Bletilla striata fermentation liquor as well as preparation method and application thereof

Abstract

The invention relates to bletilla striata fermentation liquor as well as a preparation method and application thereof, and belongs to the technical field of fermentation. According to the method, the bletilla striata is subjected to primary enzymolysis by using the compound amylase, and then the primary fermentation product is further decomposed by using the saccharomycetes, so that the obtained fermentation liquor has a good anti-inflammatory effect. The invention finds that the anti-inflammatory effect of the bletilla striata fermentation liquor obtained after the primary fermentation product is fermented by the kluyveromyces lactis, the Feibaiberg lindnella yeast and the saccharomyces cerevisiae is similar to that of an anti-inflammatory drug namely dexamethasone. In addition, the bletilla striata fermentation liquor prepared by the preparation method disclosed by the invention is relatively small in polysaccharide molecular weight and has a good moisturizing effect. Therefore, the bletilla striata fermentation liquor and the preparation method thereof have a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fermentation, in particular to a bletilla striata fermentation liquor and a preparation method and application thereof. BACKGROUND

[0002] Bletilla striata (Thuub) Rchb. f. is also known as Baiji, Baijizi, etc., and belongs to perennial orchid. The dried tuber of Bletilla striata is a traditional Chinese medicinal material. In recent years, it has been found that the active ingredients in Bletilla striata have various pharmacological effects including antibacterial, antiviral, antitumor, antioxidant, hemostatic and promoting wound healing. At present, in clinical medicine, the medicinal ingredients of Bletilla striata tuber are more, but mainly Bletilla striata polysaccharide (BSP), also known as Bletilla striata gum. Bletilla striata polysaccharide is a water-soluble polysaccharide mainly composed of β-mannose and β-glucose polymerized by 1-4 glycosidic bond, which has the effects of astringing and promoting muscle healing, antibacterial and anti-inflammatory, promoting wound healing, etc., and has a wide application potential in the field of cosmetics, especially in the field of anti-inflammatory cosmetics.

[0003] The commonly used active substance extraction methods in the field of cosmetics include water extraction, organic solvent extraction, ultrasonic extraction, supercritical fluid extraction and microbial fermentation, etc. The microbial fermentation method does not need to add other catalysts, only needs to culture a large amount of microbial strains, and then adds fermentation substrate to carry out complex biological catalytic reaction, so the microbial method is more specific and more effective than the chemical reagent reaction method; and the reaction conditions are mild, without harsh conditions such as high temperature and high pressure; the operation of the equipment is also relatively simple and safe, and the public hazard is less, generally does not cause environmental pollution, and the post-treatment is relatively simple. In the prior art, Bletilla striata is usually mixed with other ingredients with anti-inflammatory effect (such as rapeseed pollen) for fermentation, and there is a lack of research on the fermentation performance of Bletilla striata itself. In addition, Bacillus subtilis is usually used as a fermentation strain to ferment Bletilla striata in the prior art, and the obtained Bletilla striata fermentation liquor has antioxidant property, but the anti-inflammatory effect is not good, and the anti-inflammatory effect of Bletilla striata cannot be fully played. In addition, the fermentation product of Bacillus subtilis is usually acidic and has a special odor, which is limited in the field of cosmetics. Therefore, it is urgent to seek a new fermentation method to improve the anti-inflammatory property of Bletilla striata fermentation liquor. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to overcome the problem that there is a lack of Bletilla striata fermentation liquor with anti-inflammatory effect in the prior art.

[0005] To solve the above technical problems, the present application provides a white plant fermentation liquor and its preparation method and application. The present application first uses a complex amylase to preliminarily hydrolyze the white plant, and then uses yeast to further decompose the primary fermentation product, so that the obtained fermentation liquor has good anti-inflammatory effect. The present application finds that the white plant fermentation liquor obtained by fermenting the primary fermentation product with Kluyveromyces lactis, Cyberlindnera fabianii or Saccharomyces cerevisiae has an anti-inflammatory effect similar to that of the anti-inflammatory drug dexamethasone. In addition, the white plant fermentation liquor prepared by the preparation method of the present application has a small polysaccharide molecular weight, and the small molecular weight polysaccharide can penetrate into the deep layer of the epidermis through the intercellular space of the cuticle or the hair follicle channel, directly acting on the cells and better playing a role. Additionally, the white plant fermentation liquor of the present application also has good moisturizing effect, which can significantly reduce the loss of skin moisture and improve skin elasticity. In summary, the white plant fermentation liquor prepared by the preparation method of the present application has good application prospect.

[0006] The first object of the present application is to provide a preparation method of a white plant fermentation liquor, comprising the following steps:

[0007] S1, dissolving and incubating the white plant with a complex amylase to obtain a primary product;

[0008] S2, inoculating yeast into the primary product in S1 and carrying out fermentation culture to obtain a white plant fermentation liquor;

[0009] The yeast is selected from one or more of Kluyveromyces lactis, Cyberlindnera fabianii and Saccharomyces cerevisiae.

[0010] Further, the preservation number of Kluyveromyces lactis is CICC 32408, the preservation number of Cyberlindnera fabianii is CICC 31007, and the preservation number of Saccharomyces cerevisiae is CICC 1299.

[0011] Further, the solution used for dissolving in step S1 is pure water, the mass ratio of white plant to pure water is 1:(30-100), and the particle size of white plant is 30-100 mesh.

[0012] Further, the complex amylase in step S1 is selected from multiple of alpha-amylase, beta-amylase, isoamylase and saccharifying enzyme. The complex amylase can preliminarily hydrolyze the white plant to preliminarily degrade the large molecular compounds in the white plant.

[0013] Preferably, when the complex amylase is alpha-amylase, beta-amylase, isoamylase and saccharifying enzyme, the mass ratio is 1:1:1:1.

[0014] Further, the temperature of the incubation in step S1 is 50-80℃, for example, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, including but not limited to the above listed values.

[0015] Further, the incubation time is 30-60min.

[0016] Further, the temperature of the fermentation culture in step S2 is 20-35℃, for example, 20℃, 25℃, 28℃, 30℃, 35℃, including but not limited to the above listed values. The fermentation time is 48-72h.

[0017] Further, the concentration of the yeast in step S2 is 1×10 5 -1×10 8 CFU / mL, the inoculation amount of the yeast is 1%-10% of the mass of the primary product, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, including but not limited to the above listed values.

[0018] Further, the primary product in step S1 is subjected to sterilization treatment; the white plant fermentation broth in step S2 is subjected to sterilization and centrifugation treatment. The temperature of the sterilization is 15-121℃, the sterilization time is 15-20min; the centrifugal force of the centrifugation is 8000-12000g, the first centrifugation time is 10-20min, the second centrifugation time is 2-10min.

[0019] The second object of the present application is to provide a white plant fermentation broth prepared by the above preparation method.

[0020] The third object of the present application is to provide an application of the above white plant fermentation broth in preparing an anti-inflammatory product.

[0021] Further, the anti-inflammatory product inhibits the generation of inflammatory factors, including TNF-a.

[0022] The fourth object of the present application is to provide an application of the above white plant fermentation broth in preparing a moisturizing product.

[0023] The beneficial effects of the present application are:

[0024] The preparation method can fully decompose Bletilla striata, and the obtained Bletilla striata fermentation liquor has good anti-inflammatory and moisturizing effects. The Bletilla striata is first subjected to enzymatic hydrolysis by using a compound amylase to obtain a primary fermentation product, and then subjected to fermentation culture by using a yeast to obtain the Bletilla striata fermentation liquor having a good anti-inflammatory effect and capable of inhibiting the generation of inflammatory factors such as tumor necrosis factor-a. In particular, the fermentation liquor obtained by using Kluyveromyces lactis as a fermentation strain has an anti-inflammatory effect similar to that of the anti-inflammatory drug dexamethasone. In addition, the Bletilla striata fermentation liquor has small polysaccharide molecular weight, and the small-molecular-weight polysaccharide can penetrate into the deep layer of the epidermis through the intercellular space of the cuticle or the hair follicle channel, directly act on the cells, and play a stronger efficacy role. Moreover, the Bletilla striata fermentation liquor has good moisturizing effect. Therefore, the preparation method can fully decompose Bletilla striata, and the obtained Bletilla striata fermentation liquor has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which

[0026] Figure 1 is a flow chart of the preparation method of the Bletilla striata fermentation liquor;

[0027] Figure 2 is a polysaccharide molecular weight distribution diagram of the Bletilla striata fermentation liquor of Example 1;

[0028] Figure 3 is a polysaccharide molecular weight distribution diagram of the Bletilla striata fermentation liquor of Example 2;

[0029] Figure 4 is a polysaccharide molecular weight distribution diagram of the Bletilla striata fermentation liquor of Example 3;

[0030] Figure 5 is a polysaccharide molecular weight distribution diagram of the Bletilla striata fermentation liquor of Comparative Example 1;

[0031] Figure 6 is a polysaccharide molecular weight distribution diagram of the Bletilla striata fermentation liquor of Comparative Example 2;

[0032] Figure 7 is a polysaccharide molecular weight distribution diagram of the Bletilla striata fermentation liquor of Comparative Example 3;

[0033] Figure 8 is a polysaccharide molecular weight distribution diagram of the Bletilla striata fermentation liquor of Comparative Example 4;

[0034] Figure 9 is a polysaccharide molecular weight distribution diagram of the Bletilla striata fermentation liquor of Comparative Example 5;

[0035] Figure 10 is a comparison diagram of the expression amount of TNF-a inflammatory factor of Examples 1-3 and Comparative Examples 1-5. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0037] Unless otherwise specified, the Bletilla striata, enzyme preparations, culture media, reagents, etc. used in the following examples can all be obtained commercially; the bacterial strains in the examples can all be purchased from bacterial culture collection institutions such as CICC (website for purchasing bacterial strains: http: / / m.china-cicc.org / ); the mouse macrophages Raw264.7 in the examples were purchased from Wuhan Pronosai Life Science Technology Co., Ltd.; the compound amylase in the examples was prepared by compounding α-amylase, β-amylase, isoamylase and saccharifying enzyme in a mass ratio of 1:1:1:1, and was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., Xiasheng (Beijing) Biotechnology Development Co., Ltd., Zhejiang Tianhe Food Biotechnology Co., Ltd., and Henan Wanbang Chemical Technology Co., Ltd., respectively.

[0038] The detection method involved in this invention is as follows:

[0039] The determination of Bletilla striata polysaccharides was performed using the phenol-sulfuric acid method, referring to: Determination of Active Ingredients in Functional Foods, Chemical Industry Press, 1st Edition, 2005, Chapter 1, Section 2.

[0040] The culture media involved in the following examples are as follows:

[0041] YPD liquid culture medium: 10 g / L yeast extract, 10 g / L tryptone, 20 g / L glucose.

[0042] YPD solid medium: 10 g / L yeast extract, 10 g / L tryptone, 20 g / L glucose, 20 g / L agar.

[0043] The following examples illustrate the method for preparing yeast seed culture:

[0044] The preserved yeast was streaked onto YPD solid agar plates and incubated at 30°C for 48 hours to activate it. Single colonies were picked and inoculated into 50 mL of YPD liquid agar, and incubated at 30°C and 150 rpm for 24 hours. The bacterial concentration was then adjusted to 1×10⁻⁶ with sterile water. 5 1×10 7 and 1×10 8 CFU / mL was used to obtain yeast seed culture.

[0045] Example 1:

[0046] A method for preparing Bletilla striata fermentation broth, the specific steps of which are as follows:

[0047] (1) Preparation of primary product

[0048] Prepare the enzymatic substrate: mix 30 g of 100-mesh Bletilla striata tuber powder, 0.12 g of compound amylase (enzyme activity 100,000 U / g) and 1000 g of pure water, and put them into a constant-temperature water bath at 65°C for reaction for 30 min. After the reaction, sterilize at 115°C for 20 min and cool, to obtain the fermentation substrate (i.e. the primary product).

[0049] (2) Preparation of Bletilla striata fermentation broth

[0050] Inoculate the fermentation substrate obtained in step (1) with the Kluyveromyces lactis seed liquid at a concentration of 1 x 10 7 CFU / mL and an inoculum of 10% (weight ratio), and cultivate in a 28°C, 200 rpm shaking incubator for 48 h, to obtain the primary fermentation broth.

[0051] Sterilize the obtained primary fermentation broth. The sterilization temperature is 115°C, the sterilization time is 20 min, and after sterilization, centrifuge once at 10,000 g for 15 min. Centrifuge the supernatant twice at 10,000 g for 5 min, and obtain the Bletilla striata fermentation broth (S1) from the supernatant.

[0052] The Bletilla striata fermentation broth prepared in this example has a polysaccharide content of 1.96%, a pH value of 5.2, a Bletilla striata-specific smell that is significantly improved, a total number of colonies of less than 50 CFU / mL, and no pathogenic bacteria detected, meeting the quality requirements for cosmetic raw materials.

[0053] Example 2

[0054] A method for preparing a Bletilla striata fermentation broth, comprising the following steps:

[0055] (1) Preparation of primary product

[0056] Prepare the enzymatic substrate: mix 20 g of 80-mesh Bletilla striata tuber powder, 0.06 g of compound amylase (enzyme activity 100,000 U / g) and 1000 g of pure water, and put them into a constant-temperature water bath at 55°C for reaction for 60 min. After the reaction, sterilize at 110°C for 20 min and cool, to obtain the fermentation substrate (i.e. the primary product).

[0057] (2) Preparation of Bletilla striata fermentation broth

[0058] Inoculate the fermentation substrate obtained in step (1) with the Wickerhamiella siamensis seed liquid at a concentration of 1 x 10 8 CFU / mL and an inoculum of 1% (weight ratio), and cultivate in a 30°C, 200 rpm shaking incubator for 60 h, to obtain the primary fermentation broth.

[0059] The obtained primary fermentation broth was sterilized. The sterilization temperature was 121°C, the sterilization time was 20 min, and after cooling at the end of sterilization, the primary centrifugation was performed at 8000 g for 10 min. After the centrifugation, the supernatant was subjected to secondary centrifugation at 8000 g for 10 min, and the white plant fermentation broth (S2) was obtained.

[0060] The white plant fermentation broth prepared in this example had a polysaccharide content of 1.32%, a pH value of 5.5, contained a unique odor of white plant, the total number of colonies was less than 50 CFU / ml, no pathogenic bacteria were detected, and met the quality requirements of cosmetic raw materials.

[0061] Example 3

[0062] A preparation method of a white plant fermentation broth, the specific steps are as follows:

[0063] (1) Preparation of primary product

[0064] The enzyme hydrolysis substrate was prepared by mixing 10 g of 30-mesh white plant tuber powder, 0.02 g of complex amylase (enzyme activity 100,000 U / g), and 1000 g of pure water, and then placing the mixture in a 75°C constant temperature water bath for 40 min of incubation. After the reaction, the fermentation substrate (i.e., the primary product) was obtained by sterilization at 115°C for 15 min and cooling.

[0065] (2) Preparation of white plant fermentation broth

[0066] The fermentation substrate obtained in step (1) was inoculated with a yeast seed liquid at a concentration of 1 x 10 5 CFU / mL and an inoculation amount of 5% (weight ratio), and then cultured in a 25°C, 200 rpm shaking incubator for 72 h to obtain the primary fermentation broth.

[0067] The obtained primary fermentation broth was sterilized. The sterilization temperature was 115°C, the sterilization time was 15 min, and after cooling at the end of sterilization, the primary centrifugation was performed at 12000 g for 20 min. After the centrifugation, the supernatant was subjected to secondary centrifugation at 12000 g for 2 min, and the white plant fermentation broth (S3) was obtained.

[0068] The white plant fermentation broth prepared in this example had a polysaccharide content of 0.61%, a pH value of 5.1, contained a unique odor of white plant, the total number of colonies was less than 50 CFU / ml, no pathogenic bacteria were detected, and met the quality requirements of cosmetic raw materials.

[0069] Example 4

[0070] I. Safety detection of white plant fermentation broth

[0071] Human patch test is mainly used to detect the irritation of cosmetic final product or raw material. According to the "Cosmetic Safety Technical Specification" (2022), the human closed patch test of the white plant fermentation liquor obtained in examples 1-3 and comparative examples 1-5 is carried out, aiming to evaluate its potential skin irritation.

[0072] 1. Test subjects:

[0073] According to the requirements of "Diagnosis Standard and Treatment Principle of Cosmetic Contact Dermatitis", the test subjects are selected, and the test subjects with scar, fresh red nevus and other factors affecting the result determination on the skin, and highly sensitive persons cannot participate in the test. In this test, 30 suitable volunteers are selected as test subjects, with an age range of 18-60 years old.

[0074] 2. Test method:

[0075] The liquid sample (100% white plant fermentation liquor, no dilution) 0.02 mL is dropped on the filter paper, and then the filter paper is placed in the patch tester. Each sample is set with a blank control (water), and the same amount of sample solvent distilled water is added to the control patch tester hole. The test period lasts for 24 h. In order to ensure the accuracy, reliability and science of the test results, the volunteers cannot remove the patch tester during the test period, and the test site cannot be in contact with water. After 24 h, the patch tester is removed, and after 30 min (waiting for the indentation to disappear), the skin reaction is observed at 24 h and 48 h. The skin adverse reaction grading standard of human patch test is shown in Table 1.

[0076] Table 1 Skin reaction grading standard of human test

[0077] Skin reaction Classification No reaction 0 Faint erythema 1 Erythema, infiltration, papules 2 Erythema, oedema, papules, vesicles 3 Erythema, oedema, bullae 4

[0078] 3. Test results:

[0079] See Table 2, from which it can be seen that the white plant fermentation liquor (S1, S2, S3) in examples 1-3 and the white plant fermentation liquor (C1, C2, C3, C4, C5) of comparative examples 1-5 are all negative reactions, which shows that the white plant fermentation liquor and the white plant fermentation substrate provided by the present application are all safe and will not cause adverse reactions to the human body.

[0080] Table 2 Patch test of white plant fermentation liquor obtained from examples and comparative examples

[0081]

[0082]

[0083] II. Molecular weight distribution of polysaccharides in different white plant fermentation liquors

[0084] The present application determines the polysaccharide molecular weight size distribution of the Bletilla striata fermentation broth (S1, S2, S3) in Examples 1-3 and the Bletilla striata fermentation broth (C1, C2, C3, C4, C5) in Comparative Examples 1-5 by high performance gel filtration chromatography (HPGFC). The specific test is as follows:

[0085] 1. Test method

[0086] (1) Instrument: Waters 2695 high performance liquid chromatograph (with 2410 differential refractive index detector and Empower working software);

[0087] (2) Column: Ultrahydrogel™ Linear 300 mm x 7.8 mm id;

[0088] (3) Mobile phase: 0.1 N sodium nitrate;

[0089] (4) Chromatographic conditions: flow rate: 0.5 mL / min; column temperature: 40°C;

[0090] (5) Sample preparation: take about 50 mg of the Bletilla striata fermentation broth in a 10 mL volumetric flask, dilute to the mark with the mobile phase, ultrasonic for 5 min, then filter with a microporous membrane (0.45 μm) for sample injection.

[0091] Standard for molecular weight calibration curve (all purchased from China National Institute for Food and Drug Control): Dextran T-2000 (MW 2000000); Dextran T-300 (MW 300600); Dextran T-150 (MW 135030); Dextran T-10 (MW 9750); Dextran T-5 (MW 2700) glucose 180.

[0092] Table 3 Polysaccharide molecular weight size distribution of Bletilla striata fermentation broth

[0093]

[0094] The molecular weight parameters of the polysaccharide include number average molecular weight (Mn), weight average molecular weight (Mw) and peak molecular weight (Mp). The number average molecular weight (Mn) refers to the average value of the molecular weight of all molecules, taking into account the number of molecules of each molecular weight; the weight average molecular weight (Mw) refers to the total sum of the product of the molecular weight of all molecules and its corresponding molecular number, then divided by the total molecular number, which is more inclined to reflect larger molecules; the peak molecular weight (Mp) refers to the molecular weight with the highest frequency in the molecular weight distribution graph.

[0095] Bletilla striata polysaccharide is gradually concerned in cosmetics due to its natural source and diverse biological activities (such as moisturizing, antioxidant, anti-inflammatory, immune regulation, etc.). The molecular weight of Bletilla striata polysaccharide significantly affects its cosmetic efficacy. As can be seen from Table 3, microbial fermentation can significantly change the molecular weight distribution of Bletilla striata polysaccharide in the fermentation broth, and significantly increase the proportion of small molecular weight polysaccharide in the fermentation broth. Small molecular weight polysaccharide can penetrate into the deep layer of the epidermis through the intercellular space of the stratum corneum or the hair follicle channel, and directly act on cells (such as activating the antioxidant pathway and inhibiting inflammatory factors), thereby exerting stronger efficacy.

[0096] III. Moisturizing efficacy of different Bletilla striata fermentation broths

[0097] The Bletilla striata fermentation broths in Examples 1-3 (S1, S2, S3) were used as the experimental group, and the Bletilla striata fermentation broths in Comparative Examples 1-5 (C1, C2, C3, C4, C5) were used as the control group. The skin moisture content, skin elasticity and transepidermal water loss values before and after the action of different groups were compared. The specific test process is as follows:

[0098] On the inner side of the left and right arms of the subject at a distance of 5 cm from the base of the palm, two areas of the same size (3 cm x 3 cm) were defined on the left forearm as the use area; the same position on the right forearm was used as the blank control area, and the corresponding areas of the left and right arms were tested simultaneously. The subjects applied Bletilla striata fermentation broths (C1, C2, C3, C4, C5) and Bletilla striata fermentation broths (S1, S2, S3) to the two test areas of the arms, respectively. At 0, 2, 4 and 8 h after application, the skin moisture content was tested using a skin moisture meter (Corneometer CM825, Germany), the skin elasticity was tested using a multifunctional skin tester (Courage & Khazaka MPA580, Germany), and the amount of transepidermal water loss was tested using a transepidermal water loss meter (Tewameter TM300, Germany). Each measurement was performed 3 times, and the average value was taken. The average values of the skin moisture content, elasticity and water loss measured 3 times at 0 h were taken as the blank control group. The results are shown in Table 4.

[0099] Table 4 Test results of skin moisture content, elasticity and water loss

[0100]

[0101] As can be seen from the comparison of the examples and comparative examples in Table 4, the Bletilla striata fermentation broth of the present application can significantly improve the skin hydration state after use, and has obvious moisturizing effect. As can be seen from the comparison with Comparative Example 4, the inoculated strain during fermentation can also have a certain influence on the moisturizing effect of the Bletilla striata fermentation broth. At the same time, the Bletilla striata fermentation broth of the present application can also significantly reduce the skin water loss and improve the total elasticity of the skin, and has good moisturizing efficacy when applied to skin care cosmetics.

[0102] IV. Soothing efficacy of different Bletilla striata fermentation liquids

[0103] The soothing efficacy of different Bletilla striata fermentation liquids was tested according to the group standard T / SHRH 034-2021 “Cosmetic soothing efficacy test - Determination of TNF-α inflammatory factor content in vitro LPS-induced macrophage RAW264.7 test method”. Specifically, the LPS (bacterial lipopolysaccharide) was used to induce mouse macrophage Raw264.7, and the expression amount of TNF-α inflammatory factor was detected by ELISA method to evaluate the ability of the test substance to inhibit the expression of related cell pathway factors. The test scheme is shown in Table 5.

[0104] Table 5 Test scheme for soothing efficacy of Bletilla striata fermentation liquids

[0105]

[0106] The test results are shown in Table 6. Figure 10 Table 6 Test results of soothing efficacy of Bletilla striata fermentation liquids

[0107] Synergistic effect determination criteria: q = E A+B / (E A +E B -E A *E B ).

[0108] Wherein: E A is the efficacy data of A component alone, E B is the efficacy data of B component alone, E A+B is the actual efficacy data after A component and B component combination treatment, q = 0.85-1.15 is the effect of two components combined, q > 1.15 is the effect of two components combined, q < 0.85 is the effect of two components combined.

[0109] From the test results, it can be seen that the Bletilla striata fermentation liquids of Examples 1-4 have good soothing efficacy, and the soothing efficacy of the Bletilla striata fermentation liquids of Examples 1-4 is significantly higher than that of the Bletilla striata fermentation liquid of Comparative Example 1. Figure 10It can be seen that the blank group has a very significant difference compared with the model group (p<0.001), which indicates that the experimental model is successful and can be used to verify the anti-inflammatory effect of the related samples. Through One-way ANOVA analysis, it can be found that the dexamethasone used in the positive control and the various samples provided by the examples (S1-S3) and the comparative examples (C2-C5) can significantly reduce the content of TNF-a inflammatory factor of the model (p<0.001), which indicates that the Bletilla striata fermentation broth used in the present application has a certain anti-inflammatory effect, and after fermentation by the yeast strain provided by the present application, the ability to eliminate inflammatory factor TNF-a is significantly enhanced (p<0.001). Compared with the model group, the content of TNF-a inflammatory factor corresponding to dexamethasone and S1-S3 is reduced by 51.94%, 48.11%, 41.75% and 24.52% respectively, among which S1 is close to the anti-inflammatory effect of the same dose of dexamethasone, and the anti-inflammatory effect of the Bletilla striata fermentation broth provided in the examples and comparative examples is the best, followed by S2, and S1-S3 all have a certain market application value. It can be seen from the comparison of S3 and C1-C5 that: the C1 sample without enzyme preparation treatment and microbial fermentation has an anti-inflammatory effect of only 3.85% on TNF-a inflammatory factor; the C2 sample added with enzyme preparation treatment has part of the functional polysaccharides in Bletilla striata free, so that the anti-inflammatory effect is improved to 5.92%; the C3 sample added with the yeast fermentation provided by the present application has more functional polysaccharides in Bletilla striata free, so that the anti-inflammatory effect is improved to 13.53%; the S3 sample added with the same dose of enzyme preparation and the same kind of yeast treatment has an anti-inflammatory effect of 24.52% on TNF-a inflammatory factor, which meets the synergistic effect determination standard, q=4.14>1.15, indicating that the enzyme preparation and the yeast provided by the present application have a synergistic effect in preparing the Bletilla striata fermentation broth with anti-inflammatory effect; the C4 sample added with the same dose of enzyme preparation and the same inoculation amount of other yeast has an anti-inflammatory effect of only 8.45% on TNF-a inflammatory factor, which is 65.54% lower than that of S3, indicating that the Bletilla striata fermentation broth prepared by using different yeasts has a large difference in anti-inflammatory effect, and the Bletilla striata fermentation broth prepared by using the yeast provided by the present application has a stronger effect. The C5 sample added with the same dose of enzyme preparation and the same inoculation amount of Bacillus subtilis has an anti-inflammatory effect of only 18.49% on TNF-a inflammatory factor, which is 24.59% lower than that of S3, indicating that the Bletilla striata fermentation broth prepared by using Bacillus subtilis has a weaker anti-inflammatory effect than the Bletilla striata fermentation broth prepared by using the yeast provided by the present application.

[0110] Comparative Example 1

[0111] (1) Preparation of primary product

[0112] Preparation of enzyme hydrolysis substrate: 10 g of 30-mesh Bletilla striata tuber powder was mixed with 1000 g of pure water, and the mixture was placed in a 75°C constant-temperature water bath for reaction for 40 min. After reaction, the mixture was sterilized at 115°C for 15 min and then cooled to obtain the fermentation substrate.

[0113] (2) Preparation of Bletilla striata fermentation liquor

[0114] The fermentation substrate obtained in step (1) was inoculated with sterile water at a weight ratio of 5%, and then cultured in a 25°C, 200 rpm shaking incubator for 72 h to obtain the primary fermentation liquor.

[0115] The obtained primary fermentation liquor was subjected to sterilization treatment at a sterilization temperature of 115°C and a sterilization time of 15 min. After sterilization, the mixture was cooled and then subjected to centrifugation at 12000 g for 20 min. The supernatant was then subjected to centrifugation at 12000 g for 2 min to obtain the Bletilla striata fermentation liquor (C1).

[0116] The Bletilla striata fermentation liquor obtained in Comparative Example 1 had a polysaccharide content of 0.12%, a pH value of 6.7, a specific odor of Bletilla striata, a total number of colonies of less than 50 CFU / ml, and no pathogenic bacteria were detected, which met the quality requirements of cosmetic raw materials.

[0117] Comparative Example 2:

[0118] (1) Preparation of primary product

[0119] Preparation of enzyme hydrolysis substrate: 10 g of 30-mesh Bletilla striata tuber powder, 0.02 g of complex amylase (enzyme activity: 100000 U / g) and 1000 g of pure water were mixed, and the mixture was placed in a 75°C constant-temperature water bath for reaction for 40 min. After reaction, the mixture was sterilized at 115°C for 15 min and then cooled to obtain the fermentation substrate.

[0120] (2) Preparation of Bletilla striata fermentation liquor

[0121] The fermentation substrate obtained in step (1) was inoculated with sterile water at a weight ratio of 5%, and then cultured in a 25°C, 200 rpm shaking incubator for 72 h to obtain the primary fermentation liquor.

[0122] The obtained primary fermentation liquor was subjected to sterilization treatment at a sterilization temperature of 115°C and a sterilization time of 15 min. After sterilization, the mixture was cooled and then subjected to centrifugation at 12000 g for 20 min. The supernatant was then subjected to centrifugation at 12000 g for 2 min to obtain the Bletilla striata fermentation liquor (C2).

[0123] The Bletilla striata fermentation liquor obtained in Comparative Example 2 had a polysaccharide content of 0.26%, a pH value of 5.1, a specific odor of Bletilla striata, a total number of colonies of less than 50 CFU / ml, and no pathogenic bacteria were detected, which met the quality requirements of cosmetic raw materials.

[0124] Comparative Example 3

[0125] (1) Preparation of primary product

[0126] Preparation of enzymatic substrate: 10 g of 30-mesh Bletilla striata tuber powder was mixed with 1000 g of pure water, and the mixture was placed in a 75°C constant-temperature water bath for reaction for 40 min. After reaction, the mixture was sterilized at 115°C for 15 min and then cooled to obtain a fermentation substrate.

[0127] (2) Preparation of Bletilla striata fermentation broth

[0128] The fermentation substrate obtained in step (1) was inoculated with a Saccharomyces cerevisiae seed solution at a concentration of 1×10 5 CFU / mL and an inoculum of 5% (weight ratio), and was cultured in a 25°C, 200 rpm shaking incubator for 72 h to obtain a primary fermentation broth.

[0129] The obtained primary fermentation broth was subjected to sterilization treatment. The sterilization temperature was 115°C, the sterilization time was 15 min, and after sterilization and cooling, the broth was centrifuged at 12000 g for 20 min. The supernatant was then centrifuged at 12000 g for 2 min to obtain a Bletilla striata fermentation broth (C3).

[0130] The Bletilla striata fermentation broth obtained in Comparative Example 3 had a polysaccharide content of 0.47%, a pH value of 5.3, a Bletilla striata odor that was significantly improved, a total number of colonies of less than 50 CFU / mL, and no pathogenic bacteria were detected, which met the quality requirements for cosmetic raw materials.

[0131] Comparative Example 4

[0132] (1) Preparation of primary product

[0133] Preparation of enzymatic substrate: 10 g of 30-mesh Bletilla striata tuber powder, 0.02 g of complex amylase (enzyme activity 100,000 U / g) and 1000 g of pure water were mixed, and the mixture was placed in a 75°C constant-temperature water bath for reaction for 40 min. After reaction, the mixture was sterilized at 115°C for 15 min and then cooled to obtain a fermentation substrate.

[0134] (2) Preparation of Bletilla striata fermentation broth

[0135] The fermentation substrate obtained in step (1) was inoculated with an abnormal Hansenula polymorpha seed solution (preservation number: CICC 31079) at a concentration of 1×10 5 CFU / mL and an inoculum of 5% (weight ratio), and was cultured in a 37°C, 200 rpm shaking incubator for 72 h to obtain a primary fermentation broth.

[0136] The obtained primary fermentation broth was sterilized. The sterilization temperature was 121°C, the sterilization time was 30 min, and after sterilization, the primary fermentation broth was centrifuged at 12000 g for 20 min. The supernatant was centrifuged at 12000 g for 2 min, and the white plant fermentation broth (C4) was obtained.

[0137] The white plant fermentation broth prepared in Comparative Example 4 had a polysaccharide content of 0.35%, a pH value of 6.6, a white plant specific smell and other smells, a total number of colonies less than 50 CFU / ml, and no pathogenic bacteria detected.

[0138] Comparative Example 5

[0139] (1) Preparation of primary product

[0140] The enzyme hydrolysis substrate was prepared by mixing 10 g of 30-mesh white plant tuber powder, 0.02 g of complex amylase (enzyme activity 100,000 U / g), and 1000 g of pure water, and then placing the mixture in a 75°C constant temperature water bath for 40 min. The fermentation substrate was prepared by sterilizing the reaction mixture at 115°C for 15 min and then cooling.

[0141] (2) Preparation of white plant fermentation broth

[0142] The fermentation substrate obtained in step (1) was inoculated with Bacillus subtilis seed liquid (preservation number: CICC 24713) at a concentration of 1 x 10 5 CFU / mL and an inoculation amount of 5% (weight ratio), and then cultured in a 37°C, 200 rpm shaking incubator for 72 h to obtain the primary fermentation broth.

[0143] The obtained primary fermentation broth was sterilized. The sterilization temperature was 121°C, the sterilization time was 30 min, and after sterilization, the primary fermentation broth was centrifuged at 12000 g for 20 min. The supernatant was centrifuged at 12000 g for 2 min, and the white plant fermentation broth (C4) was obtained.

[0144] The white plant fermentation broth prepared in Comparative Example 5 had a polysaccharide content of 0.58%, a pH value of 4.8, a white plant specific smell not obvious and a strong fermentation smell, a total number of colonies less than 50 CFU / ml, and no pathogenic bacteria detected.

[0145] Obviously, the above examples are merely examples for the purpose of clarity, and are not intended to limit the embodiments. Based on the above description, those skilled in the art can make other different forms of changes or variations. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for preparing Bletilla striata fermentation broth, characterized in that, Includes the following steps: S1. Mix and dissolve Bletilla striata with compound amylase and incubate to obtain the primary product; S2. Yeast is inoculated into the primary product in S1 and fermented to obtain Bletilla striata fermentation broth; The yeast strain is selected from one or more of Kluyveromyces lactis, Cyberlindnera fabianii, and Saccharomyces cerevisiae.

2. The preparation method according to claim 1, characterized in that, The complex amylase mentioned in step S1 is selected from two or more of α-amylase, β-amylase, isoamylase and saccharifying enzyme.

3. The preparation method according to claim 1, characterized in that, The incubation temperature in step S1 is 50-80℃, and the fermentation temperature in step S2 is 20-35℃.

4. The preparation method according to claim 1, characterized in that, The preservation number of the *Kluyveromyces lactis* is CICC 32408; the preservation number of the *Fibrians cerevisiae* is CICC 31007; and the preservation number of the *Saccharomyces cerevisiae* is CICC 1299.

5. The preparation method according to claim 1, characterized in that, The concentration of yeast in step S2 is 1×10⁻⁶. 5 -1×10 8 The yeast inoculum concentration is CFU / mL, and the inoculum size is 1%-10% of the mass of the primary product.

6. The preparation method according to claim 1, characterized in that, The primary product described in step S1 is sterilized; the Bletilla striata fermentation broth described in step S2 is sterilized and centrifuged.

7. The Bletilla striata fermentation broth prepared by the preparation method according to any one of claims 1-6.

8. The application of the Bletilla striata fermentation broth according to claim 7 in the preparation of anti-inflammatory products.

9. The application according to claim 8, characterized in that, The anti-inflammatory product inhibits the generation of inflammatory factors, including tumor necrosis factor-α.

10. The use of the Bletilla striata fermentation broth according to claim 7 in the preparation of moisturizing products.

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