Preparation method of white truffle fermentation product with anti-oxidation effect as well as product and application of white truffle fermentation product

Abstract

The invention relates to a preparation method of a white truffle fermentation product with an anti-oxidation effect as well as a product and application of the white truffle fermentation product. The preparation method comprises the following steps: mixing a composite plant extract, inorganic salt, water and a composite probiotic agent for fermentation culture, and centrifuging, filtering and sterilizing a culture product to obtain the white truffle fermentation product, the strains in the compound probiotic agent comprise a combination of streptococcus thermophilus, saccharomyces cerevisiae boulardii, a plant lactobacillus YK-L002 strain and a saccharomyces cerevisiae YK-S001 strain. According to the invention, specific strains are subjected to compounding and combined fermentation, and the fermentation product filtrate obtained after fermentation treatment of four bacteria has an excellent skin conditioning effect, can effectively improve the skin state, significantly resist oxidation and resist and weaken wrinkles, and has an excellent effect when being applied to moisturizing and anti-oxidation cosmetics.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics and relates to a preparation method of a white truffle fermentation product with antioxidant effect, as well as a product and application thereof. Background Art

[0002] Long-term exposure to sunlight, air pollution, and computer radiation increases the number of free radicals in the body. These free radicals react with collagen, preventing the skin from receiving adequate nutrition. This leads to decreased vitality and elasticity, wrinkles, and can cause skin aging and dull complexion. Skin aging primarily begins when free radicals attack healthy cells.

[0003] Plant extracts are active ingredients extracted from plants in skin care products. After scientific extraction and processing, they can have multiple effects such as whitening, freckle removal, anti-oxidation, skin cell activation, anti-inflammatory and skin soothing. Moreover, because their components are naturally green, environmentally friendly and milder, they will not irritate the skin or cause allergic reactions. They can be more easily absorbed and metabolized by the body and will not cause adverse effects on the body. The active ingredients produced by the fermentation of probiotic strains, such as polysaccharides, biological enzymes or collagen, play an important role in delaying skin aging, providing skin nutrients, promoting skin cell regeneration, and eliminating skin wrinkles.

[0004] Therefore, how to provide a plant-microorganism co-culture fermentation product that can effectively improve skin condition and have significant antioxidant effects has important application value. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a preparation method of a white truffle fermentation product with antioxidant efficacy, as well as its product and application.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a white truffle fermentation product having antioxidant efficacy, the preparation method comprising the following steps:

[0008] The composite plant extract, inorganic salt, water and composite probiotic agent are mixed and fermented, and the culture product is centrifuged, filtered and sterilized to obtain a white truffle fermentation product;

[0009] The plants in the composite plant extract include white truffle, white willow bark and chrysanthemum; the strains in the composite probiotic include a combination of Streptococcus thermophilus, Saccharomyces boulardii, Lactobacillus plantarum YK-L002 strain with a preservation number of CGMCC No. 29289, and Saccharomyces cerevisiae YK-S001 strain with a preservation number of CGMCC No. 28276.

[0010] In the present invention, it was found that thermophilic Streptococcus, boulardii yeast, Lactobacillus plantarum YK-L002 strain and Saccharomyces cerevisiae YK-S001 strain were compounded, and there was potential interaction between the strains, which could cooperate with each other and synergize. The fermentation product filtrate after fermentation by the four bacteria had an excellent skin conditioning effect, could effectively improve skin condition, resist and weaken wrinkles, and had excellent effects when applied to moisturizing and antioxidant cosmetics.

[0011] Moreover, the present invention further discovered that the fermentation matrix prepared from pure plant raw materials, in which the natural bioactive ingredients rich in the three plant ingredients of white truffle, white willow bark and chrysanthemum can not only enhance the relevant skin condition conditioning effect of the final fermented product, but also the plant polysaccharides or fungal polysaccharides, terpenoids, proteins and fibers contained therein can further promote the fermentation of probiotics and increase the content of active ingredients in the fermentation product. There is also a potential synergistic relationship between the three plant ingredients.

[0012] Preferably, the inorganic salt includes any one of dipotassium hydrogen phosphate, potassium dihydrogen phosphate or anhydrous magnesium sulfate, or a combination of at least two thereof.

[0013] Preferably, the mass ratio of the white truffle, white willow bark and chrysanthemum is (2-6):(0.5-3):(1-4);

[0014] The above “2-6” may be, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, etc.;

[0015] The above-mentioned “0.5-3” can be, for example, 0.5, 1, 1.5, 2, 2.5, 3, etc.;

[0016] The above-mentioned "1-4" can be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, etc. Other specific point values ​​within this numerical range can be selected, and they will not be detailed here.

[0017] In the present invention, when the dosage ratio of the three plant ingredients is within the above-mentioned specific range, the fermentation product finally obtained has better skin conditioning effects such as anti-oxidation.

[0018] Preferably, the mass ratio of the composite plant extract, the inorganic salt and water is (4-10):(0.1-1):(8-16);

[0019] Wherein, the "4-10" may be, for example, 4, 5, 6, 7, 8, 9, 10, etc.;

[0020] The “0.1-1” may be, for example, 0.1, 0.2, 0.5, 0.6, 0.8, 0.9, 1, etc.;

[0021] The "8-16" can be, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, etc.

[0022] The composite plant extracts involved in this invention are not limited to their specific preparation process and source. Products produced by conventional extraction and preparation processes and regular commercially available products can be used in the present invention. For example, the plant material can be crushed and mixed with water, heated and extracted at 70-95°C for 0.5-3 hours, and the extract is filtered and the filtrate or residue is collected. When prepared using the following process, the resulting fermentation matrix has a more effective effect for culturing fermentation-related probiotics, and the resulting fermentation product has a more excellent skin-improving effect.

[0023] Preferably, the composite plant extract is prepared by a preparation method comprising the following steps:

[0024] mixing white truffle, white willow bark and chrysanthemum with a composite enzyme and water for enzymatic hydrolysis, performing high-temperature extraction after the enzymatic hydrolysis, filtering the extract and collecting the filtrate and filter residue respectively to obtain the composite plant extract;

[0025] The complex enzyme comprises a combination of lysase, β-glucanase and papain.

[0026] In the present invention, it was unexpectedly discovered that when a composite enzyme of three enzymes, namely, cleavage enzyme, β-glucanase and papain, is used to treat plant raw materials, the combined use of the three enzymes can synergistically enhance efficiency and obtain a better extraction effect. The prepared extract has higher activity and better effect. In addition, high-temperature extraction after enzymatic hydrolysis can not only achieve mild inactivation of the biological enzymes, but also further perform secondary extraction of the active ingredients in the plant to obtain a plant extract with better effect.

[0027] Preferably, the mass ratio of the collapsinase, β-glucanase and papain is (0.5-2):(2-6):(1-3);

[0028] Here, “0.5-2” can be, for example, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, etc.;

[0029] “2-6” can be, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, etc.;

[0030] “1-3” can be, for example, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, etc. Other specific point values ​​within this numerical range can be selected and will not be listed here one by one.

[0031] Preferably, the added amount of the complex enzyme is 0.5‰-2‰ of the total mass of the plant raw material, for example, it can be 0.5‰, 0.6‰, 0.7‰, 0.8‰, 0.9‰, 1‰, 1.2‰, 1.4‰, 1.6‰, 1.8‰, 2‰, etc. Other specific point values ​​within this numerical range can be selected, and they will not be repeated here.

[0032] Preferably, the material-liquid ratio of the plant raw material to water is 1g:(50-80)mL, for example, it can be 1g:50mL, 1g:55mL, 1g:60mL, 1g:65mL, 1g:70mL, 1g:72mL, 1g:75mL, 1g:80mL, etc. Other specific point values ​​within this numerical range can be selected, and they will not be repeated here.

[0033] Preferably, the enzymatic hydrolysis temperature is 50-60°C, for example, it can be 50°C, 52°C, 55°C, 58°C, 60°C, etc.; the time is 0.5-2 h, for example, it can be 0.5 h, 0.8 h, 1 h, 1.2 h, 1.5 h, 1.8 h, 2 h, etc. Other specific point values ​​within this numerical range can be selected, and they will not be repeated here.

[0034] Preferably, the temperature of the high-temperature extraction is 75-90°C, for example, 75°C, 78°C, 80°C, 85°C, 90°C, etc.; the time is 1-3 h, for example, 1 h, 1.2 h, 1.5 h, 1.8 h, 2 h, 2.5 h, 3 h, etc. Other specific point values ​​within this numerical range can be selected, and they will not be repeated here.

[0035] Preferably, the ratio of the viable counts of the thermophilic Streptococcus, boulardii Saccharomyces, Lactobacillus plantarum YK-L002 strain and Saccharomyces cerevisiae YK-S001 strain is (0.5-5):(8-15):(2-8):(10-20);

[0036] The above “0.5-5” may be, for example, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.;

[0037] The above “8-15” can be, for example, 8, 9, 10, 11, 12, 13, 14, 15, etc.;

[0038] The above-mentioned "2-8" can be, for example, 2, 3, 4, 5, 5.5, 6, 7, 8, etc.;

[0039] The above-mentioned "10-20" can be, for example, 10, 12, 14, 15, 17, 18, 20, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0040] In the present invention, when Streptococcus thermophilus, Saccharomyces boulardii, Lactobacillus plantarum YK-L002 strain and Saccharomyces cerevisiae YK-S001 strain are inoculated and fermented at the above-mentioned specific live bacteria ratio, the fermentation product obtained has better efficacy.

[0041] Preferably, the concentration of viable bacteria of the thermophilic Streptococcus is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g, for example, can be 1×10 6 CFU / mL (CFU / g), 2×10 6 CFU / mL (CFU / g), 5×10 6 CFU / mL (CFU / g), 8×10 6 CFU / mL (CFU / g), 1×10 7 CFU / mL (CFU / g), 5×10 7 CFU / mL (CFU / g), 1×10 8 CFU / mL (CFU / g), etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0042] Preferably, the inoculation amount of the composite probiotic in the culture matrix is ​​5%-12%, for example, it can be 5%, 6%, 7%, 8%, 9%, 9.5%, 10%, 12%, etc. Other specific point values ​​within this numerical range can be selected and will not be repeated here.

[0043] Preferably, the fermentation culture temperature is 25-38°C, for example, 25°C, 28°C, 30°C, 32°C, 34°C, 36°C, 37°C, 38°C, etc.; the fermentation time is 70-120 h, for example, 70 h, 80 h, 90 h, 100 h, 105 h, 110 h, 115 h, 120 h, etc.

[0044] Preferably, the preparation method specifically comprises the following steps:

[0045] The filter residue of the first composite plant extract and inorganic salts are mixed and sterilized to obtain a sterilized culture matrix, and thermophilic Streptococcus and Lactobacillus plantarum YK-L002 strains are inoculated into the matrix for a first fermentation culture. The first fermentation culture product, the filtrate of the first composite plant extract, and the filtrate of the second composite plant extract are mixed and sterilized, and then inoculated with Saccharomyces boulardii and Saccharomyces cerevisiae YK-S001 strains for a second fermentation culture. The second culture product is centrifuged, filtered, and sterilized to obtain a white truffle fermentation product.

[0046] Preferably, the plants in the first composite plant extract include white truffle and white willow bark.

[0047] Preferably, the plant in the second complex plant extract includes chrysanthemum.

[0048] The first fermentation culture temperature is 32-38°C, for example, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 37.5°C, 38°C, etc.; the time is 48-72 h, for example, 48 h, 50 h, 55 h, 60 h, 65 h, 70 h, 72 h, etc.;

[0049] Preferably, the temperature of the second fermentation culture is 25-35°C, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 32°C, 35°C, etc.; the time is 25-40 h, for example, 25 h, 28 h, 30 h, 32 h, 35 h, 40 h, etc.

[0050] In a second aspect, the present invention provides a white truffle fermentation product prepared according to the preparation method described in the first aspect.

[0051] In a third aspect, the present invention provides a use of the white truffle fermentation product according to the second aspect in the preparation of cosmetics.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] In the present invention, four strains of bacteria—Streptococcus thermophilus, Saccharomyces boulardii, Lactobacillus plantarum YK-L002, and Saccharomyces cerevisiae YK-S001—were added to a pure plant culture matrix prepared from white truffles, white willow bark, and chrysanthemums to produce related fermentation products. It was found that the four strains interacted with each other, positively influencing each other, and exhibited a synergistic effect that was difficult for those skilled in the art to predict in improving the anti-wrinkle, moisturizing, and antioxidant effects of the final product, further enhancing the relevant efficacy of the product when applied to cosmetics. Furthermore, the three plant components in the culture matrix also exhibited a potential interaction, which could further improve fermentation efficiency and optimize product efficacy.

[0054] The YK-L002 strain involved in the present invention is classified as Lactiplantibacillus plantarum, and the depository is the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with the deposit number CGMCC No. 29289, the deposit date is December 11, 2023, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0055] The YK-S001 strain involved in the present invention is classified as Saccharomyces cerevisiae, and the depository unit is the General Microbiology Center of the China Culture Collection Administration, the deposit number is CGMCC No. 28276, the deposit date is August 28, 2023, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0056] In order to further illustrate the technical means and effects adopted by the present invention, the technical solutions of the present invention are further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0057] The following bacterial species information is:

[0058] ① Streptococcus thermophilus: ATCC 19258, ATCC 14485, and CICC 22561;

[0059] ② The YK-L002 strain is named Lactiplantibacillus plantarum and its deposit number is CGMCC No. 29289;

[0060] ③ The YK-S001 strain is named Saccharomyces cerevisiae and its deposit number is CGMCC No. 28276;

[0061] ④ Saccharomyces boulardii 01 strain was purchased from Guopeptide Biotechnology (Shanxi) Co., Ltd.

[0062] Saccharomyces boulardii 02 strain was purchased from Ningke (Xi'an) Biopharmaceutical Technology Co., Ltd.

[0063] The S. boulardii SHBCC D25151 strain was purchased from Shanghai Microbiological Collection Center.

[0064] The following biological enzyme information is involved: collapsin (S10112), β-glucanase (S23089) and papain (S10011), all of which were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0065] Preparation Example 1

[0066] This preparation example provides a composite plant extract, which is prepared by the following method:

[0067] (1) The first composite plant extract: fresh white truffle was homogenized into a paste, white willow bark was crushed and sieved, and then mixed with white truffle paste, composite enzyme, and pure water. The mass ratio of white truffle to white willow bark was 4:1, and the amount of composite enzyme added was 1‰ of the total mass of white truffle and white willow bark. The material-liquid ratio of white truffle and white willow bark to water was 1g:65mL. Among them, the composite enzyme was composed of cleavage enzyme, β-glucanase, and papain in a mass ratio of 1:4:2. After enzymatic hydrolysis at 55℃ for 1 hour, the temperature was raised to 85℃ and the extraction was continued for 2 hours. The extract was filtered, and the filtrate and filter residue were collected respectively. The filtrate was concentrated and freeze-dried.

[0068] (2) Second composite plant extract: The chrysanthemum raw material was dried and crushed, and then mixed with composite enzyme and pure water. The mass ratio of the chrysanthemum raw material to the white willow bark raw material in the first composite extract was 1:3. The amount of composite enzyme added was 1‰ of the mass of chrysanthemum. The material-liquid ratio of chrysanthemum to water was 1g:65mL. The composite enzyme was composed of cleavage enzyme, β-glucanase and papain in a mass ratio of 1:4:2. After enzymatic hydrolysis at 55℃ for 1 hour, the temperature was raised to 85℃ and the extraction was continued for 2 hours. The extract was filtered, and the filtrate was collected, concentrated and freeze-dried.

[0069] Preparation Example 2

[0070] This preparation example provides a composite plant extract, which is prepared by the following method:

[0071] (1) First extract: Fresh white truffle was homogenized into a paste, white willow bark was crushed and sieved, and then mixed with white truffle paste, complex enzyme, and pure water. The mass ratio of white truffle to white willow bark was 2:3, and the amount of complex enzyme added was 0.5‰ of the total mass of white truffle and white willow bark. The material-liquid ratio of white truffle and white willow bark to water was 1g:80mL. The complex enzyme was composed of lyase, β-glucanase, and papain in a mass ratio of 2:2:3. After enzymatic hydrolysis at 58℃ for 0.8h, the temperature was raised to 72℃ and the extraction was continued for 2.5h. The extract was filtered, and the filtrate and filter residue were collected separately. The filtrate was concentrated and freeze-dried.

[0072] (2) Second extract: The chrysanthemum raw material was dried and crushed, and then mixed with a composite enzyme and pure water. The mass ratio of the chrysanthemum raw material to the white willow bark raw material in the first composite extract was 3:1. The amount of composite enzyme added was 0.5‰ of the mass of chrysanthemum. The material-liquid ratio of chrysanthemum to water was 1g:80mL. The composite enzyme was composed of cleavage enzyme, β-glucanase and papain in a mass ratio of 2:2:3. After enzymatic hydrolysis at 58℃ for 0.8h, the temperature was raised to 72℃ and the extraction was continued for 2.5h. The extract was filtered, and the filtrate was collected, concentrated and freeze-dried.

[0073] Preparation Example 3

[0074] This preparation example provides a composite plant extract, which is prepared by the following method:

[0075] (1) First extract: Fresh white truffle was homogenized into a paste, white willow bark was crushed and sieved, and then mixed with white truffle paste, complex enzyme, and pure water. The mass ratio of white truffle to white willow bark was 6:0.5, and the amount of complex enzyme added was 2‰ of the total mass of white truffle and white willow bark. The material-liquid ratio of white truffle and white willow bark to water was 1g:55mL. Among them, the complex enzyme was composed of catalytic enzyme, β-glucanase, and papain in a mass ratio of 0.5:5:1. After enzymatic hydrolysis at 52℃ for 1.8 h, the temperature was raised to 88℃ and the extraction was continued for 1.2 h. The extract was filtered, and the filtrate and filter residue were collected separately. The filtrate was concentrated and freeze-dried.

[0076] (2) Second extract: The chrysanthemum raw material was dried and crushed, and then mixed with a composite enzyme and pure water. The mass ratio of the chrysanthemum raw material to the white willow bark raw material in the first composite extract was 2:1. The amount of composite enzyme added was 2‰ of the mass of chrysanthemum. The material-liquid ratio of chrysanthemum to water was 1g:55mL. The composite enzyme was composed of cleavage enzyme, β-glucanase and papain in a mass ratio of 0.5:5:1. After enzymatic hydrolysis at 52℃ for 1.8 h, the temperature was raised to 88℃ and the extraction was continued for 1.2 h. The extract was filtered, and the filtrate was collected, concentrated and freeze-dried.

[0077] Preparation Example 4

[0078] This preparation example provides a composite plant extract (containing a first extract and a second extract), using the plant raw materials (white truffle, white willow bark and chrysanthemum) in Preparation Example 1. The preparation method thereof differs from that in Preparation Example 1 only in the different enzyme compositions in the composite enzymes in step (1) and step (2), namely, the absence of cleavage enzyme, and the reduced amount thereof is proportionally distributed to β-glucanase and papain. The composite enzymes used in the first extract and the second extract are the same, and other processing processes and related parameters are referred to Preparation Example 1.

[0079] Preparation Example 5

[0080] This preparation example provides a composite plant extract (containing a first extract and a second extract), using the plant raw materials (white truffle, white willow bark and chrysanthemum) in Preparation Example 1. The preparation method thereof differs from that in Preparation Example 1 only in the different enzyme compositions in the composite enzymes in step (1) and step (2), namely, the absence of β-glucanase, and the reduced amount thereof is proportionally distributed to β-glucanase and papain. The composite enzymes used in the first extract and the second extract are the same, and other processing processes and related parameters are referred to Preparation Example 1.

[0081] Preparation Example 6

[0082] This preparation example provides a composite plant extract (containing a first extract and a second extract), using the plant raw materials (white truffle, white willow bark and chrysanthemum) in Preparation Example 1. The preparation method thereof differs from that in Preparation Example 1 only in the different enzyme compositions in the composite enzymes in step (1) and step (2), namely, papain is not contained, and the reduced amount thereof is proportionally distributed to catabolase and β-glucanase. The composite enzymes used in the first extract and the second extract are the same, and other processing processes and related parameters are referred to Preparation Example 1.

[0083] Preparation Example 7

[0084] This preparation example provides a composite plant extract, which differs from Example 1 only in that no enzymatic hydrolysis treatment is performed in step (1) and step (2), and the plant raw material is directly subjected to high-temperature extraction after being mixed with water. The extraction conditions and other parameters and treatments are all referred to Example 1, and the first extract and the second extract are respectively prepared.

[0085] Comparative Preparation Example 1

[0086] This comparative preparation example provides a composite plant extract, which differs from Preparation Example 1 only in that no white truffle raw material is added, and the reduced amount is proportionally distributed to the white willow bark and chrysanthemum components. The other ingredients and contents remain unchanged. The preparation method refers to Preparation Example 1.

[0087] Comparative Preparation Example 2

[0088] This comparative preparation example provides a composite plant extract, which differs from Preparation Example 1 only in that no white willow bark raw material is added, and the reduced amount is proportionally distributed to the white truffle and chrysanthemum components. The other ingredients and contents remain unchanged. The preparation method refers to Preparation Example 1.

[0089] Comparative Preparation Example 3

[0090] This comparative preparation example provides a composite plant extract, which differs from Preparation Example 1 only in that no chrysanthemum raw material is added, and the reduced amount is proportionally distributed to the white truffle and white willow bark components. The other ingredients and contents remain unchanged. The preparation method refers to Preparation Example 1.

[0091] Example 1

[0092] This embodiment provides a white truffle fermentation product, and the preparation method thereof is as follows:

[0093] (1) The filter residue of the first extract (Preparation Example 1) was mixed with potassium dihydrogen phosphate and sterilized (121°C, 30 min), and then Streptococcus thermophilus ATCC 19258 and Lactobacillus plantarum YK-L002 were inoculated therein for solid-state fermentation. The mixture was cultured at 36°C for 60 h. After the culture was completed, the mixture was sterilized at 80°C to obtain a fermentation medium.

[0094] (2) The fermentation substrate, the filtrate (lyophilized powder) of the first extract (Preparation Example 1), the filtrate (lyophilized powder) of the second extract (Preparation Example 1) and water were mixed, the pH was adjusted to 7, and the Saccharomyces boulardii 01 strain and the Saccharomyces cerevisiae YK-S001 strain were inoculated and cultured at 32°C and 100 rpm for 36 h to obtain a fermentation culture product.

[0095] The addition amount of each component is: 7 parts of composite plant extract (first extract filtrate + first extract residue + second extract filtrate), 0.5 parts of dipotassium hydrogen phosphate, and 10 parts of water; the total inoculation amount of the four strains in the culture matrix is ​​8% (the mass ratio of the first inoculation to the second inoculation is 1:2), the ratio of the viable cell counts of Streptococcus thermophilus, Saccharomyces boulardii 01 strain, Lactobacillus plantarum YK-L002 strain, and Saccharomyces cerevisiae YK-S001 strain is 3:12:5:15, and the viable cell concentration of Streptococcus thermophilus at the time of inoculation is 1×10 6 CFU / mL.

[0096] (3) The fermentation culture product is centrifuged, filtered, sterilized (0.1 μm filtration sterilization), and freeze-dried to obtain the white truffle fermentation product.

[0097] Example 2

[0098] This embodiment provides a white truffle fermentation product, and the preparation method thereof is as follows:

[0099] (1) The filter residue of the first extract (Preparation Example 2) was mixed with anhydrous magnesium sulfate and sterilized (121°C, 30 min), and then Streptococcus thermophilus ATCC 14485 and Lactobacillus plantarum YK-L002 were inoculated therein for solid-state fermentation. The mixture was cultured at 38°C for 48 h. After the culture was completed, the mixture was sterilized at 80°C to obtain a fermentation medium.

[0100] (2) The fermentation substrate, the filtrate (lyophilized powder) of the first extract (Preparation Example 2), the filtrate (lyophilized powder) of the second extract (Preparation Example 2) and water were mixed, the pH was adjusted to 7, and the Saccharomyces boulardii 02 strain and the Saccharomyces cerevisiae YK-S001 strain were inoculated and cultured at 28°C and 150 rpm for 36 h to obtain a fermentation culture product.

[0101] The addition amount of each component is: 5 parts of composite plant extract (first extract filtrate + first extract residue + second extract filtrate), 1 part of dipotassium hydrogen phosphate, and 15 parts of water; the total inoculation amount of the four strains in the culture matrix is ​​6% (the mass ratio of the first inoculation to the second inoculation is 1:1), and the ratio of the viable cell counts of Streptococcus thermophilus, Saccharomyces boulardii 02, Lactobacillus plantarum YK-L002, and Saccharomyces cerevisiae YK-S001 is 5:8:7:10, wherein the viable cell concentration of Streptococcus thermophilus at the time of inoculation is 1×10 6 CFU / mL.

[0102] (3) The fermentation culture product is centrifuged, filtered, sterilized (0.1 μm filtration sterilization), and freeze-dried to obtain the white truffle fermentation product.

[0103] Example 3

[0104] This embodiment provides a white truffle fermentation product, and the preparation method thereof is as follows:

[0105] (1) The filter residue of the first extract (Preparation Example 3) was mixed with anhydrous magnesium sulfate and sterilized (121°C, 30 min), and then Streptococcus thermophilus CICC 22561 and Lactobacillus plantarum YK-L002 were inoculated therein for solid-state fermentation. The mixture was cultured at 33°C for 68 h. After the culture was completed, the mixture was sterilized at 80°C to obtain a fermentation medium.

[0106] (2) The fermentation substrate, the filtrate (lyophilized powder) of the first extract (Preparation Example 3), the filtrate (lyophilized powder) of the second extract (Preparation Example 3) and water were mixed, the pH was adjusted to 6.5, and the Saccharomyces boulardii SHBCC D25151 strain and the Saccharomyces cerevisiae YK-S001 strain were inoculated and cultured at 32°C and 125 rpm for 28 h to obtain a fermentation culture product.

[0107] The addition amount of each component is: 9 parts of composite plant extract (first extract filtrate + first extract residue + second extract filtrate), 0.2 parts of dipotassium hydrogen phosphate, and 8 parts of water; the total inoculation amount of the four strains in the culture matrix is ​​10% (the mass ratio of the first inoculation to the second inoculation is 1:2), the ratio of the viable cell counts of Streptococcus thermophilus, Saccharomyces boulardii SHBCC D25151 strain, Lactobacillus plantarum YK-L002 strain, and Saccharomyces cerevisiae YK-S001 strain is 1:14:3:18, and the viable cell concentration of Streptococcus thermophilus at the time of inoculation is 1×10 6 CFU / mL.

[0108] (3) The fermentation culture product is centrifuged, filtered, sterilized (0.1 μm filtration sterilization), and freeze-dried to obtain the white truffle fermentation product.

[0109] Examples 4-7

[0110] This example provides four white truffle fermentation products, which differ from Example 1 only in that the composite plant extract prepared in Preparation Example 1 is replaced by equal amounts of extracts prepared in Preparation Examples 4-7, respectively, and other components and contents remain unchanged. The preparation method refers to Example 1.

[0111] Comparative Examples 1-3

[0112] This comparative example provides three white truffle fermentation products. The culture medium formula thereof differs from that of Example 1 only in that the composite plant extract prepared in Preparation Example 1 is replaced by equal amounts of extracts prepared in Comparative Preparation Examples 1-3, respectively. Other components and contents remain unchanged. The preparation method refers to Example 1.

[0113] Comparative Example 4

[0114] This comparative example provides a white truffle fermentation product, which uses the composite plant extract raw material of Example 1. The preparation method thereof differs from that of Example 1 only in that the thermophilic Streptococcus strain is not added during the fermentation process, and the reduced amount thereof is proportionally distributed to the Lactobacillus plantarum YK-L002 strain, the boulardii yeast 01 strain, and the Saccharomyces cerevisiae YK-S001 strain. The remaining raw materials, processes, and parameters are all referred to Example 1.

[0115] Comparative Example 5

[0116] This comparative example provides a white truffle fermentation product, which uses the composite plant extract raw material of Example 1. The preparation method thereof differs from that of Example 1 only in that the Lactobacillus plantarum YK-L002 strain is not added during the fermentation process, and the reduced amount thereof is proportionally distributed to the thermophilic Streptococcus strain, the boulardii yeast 01 strain, and the cerevisiae YK-S001 strain. The remaining raw materials, processes, and parameters are all referred to Example 1.

[0117] Comparative Example 6

[0118] This comparative example provides a white truffle fermentation product, which uses the composite plant extract raw material of Example 1. The preparation method thereof differs from that of Example 1 only in that the boulardii Saccharomyces cerevisiae 01 strain is not added during the fermentation process, and the reduced amount thereof is proportionally distributed to the thermophilic Streptococcus strain, the plant extract of Lactobacillus plantarum YK-L002 strain, and the cerevisiae YK-S001 strain. The remaining raw materials, processes, and parameters are all referred to Example 1.

[0119] Comparative Example 7

[0120] This comparative example provides a white truffle fermentation product, which uses the composite plant extract raw material of Example 1. The preparation method thereof differs from that of Example 1 only in that the Saccharomyces cerevisiae YK-S001 strain is not added during the fermentation process, and the reduced amount thereof is proportionally distributed to the thermophilic Streptococcus strain, the plant extract strain YK-L002 strain, and the boulardii yeast strain 01 strain. The remaining raw materials, processes, and parameters are all referred to Example 1.

[0121] Test Example 1

[0122] Anti-wrinkle efficacy test

[0123] (1) Sample treatment: The white truffle fermentation products obtained in Examples 1-7 and Comparative Examples 1-7 were diluted with pure water to a mass concentration of 0.5% for later use; the positive control group was diluted with water to a concentration of 0.1% of the positive control substance (epigallocatechin gallate, EGCG); the negative control group was diluted with pure water.

[0124] (2) Test method: The elastase inhibition effect of the sample was tested according to the laboratory method (HMC-WI-028 elastase inhibition rate). The reagent involved was elastase (porcine pancreas, BR); substrate: N-succinyl-L-alanyl-L-alanyl-L-alanine (98%). A sample group, a sample background group, a solvent group, and a solvent background group were set up. Each group required 3 parallels. The solution of the corresponding reagent was added to a 96-well plate, gently shaken, and incubated at 25°C for 15 minutes. The plate was then placed in a microplate reader and the absorbance was measured at 410 nm.

[0125] (3) Calculation formula: ;

[0126] Wherein: A is the absorbance of the reaction solution without sample; B is the absorbance of the reaction solution without sample and enzyme; C is the absorbance of the reaction solution without sample and enzyme; D is the absorbance of the reaction solution with sample and without enzyme.

[0127] The test results are shown in Table 1.

[0128] Table 1

[0129] From the data in the table, we can see that

[0130] (1) It can be seen from Examples 1 to 3 that the white truffle fermentation product provided by the present invention can effectively inhibit elastase, wherein elastase is mainly synthesized and secreted by fibroblasts, and can degrade elastin in the skin, causing skin aging. Therefore, the anti-wrinkle effect of the product can be reflected by evaluating the inhibition rate of the experimental sample on elastase.

[0131] (2) By comparing Example 1 with Examples 4-7, it can be seen that the plant extract prepared by the method of composite enzymatic hydrolysis combined with high-temperature extraction in the present invention has a better anti-wrinkle effect when used in the subsequent culture extraction of fermentation products. There is a potential synergistic relationship between the three enzymes, namely, β-glucanase and papain. When used in combination, they can synergize and enhance the effect to obtain a better extraction effect. The prepared extract has higher activity and better effect.

[0132] (3) By comparing Example 1 with Comparative Examples 1-3, it can be seen that after adding white truffle, white willow bark and chrysanthemum plant extracts to the fermentation matrix of probiotics, the natural bioactive ingredients rich in the three plants are added to the fermentation matrix of probiotics, which can further promote the fermentation of probiotics and increase the content of active ingredients in the fermentation product. There is a potential synergistic relationship between the three plant components, which can jointly enhance the anti-wrinkle effect of the fermentation product.

[0133] (4) By comparing Example 1 with Comparative Examples 4-7, it can be seen that the fermentation product prepared by the combined fermentation of four bacteria, namely, Streptococcus thermophilus, Saccharomyces boulardii, Lactobacillus plantarum YK-L002 strain and Saccharomyces cerevisiae YK-S001 strain, has a synergistic promoting effect on improving the anti-wrinkle efficacy of the final product that is difficult for those skilled in the art to predict.

[0134] Test Example 2

[0135] Antioxidant effect test

[0136] (1) DPPH-free radical scavenging

[0137] A 96-well plate was used, with three replicates per group, and the reaction volume was 200 μL. Sample group: 100 μg of each extract sample prepared in Examples 1-7 and Comparative Examples 1-7 were mixed with 100 μL of purified water, and 100 μL of 0.1 mM DPPH solution was added to the reaction system. Control group: 100 μL of purified water was added to 100 μL of 0.1 mM DPPH solution. After the reaction system was established, shaken in the dark for 10 minutes, and the absorbance at 517 nm was measured using a microplate reader.

[0138] The calculation method of clearance rate is: clearance rate (%) = [(A0-Ax) / A0] × 100%, A0 is the absorbance of the control group, and Ax is the absorbance of the sample group.

[0139] (2) ABTS-free radical scavenging

[0140] Preparation of ABTS working solution: Mix ABTS and potassium persulfate solution to prepare a 7 mM ABTS stock solution, store in the dark for 12 h, shake well, and mix with 10 mM PBS (pH = 7.4) so ​​that the absorbance of the solution at 734 nm is 0.8 to obtain the ABTS working solution.

[0141] A 96-well plate was used, with three replicates per group, and the reaction volume was 200 μL. Sample group: 100 μg of each extract prepared in Examples 1-7 and Comparative Examples 1-7 were mixed with 10 μL of pure water, and 190 μL of ABTS working solution was added to the reaction system. Control group: 10 μL of pure water was added to 190 μL of ABTS working solution. After the reaction system was established, the mixture was thoroughly shaken and incubated for 6 minutes. The absorbance at 734 nm was measured using a microplate reader.

[0142] The calculation method of clearance rate is: clearance rate (%) = [(A0-Ax) / A0] × 100%, A0 is the absorbance of the control group, and Ax is the absorbance of the sample group.

[0143] The test results are shown in Table 2.

[0144] Table 2

[0145] As can be seen from the data in the table, the white truffle fermentation product prepared by the preparation method provided by the present invention has excellent DPPH-free radical and ABTS-free radical scavenging effects, and exhibits significant antioxidant effects. Among them, the antioxidant effect of Example 1 is better than that of Examples 4-7 and Comparative Examples 1-7, indicating that the specific preparation process conditions adopted by the present invention have different degrees of influence on the antioxidant efficacy of the final extract.

[0146] Test Example 3

[0147] Product safety testing

[0148] According to the subject inclusion criteria, 28 subjects were selected for the trial, with a male-to-female ratio of 1:1. They were randomly divided into 7 groups (2 males and 2 females in each group) and received separate trials. The fermentation products prepared in Examples 1-7 were mixed with pure water to adjust the concentration to 2%. An appropriate amount was placed in a spot tester. The spot tester containing the sample was applied to the skin on the curved side of the subject's forearm with medical tape for 24 hours. Skin reactions were observed 30 minutes (after the indentation disappeared), 24 hours, and 48 hours after removing the test substance spot tester, and the skin reaction results were recorded. The skin reaction grading standard is as follows:

[0149] The results showed that the products prepared in Examples 1-7 all showed negative reactions 30 minutes (after the indentation disappeared), 24 hours, and 48 hours after testing, with no irritation or erythema. This indicates that the white truffle fermentation products prepared in this invention are non-irritating to the skin and exhibit no positive reactions, demonstrating their high safety, minimal irritation, and mild nature.

[0150] The applicant declares that the present invention is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0151] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0152] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing a white truffle fermentation product with antioxidant efficacy, characterized in that: The preparation method comprises the following steps: The composite plant extract, inorganic salt, water and composite probiotic agent are mixed and fermented, and the culture product is centrifuged, filtered and sterilized to obtain a white truffle fermentation product; The plants in the composite plant extract include white truffle, white willow bark and chrysanthemum; the strains in the composite probiotic include a combination of Streptococcus thermophilus, Saccharomyces boulardii, Lactobacillus plantarum YK-L002 strain with a preservation number of CGMCC No. 29289, and Saccharomyces cerevisiae YK-S001 strain with a preservation number of CGMCC No. 28276.

2. The preparation method according to claim 1, characterized in that The mass ratio of the white truffle, white willow bark and chrysanthemum is (2-6):(0.5-3):(1-4).

3. The preparation method according to claim 1, characterized in that The mass ratio of the composite plant extract, inorganic salt and water is (4-10):(0.1-1):(8-16).

4. The preparation method according to claim 1, characterized in that The composite plant extract is prepared by a preparation method comprising the following steps: mixing white truffle, white willow bark and chrysanthemum with a composite enzyme and water for enzymatic hydrolysis, performing high-temperature extraction after the enzymatic hydrolysis, filtering the extract and collecting the filtrate and filter residue respectively to obtain the composite plant extract; The complex enzyme comprises a combination of lysase, β-glucanase and papain.

5. The preparation method according to claim 1, characterized in that The ratio of the viable counts of the thermophilic Streptococcus, the boulardii yeast, the Lactobacillus plantarum YK-L002 strain, and the Saccharomyces cerevisiae YK-S001 strain is (0.5-5):(8-15):(2-8):(10-20); The live bacterial concentration of the thermophilic streptococcus is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

6. The preparation method according to claim 1, characterized in that The inoculation amount of the composite probiotic in the culture matrix is ​​5%-12%.

7. The preparation method according to claim 1, characterized in that The fermentation temperature is 25-38° C. and the fermentation time is 70-120 h.

8. The preparation method according to claim 1, characterized in that The preparation method specifically comprises the following steps: The filter residue of the first composite plant extract and an inorganic salt are mixed and sterilized to obtain a sterilized culture matrix, Streptococcus thermophilus and Lactobacillus plantarum YK-L002 strain are inoculated into the matrix for a first fermentation culture, the first fermentation culture product, the filtrate of the first composite plant extract, and the filtrate of the second composite plant extract are mixed and sterilized, and then Saccharomyces boulardii and Saccharomyces cerevisiae YK-S001 strain are inoculated for a second fermentation culture, and the second culture product is centrifuged, filtered, and sterilized to obtain a white truffle fermentation product; The plants in the first composite plant extract include white truffle and white willow bark; The plants in the second composite plant extract include chrysanthemum.

9. A white truffle fermentation product obtained according to the preparation method according to any one of claims 1 to 8.

10. Use of the white truffle fermentation product according to claim 9 in the preparation of cosmetics.

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