CN113208954A

CN113208954A - Anti-inflammatory and anti-aging ganoderma lucidum composite fermentation product and preparation method and application thereof

Info

Publication number: CN113208954A
Application number: CN202010930355.6A
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-inflammatory and anti-aging ganoderma lucidum composite fermentation product, which comprises the following steps: inoculating the ganoderma lucidum to a fermentation substrate consisting of rice, purslane extract, calendula, mangnolia officinalis extract 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 proper ganoderma lucidum strain to carry out composite liquid fermentation on rice, purslane extract, calendula and mangnolia officinalis extract, has good anti-inflammatory and anti-aging effects, and can be used as an excellent raw material of an anti-inflammatory and anti-aging cosmetic or directly used as a cosmetic.

Description

Anti-inflammatory and anti-aging 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-inflammatory and anti-aging 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.

Herba Portulacae (Portulaca oleracea L.) is an annual herb of Portulacaceae of Caryophyllales, and has effects of clearing heat, promoting diuresis, removing toxic substances, relieving swelling, diminishing inflammation, quenching thirst, and promoting urination. The purslane contains rich dihydroxyethylamine, malic acid, glucose, calcium, phosphorus, iron, vitamin E, carotene, vitamin B, vitamin C and other nutrient substances, and is a good food material and medicinal material. The purslane extract is rich in a large amount of flavonoids, adrenalines, polysaccharides, various vitamins, amino acids and other compounds, has broad-spectrum antibacterial property and anti-inflammatory effect, can prevent and treat skin diseases such as skin eczema, allergic dermatitis, contact dermatitis and the like, has good oxygen radical scavenging capacity, and has obvious oxidation resistance and skin moisture retention property.

Calendula (Calendala officinalis L.) is a two-year-old herbaceous plant in Compositae, is a long-history edible medicinal plant, and has a beautifying function of petals, the main active ingredient of the Calendula is a carotenoid compound, the content of the carotenoid compound accounts for about 3 percent of dry flowers, the Calendula mainly contains lutein, zeaxanthin, a large amount of lutein esters and the like, and the Calendula further contains volatile oil, saponin, flavonol glycoside and terpenoids, so that the Calendula has good anti-inflammatory and antibacterial effects, has a proliferation effect on epidermal cells and dermal cells, has an inhibition effect on elastase, combines the antioxidation effect, and can be used for anti-aging related products.

Magnolia officinalis (Magnolia officinalis) is a plant of Magnoliaceae, and bark, root bark, flower, seed and bud can be used as the raw materials, and bark is the main ingredient of Chinese medicine, and has effects of eliminating dampness and removing food stagnation, promoting qi circulation and relieving asthma, resolving food stagnation and eliminating phlegm, dispelling pathogenic wind and relieving pain. The effective component of cortex Magnolia officinalis extract is total phenols (including honokiol and magnolol), has remarkable antifungal effect, and can be used for treating acne by combining with anti-inflammation, and the cortex Magnolia officinalis extract has antioxidant effect, can eliminate free radicals, and can be used in antiaging cosmetic.

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. Researches show that 3 materials of ganoderma lucidum sporocarp, spore powder and mycelium have similar effects of resisting tumor, regulating immunity and the like, but the content and the types 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, purslane, calendula, mangnolia officinalis and lucid ganoderma are applied to the field of cosmetics, the rice, the purslane, the calendula, the mangnolia officinalis and the lucid ganoderma are basically compounded with various 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 is still a difficult problem for 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-inflammatory and anti-aging ganoderma lucidum composite fermentation product, and a preparation method and an application thereof, wherein the anti-inflammatory and anti-aging ganoderma lucidum composite fermentation product has good moisturizing and whitening functions.

According to one aspect of the disclosure, a preparation method of an anti-inflammatory and anti-aging ganoderma lucidum composite fermentation product is provided, which comprises the following steps: inoculating the ganoderma lucidum to a fermentation substrate consisting of rice, purslane extract, calendula, mangnolia officinalis extract and water for fermentation culture, and then performing sterilization treatment to obtain the ganoderma lucidum composite fermentation product.

The rice adopted in the disclosure is a finished product prepared by rice through the processes of cleaning, rice hulling, rice milling and the like, the preferable dosage is 0.5-3%, namely the dosage of the rice accounts for 0.5-3% of the weight of water (such as 0.8%, 1.0%, 1.5%, 2.0%, 2.5% and the like), and the rice can be rice grains or rice powder. 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 purslane extract adopted in the preparation has broad-spectrum antibacterial property and anti-inflammatory effect, and can prevent and treat skin diseases such as skin eczema, allergic dermatitis, contact dermatitis and the like; has good oxygen free radical scavenging ability, obvious oxidation resistance and skin moisture retention. The purslane extract used in embodiments of the present disclosure is preferably used in an amount of 0.001% to 0.05%, i.e., the purslane extract is present in the fermentation substrate at 0.001% to 0.05% (e.g., 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.045%, etc.) by weight of water. The use amount of the purslane extract is too low to reflect the corresponding effect, and the fermentation liquor obtained by using the purslane extract is darker in overall color, is hyperpigmented on the skin and is easy to cause skin allergic reaction. More preferably, the purslane extract is extracted at a ratio of 4-20:1 (e.g., 5:1, 8:1, 10:1, 12:1, 15:1, 18:1, etc.), i.e., 1 part extract is obtained from 4-20 parts purslane raw material. The purslane extract can be purchased from the market or prepared by itself, for example, 1 part by weight of the extract is obtained by hot water extraction from 4 to 20 parts by weight of purslane raw material.

The calendula is adopted in the invention, is rich in lutein, zeaxanthin, a large amount of lutein esters and the like, and also contains volatile oil, saponin, flavonol glycoside and terpenoids, so that the calendula has good anti-inflammatory and antibacterial effects, has a proliferation effect on epidermal cells and dermal cells, has an inhibition effect on elastase, and also has an excellent antioxidation effect. The calendula used in the embodiments of the present disclosure is dry calendula, and the mass ratio of the calendula to water is 0.01-0.05%, in other words, the calendula amount is 0.01-0.05% of the water amount (such as 0.02%, 0.03%, 0.04%, etc.).

The extract of magnolia officinalis is adopted in the acne treatment cream, and the active ingredient of the extract of magnolia officinalis, namely total magnolol, has an obvious antifungal effect, can be used for treating acne by combining the anti-inflammation effect of the extract of magnolia officinalis, and also has an antioxidant effect and free radical elimination effect. Magnolia bark extract used in the examples of the present disclosure is commercially available or can be prepared by itself, and is preferably used in an amount of 0.001% to 0.05%, i.e., 0.001% to 0.05% by weight of water (e.g., 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.045%, etc.) of the fermentation substrate. The application ratio of the magnolia officinalis extract is too low to reflect the corresponding effect, and the fermentation liquor obtained by using the magnolia officinalis extract in too high ratio has darker overall color, is hyperpigmented on the skin and is easy to cause skin anaphylactic reaction. More preferably, the Magnolia bark extract is extracted at a ratio of 4-20:1 (e.g., 5:1, 8:1, 10:1, 12:1, 15:1, 18:1, etc.), i.e., 1 part extract is obtained from 4-20 parts Magnolia bark raw material. Magnolia bark extract is commercially available or may be prepared by itself, for example, by hot water extraction of 4-20 parts by weight of Magnolia bark material to obtain 1 part by weight of extract.

The fermentation substrate is prepared by adding rice, purslane extract, calendula and mangnolia officinalis extract 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; 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, preferably, the Ganoderma lucidum (Ganoderma lucidum) strain wG055 with the preservation number of CGMCC No.17789 is adopted.

Preferably, the mycelium volume percentage of the ganoderma lucidum liquid for inoculating to the fermentation substrate is more than 80% (such as 85%, 90%, 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 the anti-inflammatory and anti-aging 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) for 3-5d (such as 3.5 days, 4 days, 4.5 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-inflammatory and anti-aging Ganoderma lucidum composite fermentation product, as a preferred embodiment, the temperature of sterilization treatment after fermentation culture is 90-121 ℃ (such as 95 ℃, 100 ℃, 105 ℃, 108 ℃, 110 ℃, 115 ℃, 118 ℃, 120 ℃, etc.), and the time is 15-60min (such as 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, etc.).

In the above method for preparing the anti-inflammatory and anti-aging 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, i.e. the ganoderma lucidum composite fermentation raw stock, is obtained.

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

In the preparation method of the anti-inflammatory and anti-aging 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-inflammatory and anti-aging 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 above method for preparing the anti-inflammatory and anti-aging ganoderma lucidum composite fermentation product, as a preferred embodiment, the drying treatment may be spray drying, vacuum freeze drying, or 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 preparing cosmetics is further provided.

According to another aspect of the disclosure, the application of the ganoderma lucidum composite fermentation protoplasm in 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 purslane extract, the calendula and the mangnolia officinalis extract are subjected to composite liquid fermentation by selecting the proper ganoderma lucidum strain to prepare the novel ganoderma lucidum composite fermentation raw pulp, so that the novel ganoderma lucidum composite fermentation raw pulp has good anti-inflammatory and anti-aging effects, and can be used as an excellent raw material of an anti-inflammatory and anti-aging 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 (referred to as "water content") tests performed on the anti-inflammatory and anti-aging ganoderma lucidum composite fermentation raw stock prepared in example 1, which is prepared into 10% by volume aqueous solution (10% sample, i.e. 3# sample) and 0.1% by volume hyaluronic acid aqueous solution (0.1% HA), and is a graph showing the trend of the change of the percentage of moisture content before use in each area at different time points after use;

fig. 2 shows the results of skin moisture content (abbreviated as "water content") tests performed on the anti-inflammatory and anti-aging ganoderma lucidum composite fermentation raw stock prepared in example 1 to prepare 10% by volume aqueous solution (10% sample, i.e. 3# sample) and 0.1% by volume hyaluronic acid aqueous solution (0.1% HA), which are median variation trends of moisture content in different regions at different time points;

FIG. 3 is a graph showing the variation trend of the TEWL value before use in different time points and different areas after use, which is the result of the skin water loss (i.e. percutaneous water loss, abbreviated as "water dispersion") test performed by using the anti-inflammatory and anti-aging Ganoderma lucidum composite fermentation primary pulp prepared in example 1 to prepare 10% by volume aqueous solution (10% sample, i.e. sample No. 3) and 0.1% by volume 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% by volume aqueous solution (10% sample, i.e. sample # 3) and 0.1% by volume aqueous solution (0.1% HA) of hyaluronic acid prepared from the anti-inflammatory and anti-aging 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 purslane extract in the following embodiment is produced by Jiangxi Jiahe biological science and technology limited company, and the extraction proportion is as follows: 10:1.

The magnolia bark extract in the following embodiments is produced by Jiangxi Jiahe biological science and technology limited company in the proportion of extraction: 10:1.

The calendula in the following examples is a dry product of calendula produced by agriculture and sideline products purchasing limited of Hoodia 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 anti-inflammatory and anti-aging Ganoderma lucidum composite fermentation raw stock

1. Preparing ganoderma lucidum liquid: inoculating a 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 mycelium accounts for 80% of the volume of the liquid;

2. preparing a fermentation substrate: adding rice 5g, herba Portulacae extract 0.05g, herba Sidae Rhombifoliae 0.15g, and cortex Magnolia officinalis extract 0.05g into 500g water, sterilizing at 115 deg.C for 20min to obtain fermentation substrate; wherein the herba Portulacae extract accounts for 0.01%, the rice accounts for 1%, the calendula accounts for 0.03%, and the cortex Magnolia officinalis extract accounts for 0.01%.

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 4 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, namely antiinflammatory and antiaging Ganoderma composite fermentation raw stock.

The anti-inflammatory and anti-aging ganoderma lucidum composite fermentation raw stock prepared in the embodiment 1 is semitransparent liquid, white to light gray in color, 4.1-6.3 in pH value, free of viscosity, 1.5-5.0% in soluble solid content, less than 50CFU/ml in total number of colonies and free of pathogenic bacteria 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 anti-inflammatory and anti-aging 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 pulp prepared in the embodiment 1 contains 0.864g/kg of protein, 6.575g/kg of crude polysaccharide, 0.031g/kg of total flavone (counted by rutin) and 0.029g/kg of total phenol.

Example 2 application of Ganoderma lucidum composite fermentation protoplasm as cosmetic

Safety detection of 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 ganoderma lucidum composite fermentation raw pulp obtained in the embodiment 1 is used for negative reaction, which shows that the ganoderma lucidum composite fermentation raw pulp provided by the invention has safety and does not bring adverse reaction to human bodies.

TABLE 2 Patch test of Ganoderma lucidum composite fermentation broth obtained in example 1

Second, water content and water dispersion test of anti-inflammatory and anti-aging 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 # 3, 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 (3 # 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 sample # 3 and 0.1% HA, and FIG. 2 is a graph showing the median change of the water content of the sample # 3 and 0.1% HA. As can be seen from the figure, the median water content of 5min after use of both the 3# sample and 0.1% HA reached the maximum, and the median water content of both the 3# sample and 0.1% HA showed a decreasing trend as the time after use was extended; after 20min, a percent change in water content of 0.1% HA and 3# sample of less than 0 indicates that the water content at this time point after use was below background data, and a percent change in water content of 3# sample after 60min was closer to 0 than 0.1% HA, indicating that 3# was slightly more potent in maintaining water content than HA.

Fig. 3 is a graph showing the trend of percentage change in transdermal water loss of the sample # 3 and 0.1% HA, and fig. 4 is a graph showing the trend of median change in transdermal water loss of the sample # 3 and 0.1% HA. As can be seen from the figure, the change law of the percutaneous water loss of the sample No. 3 and the 0.1% HA is relatively similar, and both the sample No. 3 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 change percentage of TEWL of 0.1% HA is more than 0 at the time point, which means that the TEWL at the time point is higher than the background of the skin because the water-locking component (such as grease) is not further applied, and the change percentage of TEWL of the 3# sample is reduced to be less than 0 at 20min, which shows that the TEWL of the skin at the time point is lower than that before the use after the 3# sample is used, which indicates that the skin HAs certain moisturizing function.

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

Third, in vitro antioxidant free radical scavenging performance test

1. Experimental methods

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.

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. Wherein the sample solution to be tested is a rice ganoderma lucidum composite fermentation protoplasm aqueous solution with the volume concentration of 50% and 100% prepared in advance, and a supernatant (as a sample before fermentation) obtained after the substrate is sterilized in the same proportion. As can be seen from Table 3, the Ganoderma lucidum composite fermentation broth prepared in example 1 has good 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 solutions (i.e., supernatants obtained after sterilizing the substrates in the same proportion and not inoculated with fermentation, and the rice 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

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 which is not inoculated and fermented after the substrate is sterilized in the same proportion as that of the example 1 is 8.47% + -1.24%, the tyrosinase inhibition rate of the ganoderma lucidum composite fermentation raw pulp prepared in the example 1 is 31.95% + -1.28%, 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 ganoderma lucidum composite fermentation raw stock prepared in the embodiment 1 has certain tyrosinase inhibition capability and certain whitening effect.

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:

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 results are shown in Table 5, 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, indicating 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	0.63±0.05	1.77±0.03

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. It can be seen from table 7 that the inhibition rate of hyaluronidase by the post-fermentation samples is significantly higher than that of the pre-fermentation samples.

TABLE 7

Test set	100% concentration before fermentation	100% concentration after fermentation
			Hyaluronidase inhibition%	10.01±0.7	37.81±1.65

Assay of collagen protein

1. Experimental methods

Sample preparation: 1) cell blank: pure DMEM; 2) 5% before fermentation: preparing the supernatant which is not fermented after the substrate is sterilized according to the same proportion as that of the example 1 into a solution with the volume concentration of 5 percent by using a DMEM medium; 3) 5% after fermentation: sterilizing, inoculating, culturing and centrifugally sterilizing the substrate in the same proportion to obtain a fermentation sample, namely fermentation raw stock prepared in the embodiment 1, and preparing a solution with the volume concentration of 5% by using a DMEM (DMEM) culture medium; 4) positive control: ascorbyl glucoside was formulated in a 1% by volume solution with DMEM.

Collecting human skin fibroblasts (HDF-n, from ScienCell corporation) in logarithmic growth phase, digesting with trypsin, stopping digestion with complete medium, and counting; adjusting the cell concentration of the cell suspension to 1X 10⁵One/ml, and added to 6-well plates, 1ml of fresh complete medium, 1ml of cell suspension, at 37 ℃ in 5% CO per well₂Culturing in an incubator overnight; the medium was aspirated, 2ml of each sample was added to each well, 2ml of basal medium was added to the cell control wells, and the mixture was incubated at 37 ℃ with 5% CO₂Culturing in an incubator for 48 h. The cell supernatant was collected using a sterile 1.5mL centrifuge tube, centrifuged at 3000 rpm for about 2000-. The light absorption value (OD value) of the supernatant is detected by adopting a human type I Collagen (COLI) enzyme-linked immunoassay kit provided by Nanjing institute of built bioengineering.

2. Collagen content measurement results

The collagen concentration was calculated using elisa calc, a logistic curve (four parameters) was selected for the fitted model, and the regression equation of the standard curve was calculated using the concentration of the standard and the corresponding OD value. Regression equation y ═ (A-D)/[1+ (x/C)^B]+ D, wherein: a is 1.64442; b-0.98920; c-14.91037; d-0.22063; r is²＝0.96004。

Based on the OD value of the sample, ELISAcalc was used to calculate the corresponding sample concentration on the regression equation, as shown in Table 8. From table 8, it can be seen that the sample after fermentation has a significant effect of promoting the collagen content of the cells at a concentration of 5% compared with the cell control, and no significant difference compared with the positive control, indicating that the fermentation liquid has a comparable collagen synthesis promoting effect with ascorbyl glucoside.

TABLE 8

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

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

The results are shown in Table 9, from which it can be seen that sample 1 has the highest ABTS radical scavenging capacity compared to the other samples.

TABLE 9 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.

In contrast, the fermented raw stock obtained in example 1 has faint scent of Chinese herbal medicines after being smeared, and unpleasant smell is covered, and due to the addition of the Chinese herbal medicine components such as purslane, calendula, mangnolia officinalis and the like, the fermented raw stock has stronger functionality and better skin feel performance.

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-inflammatory and anti-aging ganoderma lucidum composite fermentation product is characterized by comprising the following steps: inoculating the ganoderma lucidum to a fermentation substrate consisting of rice, purslane extract, calendula, mangnolia officinalis extract and water for fermentation culture, and then performing sterilization treatment to obtain the ganoderma lucidum composite fermentation product.

Scheme 2, the preparation method according to scheme 1, characterized in that in the fermentation substrate, the rice accounts for 0.5% -3% of the weight of water; the purslane extract accounts for 0.001-0.05% of the weight of water; the calendula officinalis is a dry product and accounts for 0.01-0.05% of the weight of water; the magnolia bark extract accounts for 0.001-0.05% of the weight of the water.

Scheme 3, the preparation method according to scheme 2, wherein the extraction ratio of the purslane extract in the fermentation substrate is 4-20: 1.

Scheme 4, the preparation method according to scheme 2, wherein the extraction ratio of the magnolia bark extract in the fermentation substrate is 4-20: 1.

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 and the preparation method according to scheme 6, wherein the Ganoderma lucidum is Ganoderma lucidum (Ganoderma lucidum) strain wG055, and the preservation number is 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 3-5d, and the stirring treatment is carried out during the fermentation culture.

Scheme 10, the preparation method according to scheme 8, wherein 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 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-inflammatory and anti-aging ganoderma lucidum composite fermentation product, and preparation method and application thereof

<130> PD200213CN0282

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 646

<212> DNA

<213> Ganoderma lucidum (Ganoderma lucidum)

<400> 1

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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. The preparation method of the anti-inflammatory and anti-aging ganoderma lucidum composite fermentation product is characterized by comprising the following steps of: inoculating the ganoderma lucidum to a fermentation substrate consisting of rice, purslane extract, calendula, mangnolia officinalis extract and water for fermentation culture, and then performing sterilization treatment to obtain the ganoderma lucidum composite fermentation product.

2. The method of claim 1, wherein the rice comprises 0.5-3% of the fermentation substrate by weight of water; the purslane extract accounts for 0.001-0.05% of the weight of water; the calendula officinalis is a dry product and accounts for 0.01-0.05% of the weight of water; the magnolia bark extract accounts for 0.001-0.05% of the weight of the water.

3. The method of claim 2, wherein the purslane extract is extracted from the fermentation substrate at a ratio of 4-20: 1.

4. The method as claimed in claim 2, wherein the extraction ratio of the Magnolia bark extract in the fermentation substrate is 4-20: 1.

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 claim 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 5 days, and the stirring treatment is performed during the fermentation culture.

10. The method as claimed in claim 8, wherein the rotation speed of the stirring process is 100-200 rpm.