating mixed bacteria of lactococcus lactis and lactobacillus plantarum into ganoderma lucidum hydrolysate, and adding MRS broth culture medium and deionized water with the volume of 3-5 times that of the ganoderma lucidum hydrolysate, wherein the bacteria liquid concentration of the mixed bacteria is 10⁵～10⁸CFU/mL, wherein the adding amount ratio of the lucid ganoderma hydrolysate to the mixed bacteria liquid to the MRS broth culture medium is (1-100) mL and (1-5) mL, the (0.1-5) g is fermented for 36-120 h at 25-40 ℃, the lucid ganoderma hydrolysate is sterilized for 20-30 min at 100-121 ℃, the centrifugal radius is 6-15 cm, the lucid ganoderma broth culture medium is centrifuged for 10-30 min at 3000-4700 rpm, and the supernatant is taken to obtain the lactobacillus fermentation liquid;

(3) and mixing the yeast fermentation liquor and the lactobacillus fermentation liquor to obtain the ganoderma lucidum fermentation product.

In a second aspect, the present invention provides a ganoderma lucidum fermented product, which is prepared by the preparation method of the first aspect.

Preferably, the ganoderma lucidum fermentation product comprises a lactic acid bacteria fermentation liquor and/or a yeast fermentation liquor.

Preferably, the ganoderma lucidum fermentation product comprises 1-100 parts by weight of lactobacillus fermentation liquor and/or 1-100 parts by weight of yeast fermentation liquor.

Preferably, the ganoderma lucidum fermentation product comprises 1-100 parts by weight of lactobacillus fermentation liquor, including but not limited to 2 parts, 10 parts, 15 parts, 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts or 80 parts.

Preferably, the ganoderma lucidum fermentation product comprises 1-100 parts by weight of yeast fermentation liquid, including but not limited to 2 parts, 10 parts, 15 parts, 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, 70 parts or 80 parts.

According to the invention, the whitening and anti-aging effects of the ganoderma lucidum fermentation product can be remarkably improved by mixing the yeast fermentation liquid and the lactobacillus fermentation liquid.

Preferably, the lactic acid bacteria comprise lactococcus lactis and/or lactobacillus plantarum.

Preferably, the lactococcus lactis comprises lactococcus lactis ATCC 13675.

Preferably, the kluyveromyces lactis comprises kluyveromyces lactis ATCC 10689.

Preferably, the content of ganoderic acid A in the ganoderma lucidum fermentation product is higher than 100 mug/mL.

Preferably, the content of protein in the ganoderma lucidum fermentation product is higher than 0.61 mg/mL.

Preferably, the molecular weight of the protein in the ganoderma lucidum fermentation product is less than 5000 u.

In a third aspect, the invention provides the use of the ganoderma lucidum ferment of the second aspect in the preparation of cosmetics.

In a fourth aspect, the present invention provides a cosmetic comprising the ganoderma lucidum fermented product of the second aspect.

Preferably, the mass percentage of the ganoderma lucidum fermentation product in the cosmetic is 15-25%, including but not limited to 17%, 18%, 19%, 20%, 22% or 24%.

Preferably, the cosmetic further comprises any one or a combination of at least two of jojoba oil, glycerin, sodium benzoate, potassium sorbate, p-hydroxyacetophenone, or water.

Preferably, the formulation of the cosmetic includes any one of a water agent, an emulsion, a essence, a gel or a spray.

Compared with the prior art, the invention has the following beneficial effects:

(1) the preparation method combines the enzymolysis reaction and the fermentation reaction, fully utilizes the advantages of protease enzymolysis and microbial conversion, and can prepare the ganoderma lucidum leavening which has strong inoxidizability and good whitening effect and can inhibit the generation of inflammatory factors;

(2) according to the ganoderma lucidum fermentation product, the content of ganoderic acid A is more than 89 mu g/mL and is maximally 137 mu g/mL, the content of protein with the molecular weight of less than 5000u is more than 0.5mg/mL and is maximally 0.96mg/mL, the inhibition rate on the activity of tyrosinase is more than 62 percent and is maximally 84.6 percent, the hydroxyl radical clearance rate is more than 47 percent and is maximally 79.5 percent, the DPPH radical clearance rate is more than 50 percent and is maximally 84.6 percent, and the production of inflammatory factors IL-1 beta and TNF-alpha can be inhibited;

(3) the cosmetic disclosed by the invention has remarkable whitening and anti-aging effects and has a wide application prospect.

Detailed Description

For the purpose of facilitating an understanding of the present invention, the present invention will now be described by way of examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

In the examples of the invention and comparative examples, alpha-amylase was purchased from alatin, cat #: a109181-500 g;

beta-amylase was purchased from the summer prosperous group under the cat no: FDG-2258-1 kg; MRS broth and YPD medium were purchased from Kyork, Guangdong, Microscience, Inc.

Example 1

The embodiment provides a preparation method of a ganoderma lucidum fermented product, which comprises the following steps:

(1) taking 100g of crushed ganoderma lucidum medicinal material, adding 1500mL of deionized water, heating in 100 ℃ water bath for 2h, filtering, taking residues, adding 1500mL of deionized water, heating in 100 ℃ water bath for 2h, filtering, combining two extracting solutions, and concentrating under reduced pressure at 60 ℃ to 1000mL to obtain ganoderma lucidum extracting solution;

(2) adding 2g of alpha-amylase, 0.05g of beta-amylase and 400mL of deionized water into 100g of lucid ganoderma extract, uniformly mixing, adjusting the pH to 6.5, carrying out enzymolysis for 2h at 40 ℃ and 250rpm, and then inactivating the enzyme for 20min at 120 ℃ to obtain hydrolysate;

(3) 100mL of hydrolysate and 5mL of lactococcus lactis bacterial liquid (10)⁸CFU/mL), 5g of MRS broth culture medium and 400mL of deionized water are mixed, fermentation is carried out for 36h at 40 ℃, the fermentation broth is sterilized for 20min at 121 ℃ after the fermentation is finished, then centrifugation is carried out for 30min at the centrifugation radius of 15cm and the rotation speed of 3000rpm, and the centrifuged supernatant is taken, thus obtaining the lactococcus lactis fermentation broth.

Example 2

(2) taking 100g of lucid ganoderma extract, adding 0.05g of alpha-amylase, 2g of beta-amylase and 400mL of deionized water, uniformly mixing, adjusting the pH to 4.0, carrying out enzymolysis for 8h at 28 ℃ and 100rpm, and then carrying out enzyme deactivation for 35min at 80 ℃ to obtain hydrolysate;

(3) 1mL of hydrolysate and 1mL of lactobacillus plantarum bacterial liquid (10)⁵CFU/mL), 0.1g of MRS broth culture medium and 5mL of deionized water are mixed, fermentation is carried out for 120h at 25 ℃, the fermentation broth is sterilized for 30min at 100 ℃ after the fermentation is finished, then centrifugation is carried out for 10min at the centrifugation radius of 6cm and the rotation speed of 4700rpm, and the centrifuged supernatant is taken, thus obtaining the lactobacillus plantarum fermentation broth.

Example 3

(2) adding 1g of alpha-amylase, 1g of beta-amylase and 400mL of deionized water into 100g of lucid ganoderma extract, uniformly mixing, adjusting the pH to 5.5, carrying out enzymolysis for 4h at 32 ℃ and 200rpm, and then inactivating the enzyme for 25min at 90 ℃ to obtain hydrolysate;

(3) 20mL of hydrolysate and 1mL of lactobacillus plantarum bacterial liquid (10)⁶Mixing CFU/mL), 2g MRS broth culture medium and 80mL deionized water, fermenting at 30 deg.C for 96 hr, sterilizing the fermentation broth at 100 deg.C for 25min, centrifuging at 4500rpm with a centrifugal radius of 11cm for 20min, centrifuging, and collecting the supernatantThe lactobacillus plantarum fermentation liquor is obtained from the supernatant.

Example 4

(3) 20mL of hydrolysate and 1mL of lactococcus lactis bacterial liquid (10)⁶CFU/mL), 2g of MRS broth culture medium and 80mL of deionized water are mixed, fermentation is carried out for 96h at 30 ℃, the fermentation broth is sterilized for 25min at 100 ℃ after the fermentation is finished, then centrifugation is carried out for 20min at the centrifugation radius of 11cm and the rotation speed of 4500rpm, and the centrifuged supernatant is taken, thus obtaining the lactococcus lactis fermentation broth.

Example 5

(3) 20mL of hydrolysate and 1mL of saccharomyces cerevisiae liquid (10)⁶CFU/mL), 2g YPD culture medium and 80mL deionized water, fermenting at 30 deg.C for 96 hr, sterilizing the fermentation liquid at 100 deg.C for 25min, centrifuging at a centrifugal radius of 11cm and rotating at a speed of 11cmCentrifuging at 4500rpm for 20min, and collecting supernatant to obtain Saccharomyces cerevisiae fermentation liquid.

Example 6

(3) 20mL of hydrolysate and 1mL of Kluyveromyces lactis liquid (10)⁶CFU/mL), 2g YPD culture medium and 80mL deionized water, fermenting for 84h at 30 ℃, sterilizing the fermentation broth at 100 ℃ for 25min after the fermentation is finished, centrifuging for 20min at the centrifugal radius of 11cm and the rotating speed of 4500rpm, and taking the centrifuged supernatant to obtain the Kluyveromyces lactis fermentation broth.

Example 7

This example is different from example 3 only in that the Lactobacillus plantarum solution described in step (2) is replaced with a mixed solution of Lactobacillus plantarum and Lactobacillus lactis, and the Lactobacillus plantarum solution (10) is contained in the mixed solution⁶CFU/mL) and lactococcus lactis bacterial liquid (10)⁶CFU/mL) was 8:2, and the amount of the mixed bacterial suspension was the same as that of the lactobacillus plantarum bacterial suspension of example 3, but the other examples were the same as example 3.

Example 8

This example is different from example 3 only in that the Lactobacillus plantarum solution described in step (2) is replaced with a mixed solution of Lactobacillus plantarum and Lactobacillus lactis, and a Lactobacillus plantarum solution (10) is planted in the mixed solution⁶CFU/mL) and lactococcus lactis bacterial liquid (10)⁶CFU/mL) ratio of 5:5, the amount of the mixed bacterial liquid added and the amount of the Lactobacillus plantarum bacterial liquid added in example 3The same amounts were used, and the rest was the same as in example 3.

Example 9

This example is different from example 3 only in that the Lactobacillus plantarum solution described in step (2) is replaced with a mixed solution of Lactobacillus plantarum and Lactobacillus lactis, and a Lactobacillus plantarum solution (10) is planted in the mixed solution⁶CFU/mL) and lactococcus lactis bacterial liquid (10)⁶CFU/mL) was 2:8, the amount of the mixed bacterial suspension was the same as that of the lactobacillus plantarum bacterial suspension of example 3, and the other examples were the same as example 3.

Example 10

Compared with the embodiment 5, the difference of the embodiment is only that the saccharomyces cerevisiae liquid in the step (2) is replaced by the mixed liquid of the saccharomyces cerevisiae and the kluyveromyces lactis, and the saccharomyces cerevisiae liquid (10) in the mixed liquid⁶CFU/mL) and Kluyveromyces lactis solution (10)⁶CFU/mL) was 8:2, the amount of the mixed bacterial suspension was the same as that of the Saccharomyces cerevisiae bacterial suspension of example 5, and the other examples were the same as example 5.

Example 11

Compared with the embodiment 5, the difference of the embodiment is only that the saccharomyces cerevisiae liquid in the step (2) is replaced by the mixed liquid of the saccharomyces cerevisiae and the kluyveromyces lactis, and the saccharomyces cerevisiae liquid (10) in the mixed liquid⁶CFU/mL) and Kluyveromyces lactis solution (10)⁶CFU/mL) was 5:5, the amount of the mixed bacterial suspension was the same as that of the Saccharomyces cerevisiae bacterial suspension of example 5, and the other examples were the same as example 5.

Example 12

Compared with the embodiment 5, the difference of the embodiment is only that the saccharomyces cerevisiae liquid in the step (2) is replaced by the mixed liquid of the saccharomyces cerevisiae and the kluyveromyces lactis, and the saccharomyces cerevisiae liquid (10) in the mixed liquid⁶CFU/mL) and Kluyveromyces lactis solution (10)⁶CFU/mL) was 2:8, the amount of the mixed bacterial suspension was the same as that of the Saccharomyces cerevisiae bacterial suspension of example 5, and the other examples were the same as example 5.

Example 13

This example provides a mixed fermentation, i.e., the fermentation prepared in example 3 was mixed with the fermentation prepared in example 4 at a weight ratio of 8: 2.

Example 14

This example provides a mixed fermentation, i.e., the fermentation prepared in example 3 was mixed with the fermentation prepared in example 4 at a weight ratio of 5: 5.

Example 15

This example provides a mixed fermentation, i.e., the fermentation prepared in example 3 was mixed with the fermentation prepared in example 4 at a weight ratio of 2: 8.

Example 16

This example provides a mixed fermentation, i.e., the fermentation prepared in example 3 was mixed with the fermentation prepared in example 5 at a weight ratio of 8: 2.

Example 17

This example provides a mixed fermentation, i.e., the fermentation prepared in example 3 was mixed with the fermentation prepared in example 5 in a weight ratio of 5: 5.

Example 18

This example provides a mixed fermentation, i.e., the fermentation prepared in example 3 was mixed with the fermentation prepared in example 5 at a weight ratio of 2: 8.

Example 19

This example provides a mixed fermentation, i.e., the fermentation prepared in example 3 was mixed with the fermentation prepared in example 6 at a weight ratio of 8: 2.

Example 20

This example provides a mixed fermentation, i.e., the fermentation prepared in example 3 was mixed with the fermentation prepared in example 6 at a weight ratio of 5: 5.

Example 21

This example provides a mixed fermentation, i.e., the fermentation prepared in example 3 was mixed with the fermentation prepared in example 6 at a weight ratio of 2: 8.

Example 22

This example provides a mixed fermentation, i.e., the fermentation prepared in example 4 was mixed with the fermentation prepared in example 5 at a weight ratio of 8: 2.

Example 23

This example provides a mixed fermentation, i.e., the fermentation prepared in example 4 was mixed with the fermentation prepared in example 5 in a weight ratio of 5: 5.

Example 24

This example provides a mixed fermentation, i.e., the fermentation prepared in example 4 was mixed with the fermentation prepared in example 5 at a weight ratio of 2: 8.

Example 25

This example provides a mixed fermentation, i.e., the fermentation prepared in example 4 was mixed with the fermentation prepared in example 6 at a weight ratio of 8: 2.

Example 26

This example provides a mixed fermentation, i.e., the fermentation prepared in example 4 was mixed with the fermentation prepared in example 6 at a weight ratio of 5: 5.

Example 27

This example provides a mixed fermentation, i.e., the fermentation prepared in example 4 was mixed with the fermentation prepared in example 6 at a weight ratio of 2: 8.

Example 28

This example provides a mixed fermentation, i.e., the fermentation prepared in example 5 was mixed with the fermentation prepared in example 6 at a weight ratio of 8: 2.

Example 29

This example provides a mixed fermentation, i.e., the fermentation prepared in example 5 was mixed with the fermentation prepared in example 6 at a weight ratio of 5: 5.

Example 30

This example provides a mixed fermentation, i.e., the fermentation prepared in example 5 was mixed with the fermentation prepared in example 6 at a weight ratio of 2: 8.

Comparative example 1

(2) adding 1g of alpha-amylase and 400mL of deionized water into 100g of lucid ganoderma extract, uniformly mixing, adjusting the pH to 5.5, carrying out enzymolysis for 4h at 32 ℃ and 200rpm, and then inactivating enzymes at 90 ℃ for 25min to obtain hydrolysate;

(3) 20mL of hydrolysate, 2g of MRS broth, and 80mL of deionized water were mixed.

Comparative example 2

(2) adding 1g of beta-amylase and 400mL of deionized water into 100g of lucid ganoderma extract, uniformly mixing, adjusting the pH to 5.5, carrying out enzymolysis for 4h at 32 ℃ and 200rpm, and then inactivating enzymes at 90 ℃ for 25min to obtain hydrolysate;

(3) 20mL of the digest, 2g of YPD medium and 80mL of deionized water were mixed.

Comparative example 3

Comparative example 4

(3) mixing 20mL of hydrolysate, 2g of MRS broth culture medium and 80mL of deionized water, fermenting at 30 ℃ for 96h, sterilizing at 100 ℃ for 25min, centrifuging at 4500rpm with the centrifugal radius of 11cm for 20min, and taking supernatant.

Comparative example 5

(3) mixing 20mL of hydrolysate, 2g of YPD medium and 80mL of deionized water, fermenting at 30 deg.C for 96h, sterilizing at 100 deg.C for 25min, centrifuging at 4500rpm with a centrifugation radius of 11cm for 20min, and collecting supernatant.

Test example 1 analysis of components

The tests were carried out with the samples prepared in examples 1 to 30 and comparative examples 1 to 5.

Respectively taking 5mL of sample liquid, placing the sample liquid in a dialysis bag (MWCO, 3.5-5 Ku), placing the dialysis bag in deionized water for dialysis for 6h, concentrating the dialysate, then determining the protein concentration by using a biuret method, namely taking 1.0mL of dialysate, adding 2.0mL of biuret reagent, fully and uniformly mixing, placing for 15min at room temperature, determining the light absorption value at the wavelength of 540nm, and calculating the concentration of the protein in the sample liquid by using bovine serum albumin as a control.

Respectively putting 5mL of sample liquid into a round-bottom flask, adding concentrated hydrochloric acid to enable the concentration of hydrochloric acid in a reaction system to be 2mol/L, reacting in a boiling water bath for 3 hours, evaporating the reaction liquid under reduced pressure, dissolving with methanol, fixing the volume to 5mL, and determining the content of ganoderic acid A in the sample by adopting an HPLC-ELSD method.

The results are shown in Table 1.

TABLE 1

As shown in Table 1, compared with comparative examples 1-5, the content of ganoderic acid A and the content of small molecular protein in the ganoderma lucidum fermentations prepared in examples 1-30 are both significantly improved, which indicates that the enrichment of ganoderma lucidum active ingredients is realized by studying the fermentation reaction process and controlling the types of key factors, the concentration and the addition amount of bacteria liquid and the fermentation time.

Test example 2 evaluation of hemolysis of erythrocytes

The erythrocyte hemolysis experiment is one of the alternative methods of rabbit eye irritation experiments, the basic principle is to evaluate the injury of chemicals to eye tissue cells by measuring the dissolving-out amount and denaturation degree of hemoglobin, and the erythrocyte hemolysis experiment is mainly used for evaluating eye irritation research of chemicals such as cosmetics and raw materials internationally.

The samples prepared in examples 1 to 30 and comparative examples 1 to 5 were used in the hemolysis test of erythrocytes according to the hemolysis test method and the classification standard of the hemolysis test of erythrocytes of the European Alternatives test center, which is shown in Table 2, wherein HD is₅₀The concentration of the sample at which 50% of erythrocytes are hemolyzed, DI is the protein denaturation index, and L/D is HD₅₀Ratio to DI.

TABLE 2

L/D	Grading
		L/D>100	Has no irritation
10<L/D≤100	Micro-stimulation property
		1<L/D≤10	Mild irritation
0.1<L/D≤1	Irritation due to poisoning
		L/D≤0.1	Severe irritation

The results of the experiment are shown in table 3.

TABLE 3

Table 3 shows that the ganoderma lucidum fermentate prepared by the invention has no irritation.

Test example 3 evaluation of tyrosinase inhibitory Activity

The ability to inhibit tyrosinase activity reflects the whitening ability of the material to a certain extent, and the greater the tyrosinase activity inhibition rate, the greater the whitening ability, and the samples prepared in examples 1 to 30 and comparative examples 1 to 5 were tested.

Accurately sucking a sample solution, a Phosphate Buffer Solution (PBS) with the pH value of 6.8 and a dopa solution with the mass fraction of 0.1% according to the table 4, fully mixing, incubating at 30 ℃ for 5min, respectively adding tyrosinase solutions (the activity is 100U/mL) with corresponding volumes, incubating at 30 ℃ for 10min, quickly transferring to a cuvette, and measuring the absorbance at 475 nm.

TABLE 4

	Group 1	2 groups of	Group 3	4 groups of
					Sample solution (mL)	0	0	1.0	1.0
Phosphate buffer solution (mL)	2.0	2.5	1.0	1.5
					Dopa solution (mL)	1.0	1.0	1.0	1.0
Tyrosinase solution (mL)	0.5	0	0.5	0
					Total volume (mL)	3.5	3.5	3.5	3.5

Calculating the relative inhibition rate (I) of the sample solution to tyrosinase according to the formula (1).

I＝[(A₁-A₂)-(A₃-A₄)]/(A₁-A₂)×100％ (1)

In the formula, A₁、A₂、A₃And A₄The absorbance of the solutions of groups 1 to 4, respectively.

The results are shown in Table 5.

Test example 4 evaluation of hydroxyl radical scavenging ability

1.0mL of FeSO₄The solution (9.0mmol/L), 1mL of salicylic acid-ethanol solution (9.0mmol/mL), and 1.0mL of the sample solution were mixed, and 1.0mL of H was added₂O₂(8.8mmol/mL) was subjected to 37 ℃ water bath for 30min, and the absorbance at 510nm was measured using absolute ethanol as a blank, and the hydroxyl radical scavenging ratio (Y) of each sample solution was calculated according to the formula (2), and the results are shown in Table 5.

Y＝[1-(A₁-A₂)/A₃]×100％ (2)

In the formula, A₁Is the absorbance of the sample reaction system, A₂To add no H₂O₂Absorbance of the sample (absolute ethanol instead of H)₂O₂)，A₃The absorbance of the blank control absolute ethyl alcohol is shown.

Test example 5 evaluation of DPPH radical scavenging ability

The ability to scavenge DPPH free radicals reflects to some extent the antioxidant capacity of the material, the greater the rate of scavenging of free radicals, the greater the antioxidant and anti-aging abilities, and thus, the anti-aging effect can be judged by studying the ability of a sample to scavenge DPPH free radicals, and tests were conducted on samples prepared in examples 1-30 and comparative examples 1-5.

Respectively adding 2.0mL and 0.25mL of DPPH test solution with mass concentration of 45.8mg/L into the test tube, supplementing the total volume to 3mL with absolute ethyl alcohol, shaking up, reacting in dark for 30min, measuring absorbance at wavelength of 517nm with a 1cm cuvette, and recording as A₁(ii) a Respectively adding 2mL of absolute ethyl alcohol and an equal amount of sample to be detected into the test tube, supplementing the total volume to 3mL by the absolute ethyl alcohol, measuring the absorbance at the wavelength of 517nm and recording as A₂(ii) a Adding 2mL of DPPH test solution and 1mL of absolute ethanol into the test tube, measuring the absorbance at the wavelength of 517nm and recording as A₃The DPPH radical clearance of the sample to be tested was calculated according to the formula (3), and the results are shown in Table 5.

T＝[1-(A₁-A₂)/A₃]×100％ (3)

Wherein T is DPPH radical scavenging rate.

Test example 6 evaluation of anti-inflammatory ability

Mouse macrophage 264.7 was placed in DMEM medium supplemented with 1mL of the sample, 10% fetal bovine serum, 100. mu.g/mL of penicillin, and 100. mu.g/mL of streptomycin, respectively, incubated in a carbon dioxide incubator at 37 ℃ with 5% carbon dioxide and 95% air humidity for 1 hour, then induced with lipopolysaccharide (100ng/mL) for 24 hours, and the concentrations of IL-1. beta. and TNF-. alpha. in the cell culture supernatant were measured using an ELISA kit, and the results are shown in Table 5.

TABLE 5

As shown in Table 5, the samples prepared in examples 1-30 had higher tyrosinase activity inhibition, hydroxyl radical scavenging and DPPH radical scavenging rates, wherein the tyrosinase inhibition was 84.6% at the highest, the hydroxyl radical scavenging rate was 79.5% at the highest, the DPPH radical scavenging rate was 84.6% at the highest, and the concentrations of inflammatory factors IL-1 β and TNF- α in the cell culture system were lower, compared with the samples prepared in comparative examples 1-5, indicating that the fermentation product of the present invention has the activity of inhibiting tyrosinase activity, scavenging hydroxyl radicals and DPPH radicals, and also can inhibit the production of inflammatory factors IL-1 β and TNF- α; the comparison shows that the tyrosinase activity inhibition rate, the hydroxyl radical clearance rate and the DPPH radical clearance rate of a single fermentation product are lower than those of corresponding mixed fermentation products, and the fact that the tyrosinase activity inhibition rate, the hydroxyl radical clearance rate and the DPPH radical clearance rate of the ganoderma lucidum fermentation product can be improved by mixing the ganoderma lucidum fermentation liquid obtained by fermenting lactic acid bacteria and yeast, namely, the synergistic interaction effect exists between the lactic acid bacteria fermentation liquid and the yeast fermentation liquid, and the whitening and antioxidant effects of the ganoderma lucidum fermentation product are improved.

Application example 1

The application example provides essence containing the ganoderma lucidum fermentation product, and the specific formula of the essence is shown in table 6.

TABLE 6

Component name	Mass fraction (%)
		Water (W)	Balance of
Sample (I)	15
		Jojoba oil	20
Glycerol	5
		Sodium benzoate	0.2
Potassium sorbate	0.2
		P-hydroxyacetophenone	0.2

The sample was the sample of example 17.

The preparation method comprises the following steps: weighing the raw materials of each component according to the formula proportion in turn, and stirring and mixing the raw materials uniformly at normal temperature.

Application example 2

The application example provides essence containing the ganoderma lucidum fermentation product, and the specific formula of the essence is shown in table 7.

TABLE 7

Samples the samples prepared from example 20.

Application example 3

The application example provides essence, and the specific formula of the essence is shown in table 8.

TABLE 8

Component name	Mass fraction (%)
		Water (W)	Balance of
Sample (I)	20
		Jojoba oil	20
Glycerol	5
		Sodium benzoate	0.2
Potassium sorbate	0.2
		P-hydroxyacetophenone	0.2

The sample was the sample prepared in comparative example 3.

Application test example 1

Selecting 60 volunteers, wherein the volunteers are 20-45 years old and half male and female, equally dividing the volunteers into 2 groups, using the skin care essence prepared by application example 1 on the left face of the volunteer 1, using the skin care essence prepared by application example 3 on the right side, using the skin care essence prepared by application example 2 on the left face of the volunteer 2 on the right side, using the skin care essence prepared by application example 3 once every morning, noon and evening, respectively smearing 1g of product on the face every time, massaging for 2 minutes, continuously using for 28 days, and observing the skin colors of the left face and the right face after using for 28 days.

The improvement of skin whiteness and firmness during use was evaluated with a maximum score of 5 and a minimum score of 1, and the results are shown in table 9, which is the average of all results.

TABLE 9

Rating index	Application example 1	Application example 2	Application example 3
				Whiteness degree	4.5	4.4	3.6
Compactness degree	3.9	4.0	3.3

The results show that the user thinks that the skin whiteness and the firmness are obviously improved after the essence prepared by the application examples 1 and 2 are used, and are better than the essence prepared by the application example 3, which shows that the ganoderma lucidum fermentation product has obvious whitening and anti-aging effects when being applied to cosmetics.

In conclusion, the preparation method combines enzymolysis and fermentation, fully exerts the advantages of protease enzymolysis and microbial transformation, and can prepare the ganoderma lucidum leavening which has strong inoxidizability and good whitening effect and can inhibit the generation of inflammatory factors, wherein the content of ganoderic acid A in the leavening is more than 89 mug/mL and the highest 137 mug/mL, the content of protein with the molecular weight of less than 5000u in the leavening is more than 0.5mg/mL and the highest 0.96mg/mL, the inhibition rate of tyrosinase activity is more than 62 percent and the highest 84.6 percent, the hydroxyl radical clearance is more than 47 percent and the highest 79.5 percent, the DPPH radical clearance is more than 50 percent and the highest 84.6 percent, and the generation of inflammatory factors IL-1 beta and TNF-alpha can be inhibited.

The applicant states that the present invention is illustrated by the above examples to show the detailed process equipment and process flow of the present invention, but the present invention is not limited to the above detailed process equipment and process flow, i.e. it does not mean that the present invention must rely on the above detailed process equipment and process flow to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. A preparation method of a ganoderma lucidum fermentation product is characterized by comprising the following steps:

2. The method according to claim 1, wherein the protease of step (1) comprises an amylase;

preferably, the amylase comprises an alpha-amylase and/or a beta-amylase;

preferably, the mass ratio of the alpha-amylase to the lucid ganoderma extracting solution is (0.05-2): 100;

preferably, the mass ratio of the beta-amylase to the lucid ganoderma extracting solution is (0.05-2): 100;

preferably, the pH value of the enzymolysis reaction in the step (1) is 4.0-6.5, preferably 5.0-6.5;

preferably, the temperature of the enzymolysis reaction in the step (1) is 28-40 ℃, and preferably 28-35 ℃;

preferably, the time of the enzymolysis reaction in the step (1) is 2-8 hours, and preferably 2-5 hours;

preferably, the stirring speed of the enzymolysis reaction in the step (1) is 100-250 rpm, preferably 150-250 rpm;

preferably, the inactivation temperature in the step (1) is 80-120 ℃, and preferably 80-100 ℃;

preferably, the inactivation time in the step (1) is 20-35 min, preferably 20-30 min.

3. The method according to claim 1 or 2, wherein the microorganism of step (2) comprises lactic acid bacteria;

preferably, the lactic acid bacteria comprise lactococcus lactis and/or lactobacillus plantarum;

preferably, the lactococcus lactis comprises lactococcus lactis ATCC 13675;

preferably, the lactobacillus plantarum comprises lactobacillus plantarum ATCC 8014;

preferably, the concentration of the bacterial liquid of the lactic acid bacteria is 10⁵～10⁸CFU/mL, preferably 10⁵～10⁷CFU/mL；

Preferably, the culture medium of step (2) comprises MRS broth;

preferably, the adding amount ratio of the lucid ganoderma hydrolysate to the lactobacillus liquid to the MRS broth culture medium is (1-100) mL (1-5) g, and preferably (1-30) mL (1-5) g (1-5).

4. The method according to claim 1 or 2, wherein the microorganism of step (2) comprises yeast;

preferably, the yeast comprises saccharomyces cerevisiae and/or kluyveromyces lactis;

preferably, the saccharomyces cerevisiae comprises saccharomyces cerevisiae ATCC 9763;

preferably, the kluyveromyces lactis comprises kluyveromyces lactis ATCC 10689;

preferably, the concentration of the bacterial liquid of the yeast is 10⁵～10⁸CFU/mL, preferably 10⁵～10⁷CFU/mL；

Preferably, the medium of step (2) comprises a YPD medium;

preferably, the adding amount ratio of the ganoderma lucidum hydrolysate to the yeast strain liquid to the YPD culture medium is (1-100) mL and (1-5) g, and preferably (1-30) mL and (1-5) g.

5. The method according to any one of claims 1 to 4, wherein the temperature of the fermentation reaction in step (2) is 25 to 40 ℃, preferably 28 to 35 ℃;

preferably, the fermentation reaction time in the step (2) is 36-120 h, preferably 36-96 h;

preferably, the temperature for sterilization in the step (2) is 100-121 ℃, and preferably 115-121 ℃;

preferably, the sterilization time in the step (2) is 20-30 min;

preferably, the supernatant obtained in step (2) is obtained by centrifugation;

preferably, the centrifugal radius of the centrifugation is 6-15 cm, preferably 8-12 cm, and further preferably 8-11 cm;

preferably, the centrifugation time is 10-30 min, preferably 10-20 min;

preferably, the rotating speed of the centrifugation is 3000-4700 rpm, and preferably 3500-4700 rpm.

6. The production method according to any one of claims 1 to 5, characterized by comprising the steps of:

(2) inoculating mixed bacteria of saccharomyces cerevisiae and kluyveromyces lactis into the ganoderma lucidum hydrolysate, and adding an YPD culture medium, wherein the concentration of the mixed bacteria is 10⁵～10⁸CFU/mL, wherein the adding amount ratio of the lucid ganoderma hydrolysate to the mixed bacteria liquid to the YPD culture medium is (1-100) mL and (1-5) mL, the (0.1-5) g is used for fermenting for 36-120 h at the temperature of 25-40 ℃, sterilizing for 20-30 min at the temperature of 100-121 ℃, centrifuging for 10-30 min at the centrifugal radius of 6-15 cm and at the rpm of 3000-4700 rpm, and taking supernatant to obtain yeast fermentation liquid;

inoculating mixed bacteria of lactococcus lactis and lactobacillus plantarum into ganoderma lucidum hydrolysate, and adding MRS broth culture medium, wherein the concentration of the mixed bacteria is 10⁵～10⁸CFU/mL, wherein the adding amount ratio of the lucid ganoderma hydrolysate to the mixed bacteria liquid to the MRS broth culture medium is (1-100) mL and (1-5) mL, the (0.1-5) g is fermented for 36-120 h at 25-40 ℃, the lucid ganoderma hydrolysate is sterilized for 20-30 min at 100-121 ℃, the centrifugal radius is 6-15 cm, the lucid ganoderma broth culture medium is centrifuged for 10-30 min at 3000-4700 rpm, and the supernatant is taken to obtain the lactobacillus fermentation liquid;

7. A fermented product of Ganoderma lucidum, characterized in that it is prepared by the preparation method according to any one of claims 1 to 6;

preferably, the ganoderma lucidum fermentation product comprises lactobacillus fermentation liquor and/or yeast fermentation liquor;

preferably, the lucid ganoderma fermentation product comprises 1-100 parts by weight of lactobacillus fermentation liquor and/or 1-100 parts by weight of yeast fermentation liquor;

preferably, the lactococcus lactis comprises lactococcus lactis ATCC 13675;

preferably, the kluyveromyces lactis comprises kluyveromyces lactis ATCC 10689.

8. The fermented product according to claim 7, wherein the content of ganoderic acid A in the fermented product is higher than 100 μ g/mL;

preferably, the content of protein in the ganoderma lucidum fermentation product is higher than 0.61 mg/mL;

9. Use of the fermented product of Ganoderma lucidum according to claim 7 or 8 for the preparation of a cosmetic.

10. A cosmetic comprising the fermented product of Ganoderma lucidum according to claim 7 or 8;

preferably, the mass percentage of the ganoderma lucidum fermentation product in the cosmetic is 15-25%;

preferably, the cosmetic further comprises any one or a combination of at least two of jojoba oil, glycerin, sodium benzoate, potassium sorbate, p-hydroxyacetophenone, or water;