CN113208955A

CN113208955A - Moisturizing and relieving ganoderma lucidum composite fermentation product and preparation method and application thereof

Info

Publication number: CN113208955A
Application number: CN202010930375.3A
Authority: CN
Inventors: 王昌涛; 张佳婵; 李萌; 赵丹; 王冬冬
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2020-09-07
Filing date: 2020-09-07
Publication date: 2021-08-06

Abstract

The invention provides a preparation method of a moisturizing and relieving ganoderma lucidum composite fermentation product, which comprises the following steps: inoculating the ganoderma lucidum to a fermentation substrate consisting of rice, radix ophiopogonis, ligusticum chuanxiong hort roots, holy basil leaves and water for fermentation culture, and then performing sterilization treatment to obtain the ganoderma lucidum composite fermentation product. The novel ganoderma lucidum composite fermentation raw pulp is prepared by selecting a suitable ganoderma lucidum strain to carry out composite liquid fermentation on rice, radix ophiopogonis, ligusticum chuanxiong hort roots and holy basil leaves, has good moisturizing and relieving effects, and can be used as an excellent raw material of a moisturizing and relieving cosmetic or directly used as a cosmetic.

Description

Moisturizing and relieving ganoderma lucidum composite fermentation product and preparation method and application thereof

Technical Field

The disclosure relates to the technical field of fermentation, in particular to a moisturizing and relieving ganoderma lucidum composite fermentation product and a preparation method and application thereof.

Background

The rice is a finished product prepared by the working procedures of cleaning, hulling, milling and finishing the finished product of the rice, contains nearly 64 percent of nutrient substances in the rice and more than 90 percent of nutrient elements required by a human body, and is a main food for people in most regions of China. The rice also has the function of skin care, takes the rice extract as the main component (which is rich in the components of y-oryzanol, rice chaff sterol, procyanidine and the like), has mild and safe properties and strong whitening effect, and can supplement water missing from the skin, so that the skin is smooth and fine and is full of elasticity. With the rapid development and the widening of the application range of the rice cultivation industry, the research on the nutrient components of rice and the medical care function is also gradually and deeply carried out.

Radix Ophiopogonis is tuber of Ophiogon japonica, and is a Chinese medicinal plant. Radix Ophiopogonis contains various steroidal saponins, amino acids, glucose, vitamins A and B, sitosterol, mucilage, potassium, sodium, calcium, magnesium, zinc, chromium, etc. Modern medicine considers that radix ophiopogonis has the effects of tonifying heart, promoting urination and resisting bacteria. In addition, the health-care tea has certain effects of calming and reducing blood sugar, and also has the effects of improving the anti-hunger capability of an organism, enhancing the immunity of the organism, prolonging the antibody time, and eliminating free radicals in the body to resist aging. Mai Dong Gen is mainly used for diet therapy and health protection. The radix Ophiopogonis extract has also application in cosmetic field, and can be used as skin conditioner for relieving and resisting allergy.

Rhizoma Ligustici Chuanxiong root is dried rhizome of Umbelliferae rhizoma Ligustici Chuanxiong (Ligusticum sinense cv. Chuanxiong), and is used as Chinese medicinal plant. It is commonly used for promoting blood circulation and activating qi-flowing, dispelling pathogenic wind and relieving pain, and promoting blood circulation and removing blood stasis, and is suitable for various diseases caused by blood stasis; it has good effect in dispelling pathogenic wind and relieving pain, and can be used for treating headache, rheumatalgia, etc. The ligusticum chuanxiong hort extract has application in the field of cosmetics, and has the following effects: the product can be used as antioxidant, and can inhibit the generation of free radicals and effectively scavenge oxygen free radicals; whitening and removing freckles, can effectively inhibit the activity of melanocytes and reduce the generation of melanin under a lower concentration, but shows the cytotoxic effect when the concentration is too high; it also has moisture absorption and moisture keeping effects.

Ocimum sanctum L is a plant of Ocimum of Labiatae, and is aromatic plant used as both medicine and food, and whole plant with root can be used as medicine, and has effects of relieving pain and relieving asthma, and can be used for treating headache and asthma. The holy basil leaf extract has application in the cosmetic field, and has the following effects: the product is used as a sun-screening agent and shows better light-shielding effect in an ultraviolet irradiation experiment; has remarkable effect on controlling pruritus, and can be used as antiallergic agent; has broad-spectrum and strong bacteriostatic effect and also has better inhibition on dandruff forming bacteria.

Ganoderma Lucidum (Ganoderma Lucidum Karst) is a fungus of Polyporaceae, contains many chemical components such as ganoderan, triterpenes, nucleosides, sterols, alkaloids, amino acids, microelements, etc., and has biological activities of resisting tumor, protecting liver, regulating immunity, resisting aging, etc., so it has outstanding health promotion value, and is a cosmetic product with effects of resisting wrinkle, diminishing inflammation, and caring skin. Particularly, the ganoderma lucidum polysaccharide which is the active ingredient of ganoderma lucidum can effectively inhibit the formation of free radicals, resist oxidation and aging, increase the original collagen of skin, effectively promote the formation of hyaluronic acid, keep the skin tender and lusterless and keep the optimal state. The ganoderma lucidum is specified in the pharmacopoeia as the dry fruiting body of ganoderma lucidum or ganoderma sinensis, so the fruiting body is the main medicinal part applied in traditional Chinese medicine and health products. In the nineties, the spore powder of the ganoderma lucidum is found to have better pharmacological activity, and along with the great improvement of the yield of the spore powder, the application of the ganoderma lucidum spore powder in health-care products exceeds that of sporocarp. Meanwhile, the ganoderma lucidum fermentation mycelium has the advantages of stable quality, high yield and the like, and is increasingly applied to product development. The 3 kinds of materials of glossy ganoderma sporophore, spore powder and mycelium have similar functions of resisting tumor, regulating immunity, etc. but the contents and kinds of polysaccharide are different.

At present, the more common active substance extraction methods in the fields of food and cosmetics comprise a hot water extraction method, an acid extraction method, an alkali extraction method, an enzyme extraction method and a microbial fermentation method. The microbial fermentation method does not need to add other catalysts, only needs to culture a large number of microbial strains, and then adds a substrate to carry out reaction, so that the microbial method is more specific and effective than a chemical reagent reaction method; the reaction condition is mild, the microbial fermentation is generally carried out under the conditions of normal temperature and pH of about 7, and harsh conditions such as high temperature, high pressure and the like are not needed; the operation of the equipment is simple and safe, the produced public hazard is less, the environmental pollution is not caused generally, and the post-treatment is relatively simple; the conversion rate can be improved by screening different strains and optimizing reaction conditions, the strains for carrying out microbial conversion on the same substrate can be various, and the optimal strains can be selected by screening, so that the higher conversion rate can be ensured. At present, many researchers at home and abroad adopt a biological method to produce polysaccharide, namely, a microbial method, an enzymatic method, plant cell tissue culture and other multiple biological transformation methods are combined, and the obvious advantages are presented. Microbial fermentation technology plays an increasingly important role in the research and development of natural active substances. The application of fermentation technology in skin care products has been reported in a large number, and the most notable example is SK II Shenxian water.

Although rice, dwarf lilyturf tuber, ligusticum chuanxiong hort root and holy basil leaf are applied in the field of cosmetics, basically all the respective extracts are used as raw materials or are compounded with a plurality of raw materials, the extraction modes of active substances are also various, and the problem of improving the extraction effect of the active substances by selecting more potential material combinations still remains to be faced by researchers.

Disclosure of Invention

The following presents a simplified summary of the disclosure in order to provide a basic understanding of some aspects of the disclosure. It should be understood that this summary is not an exhaustive overview of the disclosure. It is not intended to identify key or critical elements of the disclosure or to delineate the scope of the disclosure. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is discussed later.

In view of the above defects in the prior art, the present disclosure aims to provide a moisturizing and soothing ganoderma lucidum composite fermentation product, and a preparation method and an application thereof, wherein the moisturizing and soothing ganoderma lucidum composite fermentation product has good moisturizing and soothing functions.

According to one aspect of the disclosure, a preparation method of a moisturizing and relieving ganoderma lucidum composite fermentation product is provided, which comprises the following steps: inoculating the ganoderma lucidum to a fermentation substrate consisting of rice, radix ophiopogonis, ligusticum chuanxiong hort, holy basil leaves and water for fermentation culture, and then performing sterilization treatment to obtain the ganoderma lucidum composite fermentation product.

The rice adopted in the present disclosure is a finished product made from rice after the working procedures of cleaning, rice hulling, rice milling and the like. According to the existing research practice, the rice fermentation product filtrate has the whitening effect, and can inhibit the activity of tyrosinase while inhibiting the generation of melanin; has promoting effect on the generation of cholesterol and brain amide which are important components of sebum, and the increase of the content of the cholesterol and the brain amide can help the skin to be soft and moist, thereby having the function of conditioning; in addition, it also has effects of activating glutathione reductase, and has effects of activating skin and resisting aging by combining with its oxidation resistance. The rice is preferably used in an amount of 0.5% to 3% in embodiments of the present disclosure, i.e., the rice is used in an amount of 0.5% to 3% by weight of water (e.g., 0.8%, 1.0%, 1.5%, 2.0%, 2.5%, etc.), and may be rice particles or rice flour. If the dosage ratio exceeds 3%, the system becomes too viscous after rice and water are sterilized, oxygen supply is insufficient, and microbial fermentation is not facilitated; if the dosage ratio is less than 0.5%, the fermentation can still be smoothly carried out, but the system is too dilute, and the production efficiency is lower.

The radix ophiopogonis root is adopted in the method, is rich in polyfructose active ingredients, has strong drought resistance, prevents excessive water loss by strengthening adhesion of the upper layer of epidermis and recombining the barrier function of skin, reduces disintegration of the horny layer and achieves the moisturizing effect. The radix ophiopogonis root used in the embodiment of the disclosure is a dry product, and is preferably used after being crushed; preferably, the amount is 0.01-1%, i.e. the amount of said ophiopogon root in the fermentation substrate is 0.01-1% (e.g. 0.02%, 0.03%, 0.04%, etc.) of the weight of water.

The ligusticum chuanxiong hort root adopted in the disclosure can inhibit the generation of free radicals, effectively eliminate oxygen free radicals, effectively inhibit the activity of melanocytes at a proper concentration, reduce the generation of melanin, and has the effects of absorbing moisture and preserving moisture. The rhizoma ligustici wallichii root used in the embodiment of the disclosure is a dry product, and is preferably used after being crushed; preferably, the amount is 0.01% to 0.05%, i.e., the amount of the chuanxiong rhizome root in the fermentation substrate is 0.01% to 0.05% (e.g., 0.02%, 0.03%, 0.04%, etc.) by weight of water.

The holy basil leaves adopted in the method have broad-spectrum and strong bacteriostatic effects and obvious effect on controlling pruritus, and can be used as an antiallergic agent. The holy basil leaves used in the embodiments of the present disclosure are dry products, preferably used after being pulverized; preferably, the amount is 0.01% to 0.05%, i.e., 0.01% to 0.05% (e.g., 0.02%, 0.03%, 0.04%, etc.) of the holy basil leaves by weight of water in the fermentation substrate. The use amount of the holy basil leaves is too low to reflect the corresponding effect, and the fermentation liquor obtained by using too high use amount has darker overall color, is hyperpigmented on the skin and is easy to cause skin anaphylactic reaction.

The fermentation substrate is prepared by adding rice, radix ophiopogonis, ligusticum chuanxiong hort and holy basil leaves into water and then sterilizing.

The fermentation bacteria adopted in the method are ganoderma lucidum, secondary metabolites of the ganoderma lucidum are more than 430, and the ganoderma lucidum mainly contains active ingredients such as ganoderma lucidum polysaccharide, triterpenoids, proteins and the like, and has high medicinal value and pharmacological action such as immunoregulation, antivirus, anti-tumor, blood fat reduction and the like; the active ingredients such as ganoderan, terpenoid and phenols in Ganoderma have antioxidant and free radical scavenging effects; the ganoderma lucidum can also inhibit the growth of helicobacter pylori, escherichia coli, staphylococcus aureus and other bacteria, wherein ganoderma lucidum polysaccharide plays a vital role; the Ganoderma extract has effects of promoting the generation of collagen and ceramide, enhancing skin cell metabolism, resisting wrinkle, and resisting aging; has activating effect on luciferase and shows anti-inflammatory effect; also has inhibitory effect on tyrosinase, and can whiten skin, and has good whitening effect by combining with its moisture-retaining ability. The applicant finds out through screening experiments that the Ganoderma lucidum liquid culture is carried out on the raw material formula disclosed by the invention, the Ganoderma lucidum is preferably adopted as Ganoderma lucidum (Ganoderma lucidum) strain wG055, and the preservation number is CGMCC No. 17789.

Preferably, the volume percentage of the mycelium in the ganoderma lucidum liquid for inoculating to the fermentation substrate is more than 80% (such as 85%, 95%, 100% and the like); the inoculation ratio of the ganoderma lucidum, namely the volume ratio of the bacterium liquid to the fermentation substrate is 1-10% (such as 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 9.5% and the like).

Further, the preparation method of the bacterial liquid of the Ganoderma lucidum (Ganoderma lucidum) strain wG055 sequentially comprises the following steps:

and (3) activation: inoculating strain (Ganoderma strain with preservation number of CGMCC No. 17789) to glucose potato agar culture medium, activating at 23-28 deg.C for 3-7 days;

liquid culture: inoculating the single colony 2-3 rings obtained in the activation step into 100mL of glucose potato liquid culture medium for culture at the temperature of 23-28 ℃ for 3-7d at the rotation speed of 160-200rpm to obtain the bacterial liquid of the Ganoderma lucidum (Ganoderma lucidum) strain wG055 for inoculation.

In the above method for preparing a moisturizing and soothing ganoderma lucidum composite fermentation product, as a preferred embodiment, the fermentation culture temperature is 25-30 ℃ (for example, 25.5 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 29.5 ℃ and the like) and the time is 3-7d (for example, 3.5 days, 4 days, 5 days, 6 days, 6.5 days and the like), and stirring treatment is performed during the fermentation culture process; more preferably, the rotation speed of the stirring treatment is 100-200rpm (such as 110rpm, 120rpm, 130rpm, 140rpm, 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, etc.).

In the above method for preparing the moisturizing and soothing ganoderma lucidum composite fermentation product, as a preferred embodiment, the temperature of the sterilization treatment after the fermentation culture is 90-121 ℃ (such as 90 ℃, 100 ℃, 105 ℃, 108 ℃, 110 ℃, 115 ℃, 118 ℃, 120 ℃ and the like), and the time is 15-60min (such as 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min and the like).

In the preparation method of the moisturizing and relieving ganoderma lucidum composite fermentation product, as a preferred embodiment, before the sterilization treatment, the separation treatment is further included, the precipitate is discarded, the supernatant is taken for the sterilization treatment, and finally the ganoderma lucidum composite fermentation product, namely the ganoderma lucidum composite fermentation raw stock, is obtained.

In the preparation method of the moisturizing and soothing bacteria compound fermentation product, as a preferred embodiment, the preparation method further comprises the steps of separating after the sterilization treatment, discarding the precipitate, taking the supernatant, and finally obtaining the ganoderma lucidum compound fermentation product, namely the ganoderma lucidum compound fermentation raw stock.

In the preparation method of the moisturizing and soothing ganoderma lucidum composite fermentation product, as a preferred embodiment, the separation treatment is centrifugal treatment; further preferably, the speed of the centrifugation treatment is 3500-8000r/min (such as 4000r/min, 4500r/min, 5000r/min, 5500r/min, 6500r/min, 7000r/min, 7500r/min, etc.), and the time is 20-60min (such as 25min, 30min, 40min, 50min, 55min, etc.).

In the preparation method of the moisturizing and soothing ganoderma lucidum composite fermented product, as a preferred embodiment, the preparation method further comprises drying treatment after the sterilization treatment, and finally obtaining the ganoderma lucidum composite fermented product, namely ganoderma lucidum composite fermented dry powder.

In the preparation method of the moisturizing and soothing ganoderma lucidum composite fermentation product, as a preferred embodiment, the drying treatment can be spray drying, vacuum freeze drying and the like.

According to still another aspect of the disclosure, the ganoderma lucidum composite fermented dry powder prepared by the preparation method is also provided.

According to another aspect of the disclosure, the ganoderma lucidum composite fermentation protoplasm prepared by the preparation method is also provided.

According to another aspect of the disclosure, the application of the ganoderma lucidum composite fermented dry powder in the aspect of preparing cosmetics is further provided.

According to another aspect of the disclosure, the application of the ganoderma lucidum composite fermentation protoplasm in the aspect of serving as or preparing cosmetics is further provided.

The cosmetic can be facial mask, essence or toner.

According to the technical scheme of the embodiment of the disclosure, the rice, the radix ophiopogonis, the ligusticum chuanxiong hort and the holy basil leaves are subjected to composite liquid fermentation by selecting the suitable ganoderma lucidum strains to prepare the novel ganoderma lucidum composite fermentation raw pulp, so that the novel ganoderma lucidum composite fermentation raw pulp has good moisturizing and relieving effects, and can be used as a good raw material of a moisturizing and relieving efficacy cosmetic or directly used as a cosmetic.

The preservation date of the ganoderma lucidum strain used in the disclosure is 6 months and 5 days in 2019, the preservation number is CGMCC No.17789, and the classification and the name are as follows: ganoderma (Ganoderma lucidum) strain wG055, the name of the preservation unit is China general microbiological culture Collection center (CGMCC for short), the address is: the western road No.1 Hospital No. 3, Kyoho, Beijing, is assigned a zip code of 100101.

These and other advantages of the present disclosure will become more apparent from the following detailed description of the preferred embodiments of the present disclosure when taken in conjunction with the accompanying drawings.

Drawings

The disclosure may be better understood by reference to the following description taken in conjunction with the accompanying drawings. The accompanying drawings, which are incorporated in and form a part of this specification, illustrate preferred embodiments of the present disclosure and, together with the detailed description, serve to explain the principles and advantages of the disclosure. Wherein:

fig. 1 shows the results of skin moisture content (abbreviated as "moisture content") tests performed on 10 vol% aqueous solution (sample # 2) and 0.1 vol% hyaluronic acid aqueous solution (0.1 vol% HA) prepared from the moisturizing and soothing ganoderma lucidum composite fermentation raw stock prepared in example 1, which are graphs showing the trend of the change in percentage of moisture content before use at different time points and in various areas after use;

fig. 2 shows the results of skin moisture content (abbreviated as "water content") tests performed on 10 vol% aqueous solution (sample # 2) and 0.1 vol% hyaluronic acid aqueous solution (0.1 vol% HA) prepared from the moisturizing and soothing ganoderma lucidum composite fermentation raw stock prepared in example 1, which are median variation trends of moisture content in different areas at different time points;

FIG. 3 is a graph showing the variation of the TEWL value at different time points and in different areas after use, showing the variation trend of the TEWL value before use, when 10 vol% of the aqueous solution (sample No. 2) prepared from the moisturizing and soothing Ganoderma lucidum composite fermentation raw stock prepared in example 1 and 0.1 vol% of hyaluronic acid aqueous solution (0.1% HA) are used for measuring the skin water loss (namely, the skin water loss, abbreviated as water dispersion);

fig. 4 shows the results of skin water loss (i.e. percutaneous water loss, abbreviated as "water dispersion") tests performed on 10 vol% aqueous solution (sample # 2) and 0.1 vol% hyaluronic acid aqueous solution (0.1 vol% HA) prepared from the moisturizing and soothing ganoderma lucidum composite fermentation raw stock prepared in example 1, which are median trend of TEWL values in different regions at different time points.

Detailed Description

Exemplary embodiments of the present disclosure will be described hereinafter with reference to the accompanying drawings.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The rice in the following examples is commercially available, specifically northeast wuchang rice with a floral aroma (brand: golden dragon fish).

The roots of dwarf lilyturf, roots of chuanxiong rhizome and leaves of holy basil in the following examples are dry products produced by agriculture and sideline products purchasing and selling limited of Hoodian city, Bozhou, Anhui province.

The fermentation bacteria in the following embodiments are Ganoderma lucidum, specifically Ganoderma lucidum (Ganoderma lucidum) strain wG055 with preservation number of CGMCC No.17789, and are obtained from biotransformation laboratory of chemical and material engineering college of Beijing university.

Example 1 preparation of moisturizing and soothing Ganoderma lucidum composite fermentation puree

1. Preparing ganoderma lucidum liquid: inoculating the ganoderma lucidum strain with the preservation number of CGMCC No.17789 to a glucose potato agar culture medium, culturing at 28 ℃ for 7d, activating, then inoculating the obtained single colony to 100mL of glucose potato liquid culture medium, and culturing at 28 ℃ and 180rpm for 7d to obtain ganoderma lucidum liquid, wherein the volume of the mycelium accounts for 80% of the volume of the ganoderma lucidum liquid.

2. Preparing a fermentation substrate: adding radix Ophiopogonis 0.20g, rhizoma Ligustici Chuanxiong 0.10g, folium Ocimi Pilosi 0.25g, and rice 5g into 500g water, and sterilizing at 121 deg.C for 20min to obtain fermentation substrate; wherein, the rice accounts for 1 percent, the ophiopogon root accounts for 0.04 percent, the chuanxiong rhizome accounts for 0.02 percent and the holy basil leaf accounts for 0.05 percent.

3. Obtaining ganoderma lucidum composite fermentation raw pulp: inoculating 15mL of the ganoderma lucidum liquid obtained in the step 1 into 500g of fermentation substrate (about 500mL) to obtain a fermentation system; fermenting the fermentation system in an incubator at 30 ℃ at the rotating speed of 200rpm for 6 days to obtain a fermentation product; centrifuging the fermentation product at 5000r/min for 30min, removing precipitate, collecting supernatant, sterilizing at 121 deg.C for 20min to inactivate bacteria, and obtaining sterilized fermentation product, i.e. rice Ganoderma composite fermentation raw stock.

The appearance of the ganoderma lucidum composite fermentation raw stock prepared in the embodiment 1 is semitransparent liquid, the color is light yellow to light brown, the pH value is 4.1-6.3, the ganoderma lucidum composite fermentation raw stock is free of viscosity, the content of soluble solid is 1.5-5.0%, the total number of colonies is less than 50CFU/ml, and no pathogenic bacteria are detected. According to the cosmetic hygiene standard GB7916-87, the total number of bacteria in the cosmetic is not higher than 1000CFU/ml, so that the fermentation product meets the requirement of cosmetic quality.

Performing component analysis on the ganoderma lucidum composite fermentation raw stock, wherein a protein detection method refers to GB5009.5-2010, a crude polysaccharide detection method refers to GB/T5009.8-2008, a flavone detection method refers to GB/T5009.124-2003, and a total phenol detection method refers to GB/T8313-2008; the results obtained were as follows: the composite fermentation raw pulp prepared in the embodiment 1 contains 1.105g/kg of protein, 7.045g/kg of crude polysaccharide, 0.029g/kg of total flavone (counted by rutin) and 0.030g/kg of total phenol.

Example 2 application of moisturizing and soothing ganoderma lucidum composite fermentation raw pulp as cosmetic

Safety detection of moisturizing and relieving ganoderma lucidum composite fermentation raw stock

The human body patch test is mainly used for detecting the irritation of the final cosmetic product or raw materials. The present disclosure performed a closed patch test on the complex fermented raw stock obtained in example 1 according to the cosmetic hygiene code (2015) in order to evaluate its potential skin irritation.

1. Test subjects:

according to the requirements of 'cosmetic contact dermatitis diagnostic standard and treatment principle', the selected test subject cannot participate in the test, and the test subject and the person with high physique sensitivity who have scars, nevus flammeus and other influences on the result judgment at the part to be tested of the skin cannot participate in the test. Suitable volunteers 30 were selected as subjects in this trial, and were randomly selected in the age range of 18-60 years.

2. The test method comprises the following steps:

0.02mL to 0.025mL of a liquid sample (100% ganoderma lucidum composite fermentation protoplasm without dilution) is dripped on a filter paper sheet, and then the filter paper sheet is placed in a spot tester. A blank control (water) was set for each sample and an equal amount of sample solvent distilled water was added to the control cuvette well. The test period lasted 24 h. In order to ensure the accuracy, credibility and scientificity of test results, the volunteers cannot remove the spot tester or make the tested part contact water according to the requirements during the test. After 24h, the plaque tester is removed, and after standing for 30min (waiting for the indentation to disappear), the skin reaction is observed for 24h and 48 h. The grading standard of adverse skin reactions in the human patch test is shown in Table 1.

TABLE 1 grading Standard of adverse skin reactions

3. And (3) test results:

see table 2. As can be seen from the table: the rice ganoderma lucidum composite fermentation raw pulp obtained in the embodiment 1 is used for negative reactions, which shows that the rice ganoderma lucidum composite fermentation raw pulp provided by the invention has safety and does not bring adverse reactions to human bodies.

Table 2, patch test of rice-ganoderma lucidum composite fermentation raw stock obtained in example 1

Second, testing water content and water dispersion of moisturizing and relieving ganoderma lucidum composite fermentation raw pulp

The moisturizing effect of the ganoderma lucidum composite fermentation raw stock obtained in example 1 is tested, specifically, skin moisture content test and skin moisture loss test, Hyaluronic Acid (HA) is generally considered as a positive control sample with a good moisturizing effect, and a comparison experiment is performed by using 10% ganoderma lucidum composite fermentation raw stock (marked as sample # 2, namely, an aqueous solution with a volume concentration of 10% prepared from the composite fermentation raw stock prepared in example 1) and 0.1% hyaluronic acid (marked as 0.1% HA, namely, an aqueous solution with a volume concentration of 0.1% prepared from hyaluronic acid).

1. And (3) testing environment: the temperature is 22 +/-1 ℃; humidity is 50-60%.

2. Test area: skin moisture content test, skin moisture loss test: the left and right forearms.

3. Testing time points: skin moisture content test, skin moisture loss test: before use, 5min, 20min, and 60min after use.

4. An experimental instrument: cornemeter, CM825, Tewameter, TM 300.

5. The test method comprises the following steps:

1)30 eligible volunteers participated in the test. The test place has no direct light and no wind, the room temperature is 22-24 ℃, and the humidity is 40-60%. Before detection, cleaning forearms of both sides with facial cleanser, standing for 30min, taking inner side of forearms of the subject, and drawing normal skin with area of 3.5 × 3.5cm with marking pen. The skin moisture content and the amount of skin moisture loss before use were measured in this order.

2) Cutting the facial mask soaked with the sample to be tested (2# sample, 0.1% HA) into 3 × 3cm size, respectively, sticking on the corresponding mark of forearm, taking down after 15min, lightly soaking the un-dried essence solution of the test part with cosmetic cotton, and timing.

3) The water content and TEWL value of stratum corneum of 3 parts are tested at 5min, 20min and 60min respectively. Each site measurement was averaged 3 times.

6. And (3) testing results:

FIG. 1 is a graph showing the trend of the percentage change of the water content of the sample No. 2 and 0.1% HA, and FIG. 2 is a graph showing the median change of the water content of the sample No. 2 and 0.1% HA. As can be seen from the figure, both the sample # 2 and 0.1% HA reached a maximum in median water content 5min after use; as the time after use was extended, the percent change in water content of 0.1% HA was below 0 after 20min and the percent change in water content of the 2# sample was as high as 10%, indicating that the water content at this time point after use of 0.1% HA was below the background data and the water content at this time point after use of the 2# sample was still above the background data; after 60min, the median of the water content of the 2# sample and 0.1% HA both continuously showed a decreasing trend, and the percentage of change of the water content was lower than 0, which indicates that the skin water content was lower than the background data after 60min after use; as can be seen, the sample No. 2 shows excellent water replenishing and moisturizing performances within 20min after use.

Fig. 3 is a graph showing the variation of percentage change in transdermal water loss of the sample # 2 and 0.1% HA, and fig. 4 is a graph showing the variation of median transdermal water loss of the sample # 2 and 0.1% HA. As can be seen from the figure, the change law of the percutaneous water loss of the sample No. 2 and the 0.1% HA is relatively similar, and both the sample No. 2 and the sample No. 0.1% HA reach the maximum 5min after use because the percutaneous water loss is also relatively large due to relatively large water content; the transdermal water loss gradually decreased with time after use and rebounded 60min after use, and the TEWL change percentage at the time point was all greater than 0, indicating that the TEWL at the time point was above the skin background due to no further application of water-locking components (such as oil).

Therefore, the ganoderma lucidum composite fermentation raw pulp prepared in the embodiment 1 has good water replenishing and moisture preserving performances.

Third, in vitro antioxidant free radical scavenging performance test

1. Experimental procedure

Preparing ABTS working solution: 5mL of a 7mmol/L aqueous ABTS solution and 88. mu.L of a 140mmol/L potassium persulfate solution were mixed and allowed to stand overnight at room temperature in the absence of light to form an ABTS + stock solution. Before use, the solution is diluted into a working solution by absolute ethyl alcohol, and the absorbance of the working solution at 734nm is 0.70 +/-0.02.

And (3) sample determination: adding 40 μ L of the solution to be detected into 4mL of ABTS working solution, accurately oscillating for 30s, and measuring the absorbance A at 734nm wavelength after 6min reaction_{Sample (I)}. The clearance was calculated as follows:

ABTS clearance/% (1-A)_{Sample (I)}/0.700)×100。

2. Results of the experiment

The results of the antioxidant scavenging free radical test are shown in table 3. The sample solution to be tested is a ganoderma lucidum composite fermentation raw stock aqueous solution prepared in advance from the ganoderma lucidum composite fermentation raw stock prepared in the embodiment 1 and having the volume concentration of 50% and 100%, and a supernatant (serving as a sample before fermentation) obtained after sterilizing the substrate in the same proportion. As can be seen from Table 3, the Ganoderma lucidum composite fermentation broth prepared in example 1 has a certain antioxidant property.

TABLE 3 antioxidant scavenging free radical test results

Fourth, tyrosinase inhibition assay

1. Experimental methods

Configuration: 0.1M HCl; PBS solution (pH 6.8, 0.1 mol/L); l-tyrosine solution (0.05g dissolved in 35ml of 0.1mol/L HCl, and the volume is 100ml by PBS (0.1mol/L) buffer solution with the pH value of 6.8); sample solution (namely supernatant liquid after sterilizing the substrate in the same proportion but not inoculating fermentation, and ganoderma lucidum composite fermentation raw stock prepared in example 1).

The biochemical reaction was carried out in glass test tubes, and the required PBS buffer (pH 6.8, 0.1mol/L), sample solution, L-tyrosine solution were added to each tube according to the data in Table 4 below. Reacting in 37 deg.C water bath for 10min, adding tyrosinase (with enzyme activity of 100U/mL), reacting in 37 deg.C water bath for 10min, and measuring absorbance at 475 nm.

TABLE 4

Reagent	C₁(ml)	C₀(ml)	T₁(ml)	T₀(ml)
					PBS	2	2.5	1	1.5
Sample (I)	0	0	1	1
					L-tyrosine	1	1	1	1
Tyrosinase enzyme	0.5	0	0.5	0

The formula for calculating the tyrosinase inhibition rate is as follows:

IR(％)＝[(C₁-C₀)-(T₁-T₀)]/(C₁-C₀)×100％

in the formula: IR-sample to OH. clearance; c₁-absorbance value of blank control; c₀-blank absorbance values without tyrosinase; t is₁-sample set absorbance values; t is₀-absorbance values of sample sets without tyrosinase.

2. Results of the experiment

The tyrosinase inhibition rate of the supernatant fluid which is obtained after the sterilization of the substrates in the same proportion but is not inoculated for fermentation is 10.47% + -1.14%, the tyrosinase inhibition rate of the composite fermentation raw stock prepared in the example 1 is 41.45% + -2.15%, while the vitamin C is adopted as a control, the tyrosinase inhibition rate of 0.1mg/mL vitamin C is 58.82%, the tyrosinase inhibition rate of 0.3mg/mL vitamin C is 64.71%, and the tyrosinase inhibition rate of 1.2mg/mL vitamin C is 70.59%; the tyrosinase inhibition capability of the ganoderma lucidum composite fermentation raw stock prepared in the embodiment 1 is excellent, and the whitening effect is good.

Cell MTT proliferation assay

Human skin fibroblasts are the main structural components constituting the dermis of the skin, can synthesize and secrete extracellular matrixes such as collagen fibers, elastic fibers, reticular fibers, hyaluronic acid and the like, have important effects on maintaining the strength and elasticity of the skin, repairing injuries and beautifying the skin, are the determining factors for maintaining the young state of the skin and are also important components for maintaining the stable structure of the skin. Human skin fibroblasts (HDF-n, from ScienCell) used in this experiment were tested for toxicity to cells.

1. The experimental steps are as follows:

cells were seeded in 96-well plates overnightPerforming synchronization treatment, and then performing incubation culture for 24h by using a solution to be tested (namely, a supernatant which is not fermented after the substrate in the same proportion is sterilized and the ganoderma lucidum composite fermentation raw stock prepared in the embodiment 1 are respectively diluted into a solution with a volume concentration of 5% by using a cell culture solution DMEM); after the culture is finished, replacing a new culture medium, counting MTT0.5g/L, removing the culture medium after 4h, washing the culture medium for three times by PBS, and adding 400 mu L DMSO to fully crack cells; OD was measured at 490nm using a microplate reader. Each experiment was repeated three times. The survival rate of the cells in the control group was 1, and the survival rate of the cells in the other groups was OD490_{Experimental group}/OD490_{Control group}。

2. Results of the experiment

The experimental results are shown in Table 5, and it can be seen from the table that the cell survival rate of the 5% concentration group after fermentation is higher than that of the blank control, which indicates that the sample after fermentation has the effect of promoting proliferation of cells.

TABLE 5

Experiment grouping	Blank control		5% concentration before fermentation	5% concentration after fermentation
					Cell survival rate	1	0.57±0.02	1.83±0.07

Experiment on inhibition of Hyaluronidase

The hyaluronidase is a participant of type I anaphylactic reaction, the hyaluronidase has strong correlation with inflammation and allergy, researches report that various medicaments for releasing histamine from mast cells can regulate the activity of the hyaluronidase, and some anti-allergy medicaments have strong inhibition on the activity of the hyaluronidase. The inhibition of the activity of the hyaluronic acid of the substance can be detected through a hyaluronidase inhibition experiment, so that the anti-allergic and anti-inflammatory properties of the substance can be reflected. This experiment separately tested the hyaluronidase inhibition effect of the ganoderma lucidum composite fermentation raw stock prepared in example 1 and the supernatant that has not been fermented after sterilization of the substrate in the same proportion.

1. Experimental methods

The experiments were carried out according to the experimental procedure of table 6 below.

TABLE 6 Hyaluronidase inhibition assay procedures and reagents

The hyaluronidase inhibition was calculated according to the following formula:

hyaluronidase inhibition (%) - (C-D) - (A-B)/(C-D) × 100%

In the formula: OD value of a — (hyaluronidase + sample + sodium hyaluronate) test sample solution;

OD value of blank sample B (acetic acid buffer solution + sample + acetic acid buffer solution);

OD value of C- (hyaluronidase + deionized water + sodium hyaluronate) control solution;

d- (acetic acid buffer solution + deionized water + acetic acid buffer solution) controls the blank OD value;

2. test results

The test results are shown in table 7. From table 7, it can be seen that the hyaluronidase inhibition ability of the fermented sample reaches 47.2 ± 0.59%, which is significantly higher than that of the sample before fermentation, and the sample has good anti-inflammatory and anti-allergic effects and good skin-soothing effects.

TABLE 7

Test set	100% concentration before fermentation	100% concentration after fermentation
			Hyaluronidase inhibition%	8.14±1.74	47.2±0.59

Comparative example 1 Strain screening

1. Preparation of Rice fermentation broth (samples 1-6)

Inoculating candidate No. 1-6 Ganoderma strains with 1% rice as substrate, culturing at 28 deg.C for 5 days, and centrifuging to obtain supernatant. Test after sterilization and addition of preservative. Among the candidate No. 1-6 Ganoderma strains, No. 1-3 strains are collected from Chuzhou city of Anhui province, No. 4-5 strains are collected from Changbai mountain of Jilin province, No. 6 strains are laboratory-preserved strains, and No.1 strains are Ganoderma strains wG 055.

The specific method comprises the following steps:

1) preparing a seed solution: activating the six ganoderma lucidum strains by using a PDA (personal digital Assistant) plate, transferring single colonies into a potato dextrose water liquid culture medium for amplification culture under the culture condition of 28 ℃ and 180rpm for 5 d.

2) Preparation of samples: adding 3g of rice and 297g of water into a 500ml triangular flask, sterilizing at 121 ℃ for 15min, inoculating 3ml of seed solution, and culturing at 28 ℃ and 180rpm for 5 days; respectively obtaining No. 1-6 rice fermentation liquor, namely samples 1-6. The supernatant of the non-inoculated fermented rice was used as sample No. 0.

Evaluation of ABTS radical scavenging efficacy

Preparing ABTS working solution: 5mL of a 7mmol/L aqueous LABTS solution and 88. mu.L of a 140mmol/L potassium persulfate solution were mixed and left to stand overnight at room temperature in the absence of light to form an ABTS. + stock solution. Before use, the solution is diluted into a working solution by absolute ethyl alcohol, and the absorbance of the working solution at 734nm is 0.70 +/-0.02.

ABTS clearance/% (1-A)_{Sample (I)}/0.700)×100。

The results are shown in Table 8, from which it can be seen that sample 1 has the highest ABTS radical scavenging capacity compared to the other samples.

TABLE 8 ABTS test results for rice fermentation broths obtained from different Ganoderma species

Fermentation broth numbering	ABTS clearance%
		0	28.95±5.04
1	65.41±2.34
		2	60.47±4.25
3	56.24±7.45
		4	55.85±0.87
5	57.24±2.47
		6	48.69±5.24

3. Skin feel test

The evaluation was performed according to the evaluation criteria (Table A) and the specific scores are shown in Table B, and the overall score for sample 1 was relatively high by the overall analysis.

TABLE A skin feel test evaluation criteria

Skin feel test results for samples 0-6 in Table B

Comparative example 2

In this comparative example, the fermentation substrate used only rice, and the other conditions were the same as in example 1, to obtain a fermentation raw stock, which was designated as sample a, and which had a faint unpleasant odor after spreading.

Compared with the fermented raw pulp obtained in the example 1, the fermented raw pulp has faint scent of the Chinese herbal medicines after being smeared, the faint unpleasant smell is covered, and the radix ophiopogonis, the ligusticum chuanxiong hort root and the holy basil leaf are added, so that the fermented raw pulp is superior in efficacy and has better performance in smearing smoothness and absorption degree.

In summary, in the embodiments according to the present disclosure, the present disclosure provides the following technical solutions, but is not limited thereto:

scheme 1, a preparation method of a moisturizing and relieving ganoderma lucidum composite fermentation product is characterized by comprising the following steps: inoculating the ganoderma lucidum to a fermentation substrate consisting of rice, radix ophiopogonis, ligusticum chuanxiong hort roots, holy basil leaves and water for fermentation culture, and then performing sterilization treatment to obtain the ganoderma lucidum composite fermentation product.

Scheme 2 and the preparation method according to scheme 1 are characterized in that in the fermentation substrate, the rice accounts for 0.5-3% of water, the ophiopogon root accounts for 0.01-0.05% of water, the ligusticum chuanxiong hort root accounts for 0.01-0.05% of water, and the holy basil leaf accounts for 0.01-0.05% of water.

Scheme 3 and the preparation method according to scheme 2, wherein the radix ophiopogonis, the ligusticum chuanxiong hort and the holy basil leaves in the fermentation substrate are dry products.

Scheme 4, the preparation method according to any one of schemes 1 to 3, wherein the ligusticum chuanxiong hort root and the holy basil leaf are all subjected to crushing treatment and then used.

Scheme 5, according to scheme 1-4 any preparation method, characterized in that, the fermentation substrate before inoculation is sterilized.

Scheme 6, the preparation method according to any one of schemes 1 to 5, wherein the mycelium volume percentage of the ganoderma lucidum liquid for inoculation to the fermentation substrate is 80% or more; the volume ratio of the bacterial liquid to the fermentation substrate is 1-10%.

Scheme 7, the preparation method according to scheme 1-6, characterized in that the Ganoderma lucidum is Ganoderma lucidum (Ganoderma lucidum) strain wG055, with a collection number of CGMCC No. 17789.

Scheme 8 and the preparation method according to scheme 7 are characterized in that the preparation method of the bacterial liquid of the Ganoderma lucidum (ganoderam lucidum) strain wG055 sequentially comprises the following steps:

and (3) activation: inoculating the strain to glucose potato agar culture medium, activating at 23-28 deg.C for 3-7 days;

Scheme 9, the preparation method according to any of schemes 1-8, characterized in that the temperature of the fermentation culture is 25-30 ℃ and the time is 3-7d, and the stirring treatment is carried out during the fermentation culture.

Scheme 10, the preparation method according to scheme 9, characterized in that the rotation speed of the stirring treatment is 100-200 rpm.

Scheme 11, the preparation method according to any one of schemes 1-10, characterized in that, the temperature of the sterilization treatment after the fermentation culture is 90-121 ℃, the time is 15-60 min.

Scheme 12 and the preparation method according to any one of schemes 1 to 11, wherein before the sterilization treatment, the separation treatment is further included, the precipitate is discarded, and the supernatant is taken to be subjected to the sterilization treatment, so that the ganoderma lucidum composite fermentation raw stock is finally obtained.

Scheme 13 and the preparation method according to any one of schemes 1 to 11, wherein after the sterilization treatment, the separation treatment is further included, the precipitate is discarded, and the supernatant is taken to finally obtain the rice ganoderma lucidum composite fermentation raw stock.

The production method according to claim 14, 12 or 13, wherein the separation treatment is a centrifugation treatment.

Scheme 15, the preparation method according to scheme 14, characterized in that, the speed of the centrifugal treatment is 3500-8000r/min, and the time is 20-60 min.

Scheme 16 and the preparation method according to any one of schemes 12 to 15, wherein the preparation method further comprises a drying treatment after the sterilization treatment, and finally the ganoderma lucidum composite fermented dry powder is obtained.

The method according to claim 17 or 16, wherein the drying treatment is spray drying or vacuum freeze drying.

Scheme 18, a ganoderma lucidum composite fermented dry powder prepared by the preparation method of scheme 16.

Scheme 19, a ganoderma lucidum composite fermentation raw pulp prepared by the preparation method according to any one of schemes 12-15.

20. The use of the dry powder of the complex fermentation of ganoderma lucidum as claimed in claim 18 for the preparation of cosmetics.

21. The use of the ganoderma lucidum composite fermentation protoplasm of claim 19 as or in the preparation of cosmetics.

Finally, it is also noted that, in the present disclosure, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

While the disclosure has been disclosed above by the description of specific embodiments thereof, it should be understood that various modifications, improvements or equivalents of the disclosure may be devised by those skilled in the art within the spirit and scope of the appended claims. Such modifications, improvements and equivalents are intended to be included within the scope of the present disclosure as claimed.

Sequence listing

<110> Beijing university of Industrial and commercial

<120> moisturizing and relieving ganoderma lucidum composite fermentation product and preparation method and application thereof

<130> PD200212CN0281

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 646

<212> DNA

<213> Ganoderma lucidum (Ganoderma lucidum)

<400> 1

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gcttcagatt gcgaggcacg ctctttaccg ggcttgcgga gcatatctgt gcctgcgttt 180

atcacaaact ctataaagta acagaatgtg tattgcgatg taacacatct atatacaact 240

ttcagcaacg gatctcttgg ctctcgcatc gatgaagaac gcagcgaaat gcgataagta 300

atgtgaattg cagaattcag tgaatcatcg aatctttgaa cgcaccttgc gctccttggt 360

attccgagga gcatgcctgt ttgagtgtca tgaaatcttc aacctacaag cttttgtggt 420

ttgtaggctt ggacttggag gcttgtcggc cgttatcggt cggctcctct taaatgcatt 480

agcttggttc cttgcggatc ggctctcggt gtgataatgt ctacgccgtg accgtgaagc 540

gtttggcgag cttctaaccg tcttataaga cagctttatg acctctgacc tcaaatcagg 600

taggactacc cgctgaactt aagcatatca ataaagccgg aggaaa 646

Claims

1. A preparation method of a moisturizing and relieving ganoderma lucidum composite fermentation product is characterized by comprising the following steps: inoculating the ganoderma lucidum to a fermentation substrate consisting of rice, radix ophiopogonis, ligusticum chuanxiong hort roots, holy basil leaves and water for fermentation culture, and then performing sterilization treatment to obtain the ganoderma lucidum composite fermentation product.

2. The method according to claim 1, wherein the rice accounts for 0.5-3 wt% of water, the ophiopogon root accounts for 0.01-0.05 wt% of water, the ligusticum chuanxiong hort root accounts for 0.01-0.05 wt% of water, and the holy basil leaf accounts for 0.01-0.05 wt% of water in the fermentation substrate.

3. The method according to claim 2, wherein the radix Ophiopogonis, rhizoma Ligustici Chuanxiong, and folium Ocimi Pilosi are dried.

4. The method according to any one of claims 1 to 3, wherein the Ligusticum chuanxiong Hort root and Ocimum sanctum leaf are used after being crushed.

5. The method according to any one of claims 1 to 4, wherein the fermentation substrate is sterilized before inoculation.

6. The method according to any one of claims 1 to 5, wherein the mycelia of the Ganoderma lucidum solution used for inoculation to the fermentation substrate is 80% by volume or more; the volume ratio of the bacterial liquid to the fermentation substrate is 1-10%.

7. The preparation method according to claims 1 to 6, wherein the Ganoderma lucidum is Ganoderma lucidum (Ganoderma lucidum) strain wG055 with a collection number of CGMCC No. 17789.

8. The preparation method of claim 7, wherein the preparation method of the bacterial liquid of the Ganoderma lucidum (Ganoderma lucidum) strain wG055 sequentially comprises the following steps of:

9. The method according to any one of claims 1 to 8, wherein the temperature of the fermentation culture is 25 to 30 ℃ and the time is 3 to 7 days, and the stirring treatment is performed during the fermentation culture.

10. The method as claimed in claim 9, wherein the rotation speed of the stirring process is 100-200 rpm.