CN113208987A

CN113208987A - Anti-aging and repairing rice ganoderma lucidum composite fermentation product and preparation method and application thereof

Info

Publication number: CN113208987A
Application number: CN202010931288.XA
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 an anti-aging and repairing rice ganoderma lucidum composite fermentation product, which is characterized by comprising the following steps: inoculating the ganoderma lucidum into a fermentation substrate consisting of rice, witch hazel, gallnut and water for fermentation culture, and then performing sterilization treatment to obtain the rice-ganoderma lucidum composite fermentation product. The novel rice ganoderma lucidum composite fermentation raw pulp is prepared by selecting a proper ganoderma lucidum strain to carry out composite liquid fermentation on rice, hamamelis virginiana and gallnut, has good skin-activating and anti-aging effects, and can be used as a good raw material of skin-activating anti-aging cosmetics or directly used as cosmetics.

Description

Anti-aging and repairing rice 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 an anti-aging and repairing rice 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.

Hamamelis mollis Oliver (Hamamelis mollis Oliver), Lauraceae, Hamamelidaceae, Lawsonia deciduous shrub or small tree. There are various Hamamelis virginiana extracts, and in the cosmetic field, Hamamelis virginiana extracts are most well known, and extracts can be prepared from various parts and used as cosmetic raw materials. The witch hazel extract has high cosmetic value: the skin care product contains special soft and sensitive factors, can relieve unstable skin, keep metabolic balance and eliminate discomfort of the skin; the skin care product has excellent astringency, can control the secretion of grease, help to regulate the water-oil balance of skin, effectively improve the water content of skin, balance the pH value and shrink pores; has antibacterial effect and special therapeutic effect on fungal infection; the Hamamelis virginiana extract can obviously reduce the content of malondialdehyde in tissues caused by strong ultraviolet radiation, prevent skin inflammation caused by exposure to ultraviolet rays, reduce free radical damage caused by ultraviolet radiation, and has anti-aging effect.

Gallnut (Galla chinensis), a traditional Chinese medicine name, is Rhus chinensis Mill of plants of the genus Rhus of the family Anacardiaceae, celastrus orientalis (Rhus potaninii Maxim.), or Rhus chinensis (Rhus punjabensis step. var. sinica (Diels) rehd. et Wils.) leaves gallnut mainly parasitized by aphid (melaphilis chinensis (Bell) Baker); picking in autumn, boiling in boiling water or steaming until the surface is gray, killing aphid, taking out, and drying. The main effective component of the gallnut, namely the gallnut tannin, has obvious biological activities of preventing decayed teeth, inhibiting bacteria and killing bacteria, killing sperm, inhibiting mutation, removing free radicals, resisting oxidation and the like. The gallnut extract has the functions of nourishing and moistening skin, protecting skin, diminishing inflammation and inhibiting bacteria, whitening skin, nourishing and blackening hair, preventing corrosion and oxidation, perfuming and toning.

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 witch hazel, gallnut, rice and lucid ganoderma are applied to the field of cosmetics, the witch hazel, the gallnut, the rice and the lucid ganoderma are basically compounded with various raw materials, the extraction modes of active substances are various, and the problem of improving the extraction effect of the active substances by selecting more potential material combinations is still a problem 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 defects in the prior art, the purpose of the present disclosure is to provide an anti-aging and repairing rice ganoderma lucidum composite fermentation product, a preparation method and an application thereof, which have good moisturizing and whitening functions.

According to one aspect of the disclosure, a preparation method of an anti-aging and repairing rice ganoderma lucidum composite fermentation product is provided, which comprises the following steps: inoculating the ganoderma lucidum into a fermentation substrate consisting of rice, witch hazel, gallnut and water for fermentation culture, and then performing sterilization treatment to obtain the rice-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. In embodiments of the present disclosure, the amount is preferably 0.5-3%, i.e., the amount of rice is 0.5-3% by weight of water (e.g., 0.8%, 1%, 1.5%, 2%, 2.5%, 2.8%, 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 witch hazel extract used in the present disclosure can be used as an oxidant, can activate cell aromatase and cathepsin to increase the local estrogen level of the cortex, enhance the metabolism of skin cells, have an anti-aging effect, can maintain the elasticity and wrinkle resistance of the skin, have anti-inflammatory, allergy-relieving and astringent effects, and also have good water retention capacity. Dried flowers of witch hazel are preferably used in the embodiments of the present disclosure in an amount of 0.01-0.05%, i.e., witch hazel is used in an amount of 0.01-0.05% by weight of water (e.g., 0.02%, 0.03%, 0.04%, etc.). The proper effect cannot be reflected when the dosage ratio of the witch hazel is too low, and the whole color of the fermentation liquor obtained when the dosage ratio is too high is darker, the pigment is deposited on the skin and the skin anaphylactic reaction is easily caused. More preferably, dried flowers of Hamamelis virginiana are used.

The Chinese gall is adopted in the preparation, and the extract of the Chinese gall has the effects of nourishing and moistening skin, protecting skin, diminishing inflammation and inhibiting bacteria, whitening skin, preventing corrosion and resisting oxidation. The gallnut is preferably used after being crushed in the embodiment of the disclosure; preferably, the amount is 0.01-0.05%, i.e. 0.01-0.05% (e.g. 0.02%, 0.03%, 0.04%, etc.) of Galla rhois based on the weight of water. The traditional Chinese medicine cannot reflect the existing efficacy when the dosage of the gallnut is too low, and the fermentation liquor obtained when the dosage is too high 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, witch hazel and gallnut into water and then sterilizing.

The fermentation bacteria adopted in the method are ganoderma lucidum, secondary metabolites in 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; ganoderma can also inhibit the growth of helicobacter pylori, Escherichia coli, Staphylococcus aureus, etc., wherein ganoderan plays an important 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; in addition, the whitening cream has an inhibiting effect on tyrosinase, can whiten skin, and has an obvious whitening effect by combining the moisturizing capability of the skin. 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, preferably, the Ganoderma lucidum (Ganoderma lucidum) strain wG055 with the preservation number of CGMCC No.17789 is adopted.

Preferably, the volume percentage of the mycelium in the ganoderma lucidum liquid for inoculating to the fermentation substrate is more than 80%; 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 the strain to a culture medium of glucose potato agar for activation, wherein the preservation number of the strain is CGMCC No.17789, and the activation temperature is 23-28 ℃, and the activation time is 3-7 d;

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 ℃, the activation time of 3-7d and 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 anti-aging and repairing rice ganoderma lucidum composite fermentation product, as a preferred embodiment, the temperature of the fermentation culture is 25-30 ℃ (such as 25.5 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 29.5 ℃ and the like) and the time is 3-7d (such as 3.5 days, 4 days, 5 days, 6 days, 6.5 days and the like), and the stirring treatment is carried out during the fermentation culture; 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 anti-aging and repairing rice-ganoderma lucidum composite fermentation product, as a preferred embodiment, the temperature of the sterilization treatment after the fermentation culture is 90-121 ℃ (such as 95 ℃, 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 anti-aging and repairing rice ganoderma lucidum composite fermentation product, as a preferred embodiment, the preparation method further comprises the steps of separating and processing before the sterilization processing, discarding the precipitate, taking the supernatant to perform the sterilization processing, and finally obtaining the rice ganoderma lucidum composite fermentation product, namely the rice ganoderma lucidum composite fermentation raw stock.

In the preparation method of the anti-aging and repairing rice ganoderma lucidum composite fermentation product, as a preferred embodiment, the method further comprises the steps of separation treatment after the sterilization treatment, discarding the precipitate, taking the supernatant, and finally obtaining the rice ganoderma lucidum composite fermentation product, namely the rice ganoderma lucidum composite fermentation raw stock.

In the preparation method of the anti-aging and repairing rice 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 anti-aging and repairing rice ganoderma lucidum composite fermentation product, as a preferred embodiment, the preparation method further comprises drying treatment after the sterilization treatment, and finally the rice ganoderma lucidum composite fermentation product, namely the rice ganoderma lucidum composite fermentation dry powder is obtained.

In the preparation method of the anti-aging and repairing rice 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 rice ganoderma lucidum composite fermentation dry powder prepared by the preparation method is also provided.

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

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

According to another aspect of the disclosure, the application of the rice ganoderma lucidum composite fermentation protoplasm in the aspect of 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 hamamelis virginiana and the gallnut are subjected to composite liquid fermentation by selecting the suitable ganoderma lucidum strain to prepare the novel rice ganoderma lucidum composite fermentation raw pulp, which has good skin-activating and anti-aging effects and can be used as an excellent raw material of skin-activating anti-aging cosmetics or directly used as the cosmetics.

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 "water content") tests performed on a 10 volume percent aqueous solution (10% sample, i.e., 7# sample) and a 0.1 volume percent hyaluronic acid aqueous solution (0.1% HA) prepared from the rice ganoderma lucidum composite fermentation puree prepared in example 1, which are graphs showing the percentage change trend of the moisture content before use in various regions and at various time points after use;

FIG. 2 is a graph showing the median change trend of moisture content in different regions at different time points, which is the result of skin moisture content (abbreviated as "water content") test using the rice Ganoderma lucidum composite fermentation raw stock prepared in example 1 to prepare 10% by volume of aqueous solution (10% sample, i.e. 7# sample) and 0.1% by volume of hyaluronic acid aqueous solution (0.1% HA);

FIG. 3 is a graph showing the variation of the TEWL value at different time points and before use in different areas after use, which is the result of the skin water loss (i.e. percutaneous water loss, abbreviated as "water dispersion") test using the rice Ganoderma lucidum composite fermentation slurry prepared in example 1 to prepare 10% by volume of aqueous solution (10% sample, i.e. 7# sample) and 0.1% by volume of hyaluronic acid aqueous solution (0.1% HA);

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 (10% sample, i.e. sample # 7) and 0.1 vol% hyaluronic acid aqueous solution (0.1% HA) prepared from the rice ganoderma lucidum composite fermentation puree 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).

Galla chinensis in the following examples was purchased from agriculture and sideline products purchasing Co., Ltd, Hoodia, Anhui province, and it was used as a sheet after pulverizing.

The witch hazel in the following examples is dry witch hazel flower produced by Hainan farm and sideline products purchasing Limited, Bozhou city, 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 anti-aging and repairing Rice Ganoderma Compound fermentation protoplasm

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 rice 5g, Hamamelis virginiana 0.25g, and Galla chinensis 0.25g into 500g water, and sterilizing at 115 deg.C for 20min to obtain fermentation substrate; wherein the rice accounts for 1%, the Hamamelis virginiana accounts for 0.05%, and the Galla chinensis accounts for 0.05%.

3. Obtaining rice 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 rice ganoderma lucidum composite fermentation raw stock prepared in the embodiment 1 is transparent or semitransparent yellowish or yellow liquid in appearance, has the pH value of 4.1-6.3, has no viscosity, has the soluble solid content of 1.5-5.0 percent, has the total colony count of less than 50CFU/ml, and has no pathogenic bacteria for detection. 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 fermented extract meets the quality requirement of the cosmetic.

Performing component analysis on the rice 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 compound fermentation raw stock prepared in the embodiment 1 contains 0.985g/kg of protein, 6.085g/kg of crude polysaccharide, 0.0315g/kg of total flavone (counted by rutin) and 0.0401g/kg of total phenol.

Example 2 application of anti-aging and repairing rice ganoderma lucidum composite fermentation raw stock as cosmetic, safety detection of rice 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 closed patch test of human body was performed on the rice composite fermentation raw pulp obtained in example 1 according to the cosmetic hygiene norm (2015) in order to evaluate the potential skin irritation thereof.

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% rice 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 rice ganoderma lucidum composite fermentation raw pulp

The moisturizing effect of the rice ganoderma lucidum composite fermentation raw stock obtained in example 1 is tested, specifically, a skin moisture content test and a skin moisture loss test, Hyaluronic Acid (HA) is generally regarded as a positive control sample with a good moisturizing effect, and a comparison experiment is performed by using 10% of rice ganoderma lucidum composite fermentation raw stock (marked as sample # 7, namely, an aqueous solution with a volume concentration of 10% prepared from the composite fermentation raw stock prepared in example 1) and 0.1% of hyaluronic acid (marked as 0.1% of 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 (7 # 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 on 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 7# sample and 0.1% HA, and FIG. 2 is a graph showing the median change of the water content of the 7# sample and 0.1% HA. As can be seen from the graph, the median water content of 5min after use of both the 7# sample and 0.1% HA reached the maximum, and the median water content of both the 7# sample and 0.1% HA showed a decreasing trend as the time after use was extended; after 20min, the percent change in water content of 0.1% HA was below 0 and the percent change in water content of the 7# sample was above 0, indicating that the water content at this time point after 0.1% HA was below the background data and the water content at this time point after the 7# sample was still above the background data; after 60min, the median of the water content of the 7# sample and 0.1% HA both continued to show a decreasing trend, and the percentage of change of the water content was below 0, which means that the water content at this time point after use was below the background data, but the absolute value of the percentage of change of the water content of the 7# sample was closer to 0, and thus the 7# sample had better moisturizing and moisture retention properties than 0.1% HA.

Fig. 3 is a graph showing the variation of percentage change in transdermal water loss of 7# sample and 0.1% HA, and fig. 4 is a graph showing the variation of median transdermal water loss of 7# sample and 0.1% HA. As can be seen, the amount of transdermal water loss of sample No. 7 and 0.1% HA reached a maximum 5min after use, since this time the amount of water was greater, resulting in greater transdermal water loss; the transdermal water loss gradually decreased with time after use, the water loss of sample # 7 was more stable, and the sample # 7 dropped below 0 at 60min after use, with a percentage change in TEWL of less than 0 at this time point, indicating that the TEWL at this time point was below the skin background.

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

Third, in vitro antioxidant free radical scavenging performance test

1. Experimental methods

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 rice ganoderma lucidum composite fermentation protoplasm aqueous solution prepared in advance and having the volume concentration of 50% and 100%, and a supernatant (as a sample before fermentation) obtained by sterilizing the substrate in the same proportion as that in the embodiment 1. As can be seen from Table 3, the rice ganoderma lucidum composite fermentation raw pulp prepared in example 1 has certain antioxidant performance.

TABLE 3 antioxidant scavenging free radical test results

Fourth, cell MTT proliferation experiment

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:

inoculating the cells into a 96-well plate, performing overnight synchronization treatment, and performing incubation culture for 24h with 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 is 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 4, and it can be seen 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 4

Experiment grouping	Blank control		5% concentration before fermentation	5% concentration after fermentation
					Cell survival rate	1	0.75±0.07	1.81±0.06

Hyaluronidase inhibition assay

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 5 below.

TABLE 5 Hyaluronidase inhibition assay procedures and reagents

The hyaluronidase inhibition was calculated according to the following formula:

the hyaluronidase production rate (%) - (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 of 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 OD value of blank;

2. test results

The test results are shown in table 6. From table 6, it can be seen that the hyaluronidase inhibition rate of the sample after fermentation reaches 41.24 ± 0.15%, which is significantly higher than that of the sample before fermentation.

TABLE 6

Test set	100% concentration before fermentation	100% concentration after fermentation
			Hyaluronidase inhibition%	7.18±2.47	41.24±0.15

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 7, from which it can be seen that sample 1 has the highest ABTS radical scavenging capacity compared to the other samples.

TABLE 7 ABTS test results for rice fermentation broths obtained from different Ganoderma species

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 the comparative example, only rice is used as a fermentation substrate, and the other conditions are the same as those in example 1, so that fermentation raw stock is obtained and is marked as a sample A; the application of the paste is not easy, and a light unpleasant smell is not generated.

In contrast, the fermented raw stock obtained in example 1 has faint scent of Chinese herbal medicines after being smeared, covers faint unpleasant smell, is superior in efficacy due to the addition of Hamamelis virginiana and Galla chinensis, has good skin feel, and has good expression 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 an anti-aging and repairing rice ganoderma lucidum composite fermentation product is characterized by comprising the following steps: inoculating the ganoderma lucidum into a fermentation substrate consisting of rice, witch hazel, gallnut and water for fermentation culture, and then performing sterilization treatment to obtain the rice-ganoderma lucidum composite fermentation product.

Scheme 2, the preparation method according to scheme 1, characterized in that, in the fermentation substrate, the rice amount is 0.5-3% of the weight of water; the using amount of the witch hazel accounts for 0.01 to 0.05 percent of the weight of the water; the amount of Galla chinensis is 0.01-0.05% of water.

Scheme 3, the preparation method according to scheme 2, characterized in that in the fermentation substrate, the witch hazel is a dried witch hazel flower, and the gallnut is a dried product.

Scheme 4, the preparation method according to scheme 3, wherein the hamamelis virginiana is hamamelis virginiana; the gallnut is subjected to crushing treatment before use.

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 any one of schemes 1 to 6, wherein 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;

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.

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, according to scheme 1-10 any item in the preparation method, characterized in that, the sterilization treatment temperature is 100-.

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 the sterilization treatment further comprises separation treatment, sediment removal and supernatant fluid taking, and the ganoderma lucidum composite fermentation raw stock is finally obtained.

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 according to scheme 16 or 17.

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

Scheme 20, the application of the ganoderma lucidum composite fermented dry powder in the aspect of preparing cosmetics according to scheme 18.

The application of the ganoderma lucidum composite fermentation protoplasm in the scheme 21 or the scheme 19 in the aspect of being used as or preparing 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> anti-aging and repairing rice ganoderma lucidum fungus composite fermentation product and preparation method and application thereof

<130> PD200217CN0286

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 646

<212> DNA

<213> Ganoderma lucidum (Ganoderma lucidum)

<400> 1

ccccttccgt agggtgaacc tgcggaagga tcattatcga gttttgaccg ggttgtagct 60

ggccttctga ggcatgtgca cgccctgttc atccactcta cacctgtgca cttactgtgg 120

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 an anti-aging and repairing rice ganoderma lucidum composite fermentation product is characterized by comprising the following steps: inoculating the ganoderma lucidum into a fermentation substrate consisting of rice, witch hazel, gallnut and water for fermentation culture, and then performing sterilization treatment to obtain the rice-ganoderma lucidum composite fermentation product.

2. The method according to claim 1, wherein the amount of rice in the fermentation substrate is 0.5-3% by weight of water; the using amount of the witch hazel accounts for 0.01 to 0.05 percent of the weight of the water; the amount of Galla chinensis is 0.01-0.05% of water.

3. The method of claim 2, wherein in the fermentation substrate, the witch hazel is a dried witch hazel flower and the gallnut is a dried witch hazel flower.

4. The method for preparing as claimed in claim 3, wherein the hamamelis virginiana is hamamelis virginiana; the gallnut is subjected to crushing treatment before use.

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 any one of 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.