Preparation method of truffle fermentation product with antioxidant effect, product and application thereof

Abstract

The present application relates to a kind of preparation method of truffle fermentation product with antioxidant effect and its product and application, the preparation method includes the following steps: composite plant extract, inorganic salt, water and composite probiotic agent are mixed to carry out fermentation culture, and the culture product is obtained truffle fermentation product by centrifugation, filtration, sterilization;Strain in the composite probiotic agent includes the combination of streptococcus thermophilus, brady yeast, plant lactobacillus YK-L002 strain and saccharomyces cerevisiae YK-S001 strain.The specific strain of the present application is compounded and combined fermentation, and the fermentation product filtrate after four kinds of bacteria fermentation treatment, with excellent skin conditioning effect, can effectively improve skin state, significantly antioxidant and resist weakening wrinkle, it has excellent effect when being applied in moisturizing antioxidant cosmetic.

Description

TECHNICAL FIELD

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

[0002] Due to long-term exposure of the skin to sunlight, air pollution and computer radiation, the number of free radicals in the body gradually increases, and under the action of free radicals, collagen will react, resulting in insufficient nutrient supply to the skin, leading to decreased skin tissue vitality, loss of elasticity, wrinkles, and other conditions such as skin aging and dull complexion. Skin aging mainly starts from the attack of free radicals on healthy cells.

[0003] Plant extracts in skin care products are active ingredients extracted from plants, which can play the roles of whitening, freckle removal, antioxidant, skin cell activation, anti-inflammatory and skin soothing after scientific extraction and processing. Moreover, the components are natural and green, friendly to the environment, and more mild, which will not irritate the skin or cause allergic reactions, can be more easily absorbed and metabolized by the body, and will not cause adverse effects on the body. Active ingredients such as polysaccharides, biological enzymes or collagen produced by fermentation of probiotic strains have important effects on delaying skin aging, providing skin nutrients, promoting skin cell regeneration and eliminating skin wrinkles.

[0004] Therefore, how to provide a plant-microorganism co-cultured fermentation product that can effectively improve the skin condition and has significant antioxidant effect has important application value. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a preparation method of white truffle fermentation product with antioxidant effect, and a product and application thereof.

[0006] To achieve the object of the present application, the following technical solutions are adopted:

[0007] In a first aspect, the present application provides a preparation method of white truffle fermentation product with antioxidant effect, which comprises the following steps:

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

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

[0010] In the present application, it is found that the combination of Streptococcus thermophilus, Saccharomyces boulardii, Lactiplantibacillus plantarum YK-L002 strain and Saccharomyces cerevisiae YK-S001 strain has potential interaction between the strains, and can work together and synergistically. The fermentation product filtrate after fermentation treatment of the four strains has excellent skin conditioning effect, can effectively improve the skin condition and resist weak wrinkles, and has excellent effect when applied to moisturizing and antioxidant cosmetic products.

[0011] In addition, it is further found in the present application that the fermentation substrate prepared from pure plant raw materials can not only improve the related skin condition conditioning effect of the final fermentation product, but also can further promote the fermentation of probiotics and improve the content of active ingredients in the fermentation product. The three plant components also have a potential synergistic relationship.

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

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

[0014] For example, the above "2-6" can be 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, etc.

[0015] For example, the above "0.5-3" can be 0.5, 1, 1.5, 2, 2.5, 3, etc.

[0016] For example, the above "1-4" can be 1, 1.5, 2, 2.5, 3, 3.5, 4, etc. Other specific point values within the above numerical range can also be selected, which will not be described here.

[0017] In the present application, when the amount of the three plant components is within the above specific range, the final fermentation product has better antioxidant and other skin conditioning effects. In the present application, when the amount of the three plant components is within the above specific range, the final fermentation product has better antioxidant and other skin conditioning effects.

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

[0019] For example, the "4-10" can be 4, 5, 6, 7, 8, 9, 10, etc.

[0020] For example, the "0.1-1" can be 0.1, 0.2, 0.5, 0.6, 0.8, 0.9, 1, etc.

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

[0022] The composite plant extract involved in the present application is not limited to its specific preparation process and source. The product prepared by conventional extraction process and the regular commercially available product can be used in the present application. For example, the plant raw material can be crushed and mixed with water, heated and extracted at 70-95℃ for 0.5-3h, the extraction liquid is filtered, and the filtrate or filter residue is collected, thereby obtaining the product. When the product is prepared by the following process, the fermentation substrate obtained has better effect for culturing the related probiotics, and the fermentation product has better skin improvement effect.

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

[0024] The white truffle, white willow bark and chrysanthemum are mixed with composite enzymes and water for enzymolysis, and then high-temperature extraction is carried out after the enzymolysis is completed. The filtrate and filter residue are collected respectively after the filtration of the extraction liquid, thereby obtaining the composite plant extract.

[0025] The composite enzyme comprises a combination of lyticase, beta-glucanase and papain.

[0026] In the present application, it is unexpectedly found that when the plant raw material is treated by the composite enzyme of lyticase, beta-glucanase and papain, the combination of the three enzymes can have synergistic effect, and better extraction effect can be obtained. The extract prepared has higher activity and has better effect. In addition, high-temperature extraction is carried out after enzymolysis, which not only can realize the mild inactivation of biological enzymes, but also can further extract the active ingredients in the plant for the second time, thereby obtaining the plant extract with better effect.

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

[0028] For example, the "0.5-2" can be 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, etc.

[0029] "2-6" may be 2, 2.5, 3, 3.5, 4, 4. 5, 5, 5.5, 6, etc.

[0030] "1-3" may be 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, etc., and other specific point values in the numerical range can be selected, which will not be repeated here.

[0031] Preferably, the amount of the complex enzyme added 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., and other specific point values in the numerical range can be selected, which will not be repeated here.

[0032] Preferably, the 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., and other specific point values in the numerical range can be selected, which will not be repeated here.

[0033] Preferably, the temperature of the enzymatic hydrolysis is 50-60℃, for example, it can be 50℃, 52℃, 55℃, 58℃, 60℃, etc.; the time is 0.5-2h, for example, it can be 0.5h, 0.8h, 1h, 1.2h, 1.5h, 1.8h, 2h, etc., and other specific point values in the numerical range can be selected, which will not be repeated here.

[0034] Preferably, the temperature of the high-temperature extraction is 75-90℃, for example, it can be 75℃, 78℃, 80℃, 85℃, 90℃, etc.; the time is 1-3h, for example, it can be 1h, 1.2h, 1.5h, 1.8h, 2h, 2.5h, 3h, etc., and other specific point values in the numerical range can be selected, which will not be repeated here.

[0035] Preferably, the ratio of the number of live bacteria of the Streptococcus thermophilus, the Kluyveromyces, the Lactiplantibacillus plantarum YK-L002 strain, and the Saccharomyces cerevisiae YK-S001 strain is (0.5-5):(8-15):(2-8):(10-20).

[0036] Among them, the above "0.5-5" may be 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.

[0037] The above "8-15" may be 8, 9, 10, 11, 12, 13, 14, 15, etc.

[0038] The above-mentioned "2-8" may be, for example, 2, 3, 4, 5, 5.5, 6, 7, 8, and the like; and other specific point values within the numerical range may be selected, which will not be repeated here.

[0039] The above-mentioned "10-20" may be, for example, 10, 12, 14, 15, 17, 18, 20, and the like; and other specific point values within the numerical range may be selected, which will not be repeated here.

[0040] In the present application, when the four bacteria of Streptococcus thermophilus, Saccharomyces boulardii, Lactobacillus plantarum YK-L002 strain and Saccharomyces cerevisiae YK-S001 strain are inoculated and fermented in the above-mentioned specific viable bacteria ratio, the fermented product prepared has better efficacy.

[0041] Preferably, the viable bacteria concentration of the Streptococcus thermophilus is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g, for example, 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), and the like; and other specific point values within the numerical range may be selected, which will not be repeated here.

[0042] Preferably, the inoculation amount of the complex probiotic agent in the culture medium is 5%-12%, for example, 5%, 6%, 7%, 8%, 9%, 9.5%, 10%, 12%, and the like; and other specific point values within the numerical range may be selected, which will not be repeated here.

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

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

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

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

[0047] Preferably, the plants in the second composite plant extract include chrysanthemum.

[0048] The temperature of the first fermentation culture is 32-38 DEG C, for example, 32 DEG C, 33 DEG C, 34 DEG C, 35 DEG C, 36 DEG C, 37 DEG C, 37.5 DEG C, 38 DEG C, etc., and 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 DEG C, for example, 25 DEG C, 26 DEG C, 27 DEG C, 28 DEG C, 29 DEG C, 30 DEG C, 32 DEG C, 35 DEG C, etc., and 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 application provides a white truffle fermentation product prepared by the preparation method according to the first aspect.

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

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

[0053] In the present application, the four strains of Streptococcus thermophilus, Saccharomyces boulardii, Lactobacillus plantarum YK-L002 and Saccharomyces cerevisiae YK-S001 are inoculated into the pure plant culture medium prepared from white truffle, white willow bark and chrysanthemum to prepare the related fermentation product, and it is found that the four strains can interact with each other and positively affect each other, and have a synergistic promotion effect on the efficacy of improving the skin state, such as anti-wrinkle, moisturizing and antioxidant, which is unpredictable by the skilled in the art, and the application in the cosmetic product can further improve the related efficacy of the product. And the three plant components in the culture medium also have a potential interaction relationship, which can further improve the fermentation efficiency and optimize the product efficacy.

[0054] The YK-L002 strain involved in the present application is named Lactiplantibacillus plantarum in classification, and is preserved in China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 29289, a preservation date of December 11, 2023, and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing;

[0055] The YK-S001 strain involved in the present application is named Saccharomyces cerevisiae in classification, and is preserved in China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 28276, a preservation date of August 28, 2023, and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0056] In order to further illustrate the technical means adopted by the present application and its effects, the technical solutions of the present application will be further described below in combination with preferred embodiments of the present application, but the present application is not limited in the scope of the embodiments.

[0057] The following relates to the strain information:

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

[0059] ② The YK-L002 strain is named Lactiplantibacillus plantarum in classification, and has a preservation number of CGMCC No. 29289;

[0060] ③ The YK-S001 strain is named Saccharomyces cerevisiae in classification, and has a preservation number of CGMCC No. 28276;

[0061] ④ The Blakeslea trispora 01 strain is purchased from Guopei Biotechnology (Shanxi) Co., Ltd.;

[0062] The Blakeslea trispora 02 strain is purchased from Ningke (Xi'an) Biological Medicine Technology Co., Ltd.;

[0063] The Blakeslea trispora SHBCC D25151 strain is purchased from Shanghai Collection and Preservation Center of Microorganisms.

[0064] The following relates to the biological enzyme information: lysozyme (S10112), beta-glucanase (S23089) and papain (S10011), all of which are purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.

[0065] Preparation Example 1

[0066] The present preparation example provides a composite plant extract prepared by the following method:

[0067] (1) The first composite plant extract: fresh white truffle is homogenized into a paste, white willow bark is crushed and sieved, and then mixed with the white truffle paste, a composite enzyme, and pure water. The mass ratio of white truffle to white willow bark is 4:1, the addition amount of the composite enzyme is 1 ‰ of the total mass of white truffle and white willow bark, and the solid-liquid ratio of white truffle and white willow bark to water is 1 g:65 mL. The composite enzyme is composed of lysozyme, β-glucanase, and papain at a mass ratio of 1:4:2. After enzymolysis at 55°C for 1 h, the temperature is raised to 85°C for further extraction for 2 h. The extraction liquid is filtered, and the filtrate and the residue are collected separately. The filtrate is concentrated and freeze-dried.

[0068] (2) The second composite plant extract: dried and crushed chrysanthemum raw materials are mixed with a composite enzyme and pure water. The addition mass ratio of chrysanthemum raw materials to white willow bark raw materials in the first composite extract is 1:3, the addition amount of the composite enzyme is 1 ‰ of the mass of chrysanthemum, and the solid-liquid ratio of chrysanthemum to water is 1 g:65 mL. The composite enzyme is composed of lysozyme, β-glucanase, and papain at a mass ratio of 1:4:2. After enzymolysis at 55°C for 1 h, the temperature is raised to 85°C for further extraction for 2 h. The extraction liquid is filtered, and the filtrate is concentrated and freeze-dried.

[0069] Preparation Example 2

[0070] The present preparation example provides a composite plant extract prepared by the following method:

[0071] (1) The first extract: fresh white truffle is homogenized into a paste, white willow bark is crushed and sieved, and then mixed with the white truffle paste, a composite enzyme, and pure water. The mass ratio of white truffle to white willow bark is 2:3, the addition amount of the composite enzyme is 0.5 ‰ of the total mass of white truffle and white willow bark, and the solid-liquid ratio of white truffle and white willow bark to water is 1 g:80 mL. The composite enzyme is composed of lysozyme, β-glucanase, and papain at a mass ratio of 2:2:3. After enzymolysis at 58°C for 0.8 h, the temperature is raised to 72°C for further extraction for 2.5 h. The extraction liquid is filtered, and the filtrate and the residue are collected separately. The filtrate is concentrated and freeze-dried.

[0072] (2) The second extract: dried and crushed chrysanthemum raw materials are mixed with a composite enzyme and pure water. The addition mass ratio of chrysanthemum raw materials to white willow bark raw materials in the first composite extract is 3:1, the addition amount of the composite enzyme is 0.5 ‰ of the mass of chrysanthemum, and the solid-liquid ratio of chrysanthemum to water is 1 g:80 mL. The composite enzyme is composed of lysozyme, β-glucanase, and papain at a mass ratio of 2:2:3. After enzymolysis at 58°C for 0.8 h, the temperature is raised to 72°C for further extraction for 2.5 h. The extraction liquid is filtered, and the filtrate is concentrated and freeze-dried.

[0073] Preparation Example 3

[0074] The present preparation example provides a composite plant extract prepared by the following method:

[0075] (1) First extract: fresh white truffle is homogenized into a paste, white willow bark is crushed and sieved, and then mixed with the white truffle paste, composite enzyme, and pure water. The mass ratio of white truffle to white willow bark is 6:0.5, the addition amount of composite enzyme is 2‰ of the total mass of white truffle and white willow bark, and the solid-liquid ratio of white truffle and white willow bark to water is 1g:55mL. The composite enzyme is composed of lysozyme, β-glucanase, and papain in a mass ratio of 0.5:5:1. After enzymolysis at 52℃ for 1.8h, the temperature is raised to 88℃ for further extraction for 1.2h. The extraction solution is filtered, and the filtrate and residue are collected separately. The filtrate is concentrated and freeze-dried.

[0076] (2) Second extract: dried and crushed chrysanthemum raw materials are mixed with composite enzyme and pure water. The addition mass ratio of chrysanthemum raw materials to white willow bark raw materials in the first composite extract is 2:1, the addition amount of composite enzyme is 2‰ of the mass of chrysanthemum, and the solid-liquid ratio of chrysanthemum to water is 1g:55mL. The composite enzyme is composed of lysozyme, β-glucanase, and papain in a mass ratio of 0.5:5:1. After enzymolysis at 52℃ for 1.8h, the temperature is raised to 88℃ for further extraction for 1.2h. The extraction solution is filtered, and the filtrate is concentrated and freeze-dried.

[0077] Preparation Example 4

[0078] The present preparation example provides a composite plant extract (containing first extract and second extract) using plant raw materials (white truffle, white willow bark, and chrysanthemum) in Preparation Example 1. The difference between the preparation method and Preparation Example 1 is only in the enzyme composition of the composite enzyme in steps (1) and (2). The difference is that neither contains lysozyme, and the reduced share is proportionally distributed to β-glucanase and papain. The composite enzyme used in the first extract and the second extract is the same, and other processing techniques and related parameters are referred to Preparation Example 1.

[0079] Preparation Example 5

[0080] The present preparation example provides a composite plant extract (containing first extract and second extract) using plant raw materials (white truffle, white willow bark, and chrysanthemum) in Preparation Example 1. The difference between the preparation method and Preparation Example 1 is only in the enzyme composition of the composite enzyme in steps (1) and (2). The difference is that neither contains β-glucanase, and the reduced share is proportionally distributed to lysozyme and papain. The composite enzyme used in the first extract and the second extract is the same, and other processing techniques and related parameters are referred to Preparation Example 1.

[0081] Preparation Example 6

[0082] The present 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, and the preparation method is different from that of Preparation Example 1 only in that the enzyme composition in the composite enzyme in steps (1) and (2) is different, and the difference is that neither contains papain, and the reduced share is proportionally distributed to the collapse enzyme and the beta-glucanase. The composite enzyme used in the first extract and the second extract is the same, and the other processing technology and related parameters are referred to Preparation Example 1.

[0083] Preparation Example 7

[0084] The present preparation example provides a composite plant extract, which is different from Example 1 only in that steps (1) and (2) do not perform enzymatic hydrolysis treatment, and the plant raw materials are mixed with water and then directly subjected to high-temperature extraction. The extraction conditions and the remaining parameters and treatments are referred to Example 1 to prepare the first extract and the second extract, respectively.

[0085] Comparative Preparation Example 1

[0086] The present comparative preparation example provides a composite plant extract, which is different from Preparation Example 1 only in that the white truffle raw material is not added, and the reduced share is proportionally distributed to the white willow bark and chrysanthemum components. The other ingredients and contents remain unchanged, and the preparation method is referred to Preparation Example 1.

[0087] Comparative Preparation Example 2

[0088] The present comparative preparation example provides a composite plant extract, which is different from Preparation Example 1 only in that the white willow bark raw material is not added, and the reduced share is proportionally distributed to the white truffle and chrysanthemum components. The other ingredients and contents remain unchanged, and the preparation method is referred to Preparation Example 1.

[0089] Comparative Preparation Example 3

[0090] The present comparative preparation example provides a composite plant extract, which is different from Preparation Example 1 only in that the chrysanthemum raw material is not added, and the reduced share is proportionally distributed to the white truffle and white willow bark components. The other ingredients and contents remain unchanged, and the preparation method is referred to Preparation Example 1.

[0091] Example 1

[0092] The present example provides a white truffle fermentation product, and the preparation method is as follows:

[0093] (1) The filtrate of the first extract (Preparation Example 1) is mixed with dipotassium hydrogen phosphate and sterilized (121℃, 30 min), and then Streptococcus thermophilus ATCC 19258 strain and Lactobacillus plantarum YK-L002 strain are inoculated into it for solid-state fermentation, and cultured at 36℃ for 60 h. After the culture is completed, it is sterilized at 80℃ to obtain a fermentation substrate;

[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, the Saccharomyces boulardii 01 strain and the Saccharomyces cerevisiae YK-S001 strain were inoculated, and the culture was stirred at 32°C and 100 rpm for 36 h to obtain a fermentation culture product.

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

[0096] (3) The fermentation culture product was centrifuged, filtered, sterilized (0.1 μm filtration), and lyophilized to obtain a white truffle fermentation product.

[0097] Example 2

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

[0099] (1) The 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 strain and Lactobacillus plantarum YK-L002 strain were inoculated for solid-state fermentation, and the culture was incubated at 38°C for 48 h. After the culture was completed, it was sterilized at 80°C to obtain a fermentation substrate.

[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, the Saccharomyces boulardii 01 strain and the Saccharomyces cerevisiae YK-S001 strain were inoculated, and the culture was stirred at 32°C and 100 rpm for 36 h to obtain a fermentation culture product.

[0101] The adding amount of each component is: 5 parts of the composite plant extract (first extract filtrate + first extract residue + second extract filtrate), 1 part of di-potassium hydrogen phosphate, and 15 parts of water; wherein the total inoculation amount of the four strains in the culture medium is 6% (the mass ratio of the first inoculation and the second inoculation is 1:1), and the viable cell count ratio of Streptococcus thermophilus, Saccharomyces boulardii 02 strain, Lactobacillus plantarum YK-L002 strain and Saccharomyces cerevisiae YK-S001 strain 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] The present example provides a white truffle fermentation product, and the preparation method thereof is as follows:

[0105] (1) The residue of the first extract (Preparation Example 3) is mixed with anhydrous magnesium sulfate and sterilized (121℃, 30 min), and then Streptococcus thermophilus CICC 22561 strain and Lactobacillus plantarum YK-L002 strain are inoculated for solid-state fermentation, and cultured at 33℃ for 68 h, and then sterilized at 80℃ after the culture is completed to obtain a fermentation medium;

[0106] (2) The fermentation medium, the filtrate (freeze-dried powder) of the first extract (Preparation Example 3), the filtrate (freeze-dried powder) of the second extract (Preparation Example 3) and water are mixed, the pH is adjusted to 6.5, Saccharomyces boulardii SHBCC D25151 strain and Saccharomyces cerevisiae YK-S001 strain are inoculated, and cultured at 32℃ with 125 rpm stirring for 28 h to obtain a fermentation culture product.

[0107] The adding amount of each component is: 5 parts of the composite plant extract (first extract filtrate + first extract residue + second extract filtrate), 1 part of di-potassium hydrogen phosphate, and 15 parts of water; wherein the total inoculation amount of the four strains in the culture medium is 6% (the mass ratio of the first inoculation and the second inoculation is 1:1), and the viable cell count ratio of Streptococcus thermophilus, Saccharomyces boulardii 02 strain, Lactobacillus plantarum YK-L002 strain and Saccharomyces cerevisiae YK-S001 strain is 5:8:7:10, wherein 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] The present example provides four kinds of white truffle fermentation products, which are only different from the example 1 in that the composite plant extract prepared in the preparation example 1 is respectively replaced by an equal amount of the extract prepared in the preparation examples 4-7, and the other ingredients and contents remain unchanged, and the preparation method refers to example 1.

[0111] Comparative examples 1-3

[0112] The present comparative example provides three kinds of white truffle fermentation products, which are only different from the example 1 in that the composite plant extract prepared in the preparation example 1 is respectively replaced by an equal amount of the extract prepared in the comparative preparation examples 1-3, and the other ingredients and contents remain unchanged, and the preparation method refers to example 1.

[0113] Comparative example 4

[0114] The present comparative example provides a white truffle fermentation product, which uses the composite plant extract raw material of example 1, and the difference between its preparation method and example 1 is only that no streptococcus thermophilus strain is added in the fermentation process, and the reduced share is proportionally distributed to lactobacillus plantarum YK-L002 strain, bradyzyme 01 strain and saccharomyces cerevisiae YK-S001 strain, and the rest of the raw materials, process and parameters refer to example 1.

[0115] Comparative example 5

[0116] The present comparative example provides a white truffle fermentation product, which uses the composite plant extract raw material of example 1, and the difference between its preparation method and example 1 is only that no lactobacillus plantarum YK-L002 strain is added in the fermentation process, and the reduced share is proportionally distributed to streptococcus thermophilus strain, bradyzyme 01 strain and saccharomyces cerevisiae YK-S001 strain, and the rest of the raw materials, process and parameters refer to example 1.

[0117] Comparative example 6

[0118] The present comparative example provides a white truffle fermentation product, which uses the composite plant extract raw material of example 1, and the difference between its preparation method and example 1 is only that no bradyzyme 01 strain is added in the fermentation process, and the reduced share is proportionally distributed to streptococcus thermophilus strain, lactobacillus plantarum YK-L002 strain and saccharomyces cerevisiae YK-S001 strain, and the rest of the raw materials, process and parameters refer to example 1.

[0119] Comparative example 7

[0120] The comparative example provides a white truffle fermentation product using the composite plant extract raw material of Example 1, and the difference between the preparation method and Example 1 is only that the Saccharomyces cerevisiae YK-S001 strain is not added in the fermentation process, and the reduced share is proportionally distributed to the Streptococcus thermophilus strain, the Lactobacillus plantarum YK-L002 strain and the Kluyveromyces sp. 01 strain, and the rest of the raw materials, process and parameters are referred to Example 1.

[0121] Test Example 1

[0122] Anti-wrinkle efficacy test

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

[0124] (2) Test method: the elastase inhibitory effect of the sample is tested according to the laboratory method (HMC-WI-028 elastase inhibition rate), and the reagent involved is elastase (pig pancreas, BR); substrate: N-succinyl-L-alanyl-L-alanyl-L-alanine (98%). Set up sample group, sample background group, solvent group and solvent background group, and set up 3 parallel groups for each group. Add the corresponding different reagent solutions in the 96-well plate, shake gently, incubate at 25℃ for 15 min, and then place it in the enzyme marker, and measure the absorbance at 410 nm.

[0125] (3) Calculation formula:

[0126] In the formula: A is the absorbance of the reaction solution without the sample; B is the absorbance of the reaction solution without the sample and the enzyme; C is the absorbance of the reaction solution containing the sample and the enzyme; D is the absorbance of the reaction solution containing the sample and not containing the enzyme.

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

[0128] Table 1

[0129]

[0130] From the data in the table,

[0131] (1) From Examples 1 to 3, it can be known that the white truffle fermentation product provided by the present application can effectively inhibit elastase, wherein elastase is mainly synthesized and secreted by fibroblasts, and can degrade elastin in the skin to cause skin aging. Therefore, the inhibition rate of the experimental sample on elastase can reflect the anti-wrinkle efficacy of the product. ​

[0132] (2) By comparing Example 1 with Examples 4-7, it can be seen that the plant extract prepared by the method of combined enzymatic hydrolysis and high-temperature extraction in the present application has better anti-wrinkle effect when applied to the subsequent fermentation product culture extraction, and there is a potential synergistic relationship between the three enzymes of lysozyme, beta-glucanase and papain, which can synergistically enhance the extraction effect when used together, and the prepared extract has higher activity and better effect.

[0133] (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 extract components to the fermentation substrate of probiotics, the natural bioactive components rich in the three plants can further promote the fermentation of probiotics and increase the content of active ingredients in the fermentation product, and there is a potential synergistic relationship between the three plant components, which can jointly improve the anti-wrinkle effect of the fermentation product.

[0134] (4) By comparing Example 1 with Comparative Examples 4-7, it can be seen that the fermentation product prepared by the combined fermentation of Streptococcus thermophilus, Brettanomyces yeast, plant lactobacillus YK-L002 strain and Saccharomyces cerevisiae YK-S001 strain in the present application has an unpredictable synergistic promotion effect on the anti-wrinkle effect of the final product by the four strains.

[0135] Test Example 2

[0136] Antioxidant effect test

[0137] (1) DPPH-free radical scavenging

[0138] A 96-well plate was used, with three replicates in each group, and the system was 200 μL. Sample group: 100 μg of extract samples prepared by Examples 1-7 and Comparative Examples 1-7 were mixed with 100 μL of pure water, and 100 μL of DPPH solution (0.1 mM) was added to the reaction system. Control group: 100 μL of pure water was taken, and 100 μL of DPPH solution (0.1 mM) was added. After the reaction system was constructed, it was shaken in the dark for 10 min, and the absorbance at 517 nm was tested using an enzyme marker.

[0139] Scavenging rate calculation method: scavenging rate (%) = [(A0-Ax) / A0]x100%, A0 is the absorbance of the control group, and Ax is the absorbance of the sample group.

[0140] (2) ABTS-free radical scavenging

[0141] ABTS working solution preparation: ABTS and potassium persulfate solution were mixed to prepare 7 mM ABTS stock solution, which was stored in the dark for 12 h. After shaking, 10 mM PBS (pH = 7.4) was mixed to make the solution have an absorbance of 0.8 at 734 nm, thereby obtaining the ABTS working solution.

[0142] A 96-well plate was used, and three replicates were set for each group. The system was 200 μL. Sample group: 100 μg of the extract samples prepared in Examples 1-7 and Comparative Examples 1-7 were mixed with 10 μL of pure water, and 190 μL of the ABTS working solution was added to the reaction system. Control group: 10 μL of pure water was taken, and 190 μL of the ABTS working solution was added. After the reaction system was constructed, it was incubated for 6 min after being shaken thoroughly, and the absorbance at 734 nm was tested by an enzyme marker.

[0143] Method for calculating clearance rate: clearance rate (%) = [(A0-Ax) / A0] x 100%, A0 is the absorbance of the control group, and Ax is the absorbance of the sample group.

[0144] The test results are shown in Table 2.

[0145] Table 2

[0146]

[0147] As can be seen from the data in the table, the white truffle fermentation product prepared by the preparation method has excellent DPPH-free radical and ABTS-free radical scavenging effect, and exhibits significant antioxidant effect. 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 used in the present application have different degrees of influence on the antioxidant efficacy of the final extraction product.

[0148] Test Example 3

[0149] Product safety test

[0150] According to the subject selection criteria, 28 people were selected to participate in the test, with a male to female ratio of 1:1, and were randomly divided into 7 groups (2 males and 2 females in each group), and were given the test. The fermentation product prepared in Examples 1-7 was mixed with pure water to adjust the concentration to 2%, and then an appropriate amount was placed in a patch tester. The patch tester with the sample was attached to the skin on the curved side of the forearm of the test subject with medical tape, and lasted for 24 h. After removing the patch tester, the skin reaction was observed at 30 min (after the indentation disappeared), 24 h and 48 h, respectively, and the skin reaction results were recorded. The skin reaction grading standard is as follows:

[0151]

[0152] The results show that the products prepared in Examples 1-7 show negative reaction at 30 min after the test (after the indentation disappears), 24 h and 48 h, and no irritation and erythema phenomenon. It can be seen that the white truffle fermentation product prepared by the present application has no irritation to the skin and no positive reaction, proving that the product has high safety degree, small irritation and is relatively mild.

[0153] The applicant declares that the technical solutions of the present application are illustrated by the above examples, but the present application is not limited to the above examples, that is, it does not mean that the present application must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

[0154] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above embodiments, and within the technical concept scope of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0155] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

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 first plant extract residue and inorganic salts are mixed and sterilized to obtain a sterilized culture matrix, and Streptococcus thermophilus and Lactobacillus plantarum with a preservation number of CGMCC No. 29289 are inoculated into the matrix. Lactiplantibacillus plantarum The YK-L002 strain was subjected to a first fermentation culture, and the first fermentation culture product was mixed with the lyophilized powder of the filtrate of the first plant extract, the lyophilized powder of the filtrate of the second plant extract, and solvent water, and then sterilized and inoculated with Saccharomyces boulardii and Saccharomyces cerevisiae with a preservation number of CGMCC No. 28276. Saccharomyces cerevisiae The YK-S001 strain is subjected to a second fermentation culture, and the second culture product is centrifuged, filtered, and sterilized to obtain a white truffle fermentation product; The first plant extract is prepared by a preparation method comprising the following steps: mixing white truffle and white willow bark with a first complex enzyme and water for enzymatic hydrolysis, performing high-temperature extraction after the enzymatic hydrolysis, filtering the extract, collecting a filtrate and a filter residue, and obtaining the first plant extract; The second plant extract is prepared by a preparation method comprising the following steps: mixing chrysanthemum with a second complex enzyme and water for enzymatic hydrolysis, performing high-temperature extraction after the enzymatic hydrolysis, filtering the extract and collecting the filtrate to obtain the second plant extract; The first complex enzyme and the second complex enzyme each independently comprise a combination of collapsase, β-glucanase and papain in a mass ratio of (0.5-2):(2-6):(1-3); The mass ratio of the white truffle, white willow bark and chrysanthemum is (2-6):(0.5-3):(1-4); 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.

2. The preparation method according to claim 1, characterized in that The mass ratio of the total mass of the filtrate residue of the first plant extract and the filtrate of the second plant extract to the inorganic salt and the solvent water is (4-10):(0.1-1):(8-16).

3. 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.

4. The preparation method according to claim 1, wherein The first fermentation culture temperature is 32-38° C., and the time is 48-72 h; the second fermentation culture temperature is 25-35° C., and the time is 25-40 h.

5. A white truffle fermentation product obtained according to the preparation method according to any one of claims 1 to 4.

6. Use of the white truffle fermentation product according to claim 5 in the preparation of cosmetics.

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