Preparation method and application of micromolecular oat beta-glucan

Abstract

The invention discloses a preparation method and application of micromolecular oat beta-glucan, and the preparation method comprises the following steps: inoculating lactobacillus into a fermentation substrate composed of oat bran and water, and carrying out fermentation culture and sterilization to obtain the micromolecular oat beta-glucan, wherein the lactobacillus is composed of lactobacillus helveticus and lactobacillus plantarum, and the viable count ratio of the lactobacillus helveticus to the lactobacillus plantarum is 1: (0.5-2). The lactobacillus fermentation is utilized to improve the utilization rate of the oat bran and reduce the loss of active ingredients, so that the preparation method has the advantages of low cost, low energy consumption, environmental friendliness and the like, and can be directly used as an additive for preparing facial masks, essence or toner; the prepared product is superior to a product prepared by a traditional method in the aspects of DPPH free radical scavenging capacity, total antioxidant capacity, cytotoxicity, total sugar content, anti-inflammatory performance, molecular weight and the like, and has a good market application prospect.

Description

Technical Field

[0001] This application belongs to the technical field of cosmetics, and particularly relates to a preparation method and application of small molecule oat β-glucan. Background Art

[0002] With the progress of technology, consumers' requirements for cosmetics are increasing day by day. They not only expect products to have highly targeted and scientifically verified multi-effect functions, but also emphasize the use of natural ingredients and transparent ingredient source descriptions. Empowered by technology, the cosmetics industry is realizing personalized customization, efficacy verification, and environmentally friendly and sustainable development of products through means such as biotechnology and digital technology to meet consumers' needs for high-quality, safe, and effective cosmetics.

[0003] There are various production methods for oat β-glucan, including traditional extraction, biotechnology, physical and chemical methods, etc. The most common is the water extraction method for producing oat β-glucan. The water extraction method may encounter problems such as low extraction rate and purity, more complex operation process, higher cost, and possible damage to the biological activity of glucan when extracting oat glucan.

[0004] Therefore, there is an urgent need for a preparation method that can extract glucan with higher efficiency and purity, and also shows advantages in terms of economy and environmental friendliness. Summary of the Invention

[0005] The technical problem to be solved by this application is to overcome the high cost and low extraction rate of the existing oat β-glucan extraction method, and provide a preparation method and application of small molecule oat β-glucan. This application adopts the following technical solutions to solve the above technical problems: This application can extract the active ingredients in oat bran by means of lactic acid bacteria fermentation, which can not only reduce the loss of active ingredients, but also better exert its skin care effect. The small molecule oat β-glucan prepared by this application can be used directly as a cosmetic or added to the basic formula of cosmetics, and has high safety and good antioxidant, anti-inflammatory, and skin nourishing effects.

[0006] This application provides a preparation method of small molecule oat β-glucan, which includes the following steps: inoculating lactic acid bacteria into a fermentation substrate composed of oat bran and water, culturing by fermentation, and sterilizing.

[0007] Wherein, the lactic acid bacteria are composed of Lactobacillus helveticus and Lactobacillus plantarum, and the viable bacteria number ratio of Lactobacillus helveticus to Lactobacillus plantarum is 1:(0.5 - 2).

[0008] In some embodiments, the enzyme used for enzymatic hydrolysis is a heat-resistant amylase.

[0009] In some embodiments, the temperature of the enzymatic hydrolysis is 50 to 90 °C, preferably 60 to 70 °C.

[0010] In some embodiments, the time of the enzymatic hydrolysis is 20 to 40 min, preferably 25 to 35 min.

[0011] In some embodiments, the mass-volume ratio of the enzyme to the water is 0.5 to 2 mL / L, preferably 0.8 to 1.5 mL / L.

[0012] In some embodiments, the fermentation substrate may further include a sterilization operation before use, and the sterilization may be conventional in the art, generally the high-temperature sterilization method.

[0013] When using the high-temperature sterilization method to sterilize the fermentation substrate, the temperature of the sterilization may be the conventional temperature for such operations in the art, preferably 110 to 125 °C, more preferably 115 to 121 °C.

[0014] When using the high-temperature sterilization method to sterilize the fermentation substrate, the pressure of the sterilization may be conventional in the art, preferably 0.1 to 0.14 MPa, more preferably 0.12 to 0.13 MPa.

[0015] When using the high-temperature sterilization method to sterilize the fermentation substrate, the temperature of the sterilization may be conventional in the art, preferably 15 to 35 min, more preferably 15 to 25 min, such as 20 min.

[0016] Wherein, after the operation of sterilizing the fermentation substrate, a cooling operation may further be included. Preferably, the cooling may be cooling to room temperature.

[0017] In some embodiments, the Lactobacillus helveticus includes Lactobacillus helveticus deposited in the China Center of Industrial Culture Collection with the deposit number CICC 20243.

[0018] In some embodiments, the Lactiplantibacillus plantarum includes Lactiplantibacillus plantarum BSJM23L004. The Lactiplantibacillus plantarum BSJM23L004 is deposited in the China General Microbiological Culture Collection Center (abbreviation: CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101, deposit date: April 16, 2024, deposit number: CGMCC No. 30350.

[0019] In some embodiments, the Lactobacillus helveticus is added in the form of a Lactobacillus helveticus bacterial solution, and the viable count of the Lactobacillus helveticus is 10 5 ~10 10 CFU / mL, preferably 10 6 ~10 8 CFU / mL, for example 10 7 CFU / mL.

[0020] In some embodiments, the Lactobacillus plantarum is added in the form of a Lactobacillus plantarum bacterial solution, and the viable count of the Lactobacillus plantarum is 10 5 ~10 10 CFU / mL, preferably 10 6 ~10 8 CFU / mL, for example 10 7 CFU / mL.

[0021] In some embodiments, the fermentation culture can be anaerobic fermentation.

[0022] In some embodiments, the fermentation culture can be carried out in a constant temperature incubator according to the routine in the art.

[0023] In some embodiments, the temperature of the fermentation culture can be 35 - 50 °C, preferably 40 - 45 °C, for example 43 °C.

[0024] In some embodiments, the time of the fermentation culture can be 12 - 48 h, preferably 15 - 30 h.

[0025] In some embodiments, the mass ratio of the oat bran to the water is 1:(20 - 200), preferably 1:(50 - 150).

[0026] In some embodiments, when the high-temperature sterilization method is used for the sterilization, the temperature of the sterilization can be 110 - 125 °C, preferably 115 - 121 °C. When the high-temperature sterilization method is used for the sterilization, the pressure of the sterilization can be 0.1 - 0.14 MPa, preferably 0.12 - 0.13 MPa. When the high-temperature sterilization method is used for the sterilization, the time of the sterilization can be 15 - 35 min, preferably 15 - 25 min, more preferably 20 min.

[0027] In some embodiments, after the sterilization operation, centrifugation and collection of the supernatant can be further included.

[0028] Among them, the rotation speed of the centrifugation can be 3000 - 9000 rpm, preferably 4000 - 6000 rpm, more preferably 4800 rpm.

[0029] Among them, the radius of the centrifugation is 8 - 15 cm, preferably 10 cm.

[0030] Among them, the time of the centrifugation can be 10 - 40 min, preferably 20 - 40 min, more preferably 30 min.

[0031] Among them, after the centrifugation, the operations may further include at least one of decolorizing the supernatant, performing secondary sterilization, and mixing with a preservative.

[0032] The decolorizing agent used for the decolorization can be a decolorizing agent commonly used in the art. Preferably, it includes one or more of activated carbon, bentonite, and diatomaceous earth, and more preferably activated carbon.

[0033] The mass - volume ratio of the decolorizing agent to the supernatant is preferably 10 - 30 g / L, more preferably 15 - 25 g / L.

[0034] The stirring rate of the decolorization is 300 - 400 r / min, preferably 350 r / min.

[0035] The time of the decolorization is preferably 50 - 70 min, more preferably 55 - 65 min.

[0036] The method of the secondary sterilization can be conventional in the art, generally a high - temperature sterilization method. When using the high - temperature sterilization method for the secondary sterilization, the temperature of the secondary sterilization can be the temperature conventional for such operations in the art, preferably 115 - 125 °C, more preferably 118 - 121 °C. When using the high - temperature sterilization method for the secondary sterilization, the pressure of the secondary sterilization can be conventional in the art, and the sterilization pressure can be 0.1 - 0.14 MPa, preferably 0.12 - 0.13 MPa. When using the high - temperature sterilization method for the secondary sterilization, the time of the secondary sterilization can be conventional in the art, preferably 15 - 25 min, such as 20 min.

[0037] The preservative may include p - hydroxyacetophenone and 1,2 - hexanediol. When the preservative includes p - hydroxyacetophenone, the mass percentage of p - hydroxyacetophenone in the supernatant is 0.1% - 1%. When the preservative includes 1,2 - hexanediol, the mass percentage of 1,2 - hexanediol in the supernatant is 0.5% - 2%.

[0038] During the mixing with the preservative, the temperature of the mixing can be 50 - 80 °C, preferably 60 - 80 °C, such as 75 °C.

[0039] This application also provides a small - molecule oat β - glucan, which is prepared by the preparation method of the small - molecule oat β - glucan as described above.

[0040] The present application also provides the use of the above-mentioned small molecule oat β-glucan directly as a product, as an additive or as a substrate in the preparation of topical skin agents;

[0041] Preferably, the small molecule oat β-glucan serves as an antioxidant active ingredient in the topical skin agent. More preferably, the antioxidant active ingredient is an antioxidant active ingredient having DPPH free radical scavenging effect and total antioxidant capacity of FRAP.

[0042] Preferably, the small molecule oat β-glucan serves as an anti-inflammatory active ingredient in the topical skin agent. More preferably, the anti-inflammatory active ingredient is an anti-inflammatory active ingredient having the effect of reducing IL-8 and IL-6 inflammatory factors.

[0043] The present application also provides a topical skin agent, which comprises the above-mentioned small molecule oat β-glucan.

[0044] In some embodiments, the topical skin agent may further comprise active ingredients commonly used in the art, generally including at least one of antioxidant active ingredients, anti-aging active ingredients and anti-inflammatory active ingredients.

[0045] In some embodiments, the topical skin agent used may conventionally include, but is not limited to, facial masks, essences or toners in the art.

[0046] In some embodiments, the mass percentage of the small molecule oat β-glucan in the topical skin agent is 5% to 99%, preferably 60% to 99%.

[0047] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present application.

[0048] The reagents and raw materials used in the present application are all commercially available.

[0049] The positive and progressive effects of the present application are as follows: The small molecule oat β-glucan prepared in the present application has ideal antioxidant performance, can also reduce the loss of active ingredients in oat bran, improve the extraction efficiency, has a high utilization rate of oat bran, has a simple preparation process, mild fermentation conditions, low energy consumption and low cost, and effectively reduces the waste of oat bran resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The present application can be better understood by referring to the description given below in conjunction with the accompanying drawings. The accompanying drawings, together with the following detailed description, are included in this specification and form a part of this specification, and are used to further illustrate the preferred embodiments of the present application and explain the principles and advantages of the present application.

[0051] Wherein:

[0052] Figure 1 Graph for comparing the DPPH free radical scavenging abilities of Examples 1-5 and Comparative Examples 1-5;

[0053] Figure 2 Graph for comparing the total antioxidant capacities of Examples 1-5 and Comparative Examples 1-5;

[0054] Figure 3 Graph for comparing the results of cytotoxicity experiments on human immortalized epidermal cells of Examples 1-5 and Comparative Examples 1-5;

[0055] Figure 4 Graph for comparing the total sugar contents of Examples 1-5 and Comparative Examples 1-5;

[0056] Figure 5 Effect of Examples 1-5 and Comparative Examples 1-5 on the content of IL-8 inflammatory factor in human immortalized epidermal cells;

[0057] Figure 6 Effect of Examples 1-5 and Comparative Examples 1-5 on the content of IL-6 inflammatory factor in human immortalized epidermal cells;

[0058] Figure 7 Effect of Examples 1-5 and Comparative Examples 1-5 on the relative mRNA expression level of IL-8 in human immortalized epidermal cells;

[0059] Figure 8 Effect of Examples 1-5 and Comparative Examples 1-5 on the relative mRNA expression level of IL-6 in human immortalized epidermal cells.

[0060] Figure 9 Graph for comparing the molecular weights of Examples 1-5 and Comparative Examples 1-5. Detailed implementation manners

[0061] The present application will be further illustrated below by way of examples, but the present application is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0062] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0063] The thermostable amylase in the following examples and comparative examples is commercially available.

[0064] The oat bran in the following examples and comparative examples is commercially available.

[0065] Lactiplantibacillus plantarum BSJM23L004 in the following examples and comparative examples is classified and named as Lactiplantibacillus plantarum. It is deposited with the China General Microbiological Culture Collection Center (abbreviated as CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with a postal code of 100101. The deposit date is April 16, 2024, and the deposit number is CGMCC No. 30350.

[0066] Lactobacillus helveticus in the following examples and comparative examples is purchased from the China National Center for Industrial Culture Collection of Microorganisms, with a deposit number of CICC 20243.

[0067] Lactobacillus helveticus in the following comparative examples is purchased from the China General Microbiological Culture Collection Center (abbreviated as CGMCC), with a deposit number of CGMCC No. 19090.

[0068] Schizophyllum commune in the following comparative examples is classified and named as Schizophyllum commune. It is deposited with the China General Microbiological Culture Collection Center (abbreviated as CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with a postal code of 100101. The deposit date is March 9, 2023, and the deposit number is CGMCC NO. 40388.

[0069] Lacticaseibacillus paracasei in the following comparative examples is purchased from the China National Center for Industrial Culture Collection of Microorganisms, with a deposit number of CICC 20241.

[0070] The manufacturer of 0.25% (containing EDTA) trypsin in the following examples and comparative examples is GIBCO, USA; the manufacturer of MEM medium is GIBCO, USA.

[0071] The manufacturer of double antibody in the following effect examples is Corning, USA; the manufacturer of CCK-8 is Beijing Baierdi Biotechnology Co., Ltd.; the manufacturer of fetal bovine serum is GIBCO, USA.

[0072] The manufacturer of phosphate buffer solution in the following effect examples is Beijing Bairuiji Biotechnology Co., Ltd.

[0073] The manufacturer of the WJ-80A-II type CO2 incubator used in the following effect examples is Shanghai Shengke Instrument and Equipment Co., Ltd.; the manufacturer of the Sunrise microplate reader is Tecan Trading Co., Ltd.

[0074] The manufacturer of the TL80-2 type medical centrifuge used in the following effect examples is Jiangsu Tianli Medical Instrument Co., Ltd.; the manufacturer of the NUNC 96-well cell culture plate is Thermo Fisher Scientific.

[0075] Example 1

[0076] The commercially available oat bran was crushed and sieved to a particle size of 100 mesh. 3 g of oat bran powder and 300 mL of deionized water were taken, mixed evenly, and then high-temperature resistant amylase was added. Stirring enzymatic hydrolysis was carried out at 70 °C for 30 min. The addition ratio of high-temperature resistant amylase was 1 mL / L, and the stirring speed was 300 r / min. After the enzymatic hydrolysis was completed and mixed evenly, it was sterilized in a high-pressure sterilizer at 121 °C for 20 min. After sterilization, it was cooled to room temperature to obtain the initial fermentation system;

[0077] Lactobacillus helveticus and Lactobacillus plantarum were inoculated into the initial fermentation system for fermentation. The viable cell count of the Lactobacillus helveticus CICC20243 bacterial liquid was 10 7 CFU / mL, the inoculation amount was 7.5 mL, the viable cell count of the Lactobacillus plantarum bacterial liquid was 10 7 CFU / mL, the inoculation amount was 7.5 mL. After shaking evenly, it was statically cultured in an incubator at 43 °C for 15 h. After fermentation, the obtained fermentation broth was sterilized in a high-pressure steam sterilizer at 121 °C for 20 min. After sterilization, the fermentation broth was cooled to room temperature and centrifuged at a centrifugal speed of 4800 r / min for 30 min;

[0078] After centrifugation, the supernatant was taken. The supernatant was decolorized. The decolorizing agent was activated carbon. The mass-volume ratio of activated carbon to the supernatant was 20 g / L. The decolorizing stirring rate was 300 r / min, and the decolorizing time was 60 min. After decolorization, it was filtered once on a filter paper with a pore size of 12 μm, and then filtered twice on a filter paper with a pore size of 0.45 μm to remove the decolorizing agent. After decolorization, it was sterilized again at 121 °C and a pressure of 0.13 MPa for 20 min. After sterilization, a preservative needed to be added to the fermentation broth. During the mixing process with the preservative, the mixing temperature was 75 °C. Based on the mass of the original fermentation broth, 0.5% of p-hydroxyacetophenone and 0.8% of 1,2-hexanediol were added and mixed evenly to obtain small molecule oat β-glucan.

[0079] Example 2

[0080] Compared with Example 1, the only difference is that the fermentation time is 24 h, and other conditions are the same as those in Example 1. The specific operations are as follows:

[0081] The commercially available oat bran was crushed and sieved to a particle size of 100 mesh. 3 g of oat bran powder and 300 mL of deionized water were taken, and after mixing evenly, heat-resistant amylase was added. Stirring enzymatic hydrolysis was carried out at 70 °C for 30 min. The proportion of heat-resistant amylase was 1 mL / L, and the stirring speed was 300 r / min. After the enzymatic hydrolysis, the mixture was evenly mixed and sterilized in a high-pressure sterilizer at 121 °C for 20 min. After sterilization, it was cooled to room temperature to obtain an initial fermentation system;

[0082] Lactobacillus helveticus and Lactobacillus plantarum were inoculated into the initial fermentation system for fermentation. The viable cell count of the Lactobacillus helveticus CICC20243 bacterial liquid was 10 7 CFU / mL, the inoculation amount was 7.5 mL, the viable cell count of the Lactobacillus plantarum bacterial liquid was 10 7 CFU / mL, the inoculation amount was 7.5 mL. After shaking well, it was statically cultured in an incubator at 43 °C for 24 h. After fermentation, the obtained fermentation broth was sterilized in a high-pressure steam sterilizer at 121 °C for 20 min. After sterilization, the fermentation broth was cooled to room temperature and centrifuged at a centrifugal speed of 4800 r / min for 30 min;

[0083] After centrifugation, the supernatant was taken. The supernatant was decolorized. The decolorizing agent was activated carbon. The mass-volume ratio of activated carbon to the supernatant was 20 g / L. The decolorizing stirring rate was 300 r / min, and the decolorizing time was 60 min. After decolorization, it was filtered once on a filter paper with a pore size of 12 μm, and then filtered twice on a filter paper with a pore size of 0.45 μm to remove the decolorizing agent. After decolorization, it was sterilized again at 121 °C and a pressure of 0.13 MPa for 20 min. After sterilization, a preservative needed to be added to the fermentation broth. During the mixing with the preservative, the mixing temperature was 75 °C. Based on the mass of the original fermentation broth, 0.5% of p-hydroxyacetophenone and 0.8% of 1,2-hexanediol were added and mixed evenly to obtain small-molecule oat β-glucan.

[0084] Example 3

[0085] Compared with Example 1, the only difference is that the dosage of oat bran is 5 g, and other conditions are the same as those in Example 1. The specific operations are as follows:

[0086] The commercially available oat bran was crushed and sieved to a particle size of 100 mesh. 5 g of oat bran and 300 mL of deionized water were taken, mixed evenly, and then a heat-resistant amylase was added. Stirring enzymatic hydrolysis was carried out at 70 °C for 30 min. The proportion of the heat-resistant amylase was 1 mL / L, and the stirring speed was 300 r / min. After the enzymatic hydrolysis was completed, it was mixed evenly and sterilized in a high-pressure sterilizer at 121 °C for 20 min. After sterilization, it was cooled to room temperature to obtain the initial fermentation system;

[0087] Lactobacillus helveticus and Lactobacillus plantarum were inoculated into the initial fermentation system for fermentation. Among them, the viable cell count of the Lactobacillus helveticus CICC20243 bacterial liquid was 10 7 CFU / mL, and the inoculation amount was 7.5 mL. The viable cell count of the Lactobacillus plantarum bacterial liquid was 10 7 CFU / mL, and the inoculation amount was 7.5 mL. After shaking well, it was statically cultured in an incubator at 43 °C for 15 h. After the fermentation was completed, the obtained fermentation broth was sterilized in a high-pressure steam sterilizer at 121 °C for 20 min. After sterilization was completed, the fermentation broth was cooled to room temperature and centrifuged. The centrifugation speed was 4800 r / min, and the centrifugation time was 30 min;

[0088] After centrifugation, the supernatant was taken. The supernatant was subjected to decolorization treatment. The decolorizing agent was activated carbon. The mass-to-volume ratio of the activated carbon to the supernatant was 20 g / L. The decolorization stirring rate was 300 r / min, and the decolorization time was 60 min. After decolorization was completed, it was subjected to primary suction filtration on a filter paper with a pore size of 12 μm, and then secondary suction filtration on a filter paper with a pore size of 0.45 μm to remove the decolorizing agent; After decolorization, secondary sterilization was carried out at a temperature of 121 °C and a pressure of 0.13 MPa for 20 min. After sterilization was completed, a preservative needed to be added to the fermentation broth. During the mixing process with the preservative, the mixing temperature was 75 °C. Based on the mass of the original fermentation broth, 0.5% of p-hydroxyacetophenone and 0.8% of 1,2-hexanediol were added and mixed evenly to obtain small molecule oat β-glucan.

[0089] Example 4

[0090] Compared with Example 1, the difference is only that the addition ratio of Lactobacillus helveticus and Lactobacillus plantarum is 1:2, and other conditions are the same as those in Example 1. The specific operation is as follows:

[0091] The commercially available oat bran was crushed and sieved to a particle size of 100 mesh. 300 mL of deionized water and 3 g of oat bran were taken, mixed evenly, and then a heat-resistant amylase was added. Stirring enzymatic hydrolysis was carried out at 70 °C for 30 min. The proportion of the heat-resistant amylase was 1 mL / L, and the stirring speed was 300 r / min. After the enzymatic hydrolysis was completed, it was mixed evenly and sterilized in a high-pressure sterilizer at 121 °C for 20 min. After sterilization, it was cooled to room temperature to obtain the initial fermentation system;

[0092] Inoculate Lactobacillus helveticus and Lactobacillus plantarum into the initial fermentation system for fermentation. Among them, the viable count of Lactobacillus helveticus CICC20243 bacterial liquid is 10 7 CFU / mL, the inoculation amount is 7.5 mL, and the viable count of Lactobacillus plantarum bacterial liquid is 10 7 CFU / mL, the inoculation amount is 15 mL. After shaking well, statically culture in an incubator at 43 °C for 15 h. After the fermentation is completed, sterilize the obtained fermentation broth in a high-pressure steam sterilizer at 121 °C for 20 min. After sterilization is completed, cool the fermentation broth to room temperature and centrifuge it. The centrifugation speed is 4800 r / min and the centrifugation time is 30 min.

[0093] After centrifugation, take the supernatant. The supernatant is subjected to decolorization treatment. The decolorizing agent is activated carbon. The mass-volume of activated carbon to the supernatant is 20 g / L. The decolorization stirring rate is 300 r / min, and the decolorization time is 60 min. After decolorization is completed, perform a primary suction filtration on a filter paper with a pore size of 12 μm, and then perform a secondary suction filtration on a filter paper with a pore size of 0.45 μm to remove the decolorizing agent; after decolorization, perform secondary sterilization at a temperature of 121 °C and a pressure of 0.13 MPa for 20 min. After sterilization is completed, a preservative needs to be added to the fermentation broth. During the mixing process with the preservative, the mixing temperature is 75 °C. Based on the mass of the original fermentation broth, add 0.5% of p-hydroxyacetophenone and 0.8% of 1,2-hexanediol, and mix evenly to obtain small-molecule oat β-glucan.

[0094] Example 5

[0095] Compared with Example 1, the difference is only that the addition amount of amylase is 1.2 mL / L, the enzymatic hydrolysis temperature is 60 °C, and the enzymatic hydrolysis time is 35 min. Other conditions are the same as those in Example 1. The specific operations are as follows:

[0096] Crush and sieve the commercially available oat bran, and the particle size mesh number is 100 mesh. Take 3 g of oat bran and 300 mL of deionized water, mix evenly, add heat-resistant amylase, and perform stirring enzymatic hydrolysis at 60 °C for 35 min. The addition ratio of heat-resistant amylase is 1.2 mL / L, and the stirring speed is 300 r / min; after enzymatic hydrolysis is completed, mix evenly, sterilize in a high-pressure sterilizer at 121 °C for 20 min, and cool to room temperature after sterilization to obtain the initial fermentation system;

[0097] Inoculate Lactobacillus helveticus and Lactobacillus plantarum into the initial fermentation system for fermentation. Among them, the viable count of Lactobacillus helveticus CICC20243 bacterial liquid is 10 7 CFU / mL, the inoculation amount is 7.5 mL, and the viable count of Lactobacillus plantarum bacterial liquid is 10 7CFU / mL, with an inoculation volume of 7.5 mL. After shaking well, it was statically cultured in an incubator at 43 °C for 15 h. After the fermentation ended, the obtained fermentation broth was sterilized at 121 °C for 20 min in a high-pressure steam sterilizer. After sterilization was completed, the fermentation broth was cooled to room temperature and centrifuged at a centrifugation speed of 4800 r / min for 30 min;

[0098] After centrifugation, the supernatant was taken. The supernatant was subjected to decolorization treatment. The decolorizing agent was activated carbon. The mass-volume ratio of activated carbon to the supernatant was 20 g / L. The decolorization stirring rate was 300 r / min, and the decolorization time was 60 min. After decolorization was completed, it was subjected to a primary suction filtration on a filter paper with a pore size of 12 μm, and then a secondary suction filtration on a filter paper with a pore size of 0.45 μm to remove the decolorizing agent; After decolorization, it was subjected to secondary sterilization at a temperature of 121 °C and a pressure of 0.13 MPa for 20 min. After sterilization ended, a preservative needed to be added to the fermentation broth. During the mixing with the preservative, the mixing temperature was 75 °C. Based on the mass of the original fermentation broth, 0.5% of p-hydroxyacetophenone and 0.8% of 1,2-hexanediol were added and mixed evenly to obtain small molecule oat β-glucan.

[0099] Comparative Example 1

[0100] Compared with Example 1, the difference was only that inoculation fermentation was not carried out and only extraction was performed, and other conditions were the same as those in Example 1. The specific operation was as follows:

[0101] The commercially available oat bran was pulverized and sieved to a particle size of 100 mesh. 3 g of oat bran powder and 300 mL of deionized water were taken, mixed evenly, and then heat-resistant amylase was added. It was stirred and enzymatically hydrolyzed at 70 °C for 30 min. The proportion of heat-resistant amylase was 1 mL / L, and the stirring speed was 300 r / min. After the enzymatic hydrolysis ended, the pH was adjusted to 7, and it was stirred and extracted in a water bath at 70 °C for 120 minutes with a stirring speed of 300 r / min to obtain an extract. Then, it was centrifuged at a centrifugation speed of 4800 r / min for 30 min. After centrifugation, the supernatant was taken. Then, the supernatant was decolorized in activated carbon at a stirring rate of 300 r / min for 60 min. The mass-volume ratio of activated carbon to the supernatant was 20 g / L. After decolorization was completed, it was subjected to a primary suction filtration on a filter paper with a pore size of 12 μm and a secondary suction filtration on a filter paper with a pore size of 0.45 μm to remove the decolorizing agent; After decolorization, it was subjected to secondary sterilization at a temperature of 121 °C and a pressure of 0.12 MPa for 20 min. After sterilization ended, a preservative needed to be added to the fermentation broth. During the mixing with the preservative, the mixing temperature was 75 °C; Based on the mass of the original fermentation broth, 0.5% of p-hydroxyacetophenone and 0.8% of 1,2-hexanediol were added and mixed evenly.

[0102] Comparative Example 2

[0103] Compared with Example 1, the difference is only that Lactobacillus helveticus CGMCC No. 19090 was inoculated, and other conditions were the same as those in Example 1. The specific operations are as follows:

[0104] The commercially available oat bran was pulverized and sieved to a particle size of 100 mesh. 3 g of oat bran powder and 300 mL of deionized water were taken, and after mixing evenly, heat-resistant amylase was added. Stirring enzymatic hydrolysis was carried out at 70 °C for 30 min. The proportion of heat-resistant amylase was 1 mL / L, and the stirring speed was 300 r / min; after the enzymatic hydrolysis was completed, it was mixed evenly and sterilized in a high-pressure sterilizer at 121 °C for 20 min. After sterilization, it was cooled to room temperature to obtain an initial fermentation system;

[0105] Lactobacillus helveticus CGMCC No. 19090 was inoculated into the initial fermentation system for fermentation. The viable cell count of Lactobacillus helveticus CGMCC No. 19090 was 10 7 CFU / mL, the inoculation amount was 15 mL. After shaking evenly, it was statically cultured in an incubator at 43 °C for 15 h. After the fermentation was completed, the obtained fermentation broth was sterilized in a high-pressure steam sterilizer at 121 °C for 20 min. After sterilization was completed, the fermentation broth was cooled to room temperature and centrifuged at a centrifugal speed of 4800 r / min for 30 min;

[0106] After centrifugation, the supernatant was taken. The supernatant was decolorized. The decolorizing agent was activated carbon. The mass-volume ratio of activated carbon to the supernatant was 20 g / L. The decolorizing stirring rate was 300 r / min, and the decolorizing time was 60 min. After decolorization, it was filtered once on a filter paper with a pore size of 12 μm, and then filtered twice on a filter paper with a pore size of 0.45 μm to remove the decolorizing agent; after decolorization, it was sterilized for 20 min under the conditions of a temperature of 121 °C and a pressure of 0.13 MPa. After sterilization, a preservative needed to be added to the fermentation broth. During the mixing with the preservative, the mixing temperature was 75 °C. Based on the mass of the original fermentation broth, 0.5% of p-hydroxyacetophenone and 0.8% of 1,2-hexanediol were added and mixed evenly to obtain small molecule oat β-glucan.

[0107] Comparative Example 3

[0108] Compared with Example 1, the difference is only that a carbon source and a nitrogen source were added to the fermentation substrate, and other conditions were the same as those in Example 1.

[0109] The commercially available oat bran was crushed and sieved to a particle size of 100 mesh. 3 g of oat bran powder and 300 mL of deionized water were taken, and after mixing evenly, a high-temperature resistant amylase was added. Stirring enzymatic hydrolysis was carried out at 70 °C for 30 min. The proportion of the high-temperature resistant amylase was 1 mL / L, and the stirring speed was 300 r / min. After the enzymatic hydrolysis was completed, 1.5 g of soy peptide and 1.0 g of glucose were added and mixed evenly. After mixing evenly, it was sterilized in a high-pressure sterilizer at 121 °C for 20 min. After sterilization, it was cooled to room temperature to obtain the initial fermentation system.

[0110] Lactobacillus helveticus and Lactobacillus plantarum were inoculated into the initial fermentation system for fermentation. Among them, the viable count of the Lactobacillus helveticus CICC20243 bacterial liquid was 10 7 CFU / mL, the inoculation amount was 7.5 mL, the viable count of the Lactobacillus plantarum bacterial liquid was 10 7 CFU / mL, the inoculation amount was 7.5 mL. After shaking well, it was statically cultured in an incubator at 43 °C for 15 h. After the fermentation was completed, the obtained fermentation broth was sterilized in a high-pressure steam sterilizer at 121 °C for 20 min. After the sterilization was completed, the fermentation broth was cooled to room temperature and centrifuged. The centrifugation speed was 4800 r / min, and the centrifugation time was 30 min.

[0111] After centrifugation, the supernatant was taken, and the supernatant was subjected to decolorization treatment. The decolorizing agent was activated carbon. The mass volume of the activated carbon to the supernatant was 20 g / L. The decolorization stirring rate was 300 r / min, and the decolorization time was 60 min. After decolorization, it was subjected to a primary suction filtration on a filter paper with a pore size of 12 μm, and then a secondary suction filtration on a filter paper with a pore size of 0.45 μm to remove the decolorizing agent. After decolorization, it was subjected to secondary sterilization at a temperature of 121 °C and a pressure of 0.13 MPa for 20 min. After the sterilization was completed, a preservative needed to be added to the fermentation broth. During the mixing with the preservative, the mixing temperature was 75 °C. Based on the mass of the original fermentation broth, 0.5% of p-hydroxyacetophenone and 0.8% of 1,2-hexanediol were added and mixed evenly to obtain small molecule oat β-glucan.

[0112] Comparative Example 4

[0113] Compared with Example 1, the difference was only that Schizophyllum commune (CGMCC NO.40388) and Saccharomyces cerevisiae (CGMCC NO.17452) were inoculated, and other conditions were the same as those in Example 1.

[0114] The commercially available oat bran is crushed and sieved to a particle size of 100 mesh. Take 3 g of oat bran powder and 300 mL of deionized water, mix them evenly, add heat-resistant amylase, and carry out stirring enzymatic hydrolysis at 70 °C for 30 min. The addition ratio of heat-resistant amylase is 1 mL / L, and the stirring speed is 300 r / min; after the enzymatic hydrolysis is completed and mixed evenly, sterilize in a high-pressure sterilizer at 121 °C for 20 min, and cool to room temperature after sterilization to obtain an initial fermentation system;

[0115] Schizophyllum commune and Saccharomyces cerevisiae are inoculated into the initial fermentation system for fermentation. Among them, the inoculation amount of Schizophyllum commune is 7.5 mL, and the inoculation amount of Saccharomyces cerevisiae is 7.5 mL. After shaking well, statically culture in an incubator at 43 °C for 15 h. After the fermentation is completed, sterilize the obtained fermentation broth in a high-pressure steam sterilizer at 121 °C for 20 min. After sterilization is completed, cool the fermentation broth to room temperature and centrifuge it at a centrifugal speed of 4800 r / min for 30 min;

[0116] After centrifugation, take the supernatant, and the supernatant is subjected to decolorization treatment. The decolorizing agent is activated carbon. The mass-to-volume ratio of activated carbon to the supernatant is 20 g / L. The decolorization stirring rate is 300 r / min, and the decolorization time is 60 min. After decolorization is completed, perform a primary suction filtration on a filter paper with a pore size of 12 μm, and then perform a secondary suction filtration on a filter paper with a pore size of 0.45 μm to remove the decolorizing agent; after decolorization, perform secondary sterilization at a temperature of 121 °C and a pressure of 0.13 MPa for 20 min. After sterilization is completed, a preservative needs to be added to the fermentation broth. During the mixing process with the preservative, the mixing temperature is 75 °C. Based on the mass of the original fermentation broth, add 0.5% of p-hydroxyacetophenone and 0.8% of 1,2-hexanediol, and mix evenly to obtain small-molecule oat β-glucan.

[0117] Comparative Example 5

[0118] Compared with Example 1, the difference is only that Lactobacillus paracasei (CICC 20241) is inoculated, and other conditions are the same as those in Example 1.

[0119] The commercially available oat bran is crushed and sieved to a particle size of 100 mesh. Take 3 g of oat bran powder and 300 mL of deionized water, mix them evenly, add heat-resistant amylase, and carry out stirring enzymatic hydrolysis at 70 °C for 30 min. The addition ratio of heat-resistant amylase is 1 mL / L, and the stirring speed is 300 r / min; after the enzymatic hydrolysis is completed and mixed evenly, sterilize in a high-pressure sterilizer at 121 °C for 20 min, and cool to room temperature after sterilization to obtain an initial fermentation system;

[0120] Lactobacillus paracasei is inoculated into the initial fermentation system for fermentation, and the viable count of Lactobacillus paracasei is 10 7CFU / mL, with an inoculation volume of 7.5 mL. After shaking well, it was statically cultured in an incubator at 43 °C for 15 h. After the fermentation was completed, the obtained fermentation broth was sterilized at 121 °C for 20 min in a high-pressure steam sterilizer. After sterilization, the fermentation broth was cooled to room temperature and centrifuged at a rotation speed of 4,800 r / min for 30 min;

[0121] After centrifugation, the supernatant was taken. The supernatant was subjected to decolorization treatment. The decolorizing agent was activated carbon. The mass-volume ratio of activated carbon to the supernatant was 20 g / L. The decolorization stirring rate was 300 r / min, and the decolorization time was 60 min. After decolorization, it was subjected to primary suction filtration on a filter paper with a pore size of 12 μm, and then secondary suction filtration on a filter paper with a pore size of 0.45 μm to remove the decolorizing agent; after decolorization, it was subjected to secondary sterilization at a temperature of 121 °C and a pressure of 0.13 MPa for 20 min. After sterilization, a preservative needed to be added to the fermentation broth. During the mixing with the preservative, the mixing temperature was 75 °C. Based on the mass of the original fermentation broth, 0.5% of p-hydroxyacetophenone and 0.8% of 1,2-hexanediol were added and mixed evenly to obtain small molecule oat β-glucan.

[0122] Effect Example 1: DPPH free radical scavenging experiment

[0123] DPPH is a stable nitrogen-centered free radical, which is purple in organic solvents and has a maximum absorption at 517 nm. In the DPPH experiment, when antioxidants are added to the DPPH solution, they can react with DPPH free radicals, capture the unpaired electrons of the free radicals, so that the DPPH free radicals lose their free radical properties and change from purple to colorless. By comparing the absorbance changes before and after the reaction, the antioxidant ability of the sample can be quantitatively evaluated. This method is simple, rapid, and widely used in industries such as food, medicine, and cosmetics to evaluate the antioxidant performance of products. Therefore, the scavenging effect of the sample on DPPH free radicals can be evaluated by measuring the change in absorbance value.

[0124] The specific experimental steps of the DPPH free radical scavenging experiment are as follows:

[0125] (1) Take an equal volume (1 mL) of the test solution and mix it evenly with 2×10 -4 mol / L DPPH solution (tube A1);

[0126] (2) Take an equal volume (1 mL) of absolute ethanol (solvent of the test substance) and mix it evenly with 2×10 -4 mol / L DPPH solution (tube A2);

[0127] (3) Take an equal volume (1 mL) of absolute ethanol and mix it evenly with the test solution (tube A3);

[0128] After reacting in the dark for 30 min, measure the absorbance values of tubes A1, A2, and A3 at 517 nm; the clearance rate calculation formula is: Clearance rate = [(A2 + A3) - A1] / A2 × 100%.

[0129] This experiment tested the DPPH radical scavenging experiment on the examples and comparative examples diluted 5 times ( ns p > 0.05, no significant difference; * p < 0.05, ** p < 0.01, *** p < 0.001, there is a significant difference), and the results are shown in Table 1 and Figure 1 .

[0130] Table 1

[0131]

[0132]

[0133] From Table 1 and Figure 1 the results show that the DPPH radical scavenging ability of the products prepared in Examples 1-5 is better than that of the products prepared in Comparative Examples 1-5. It can be seen that the products prepared in the examples of this application have ideal antioxidant effects, and the components of the fermentation strains and fermentation substrates have great effects on the antioxidant performance of the final products.

[0134] Effect Example 2: Determination of total antioxidant capacity

[0135] Use the total antioxidant capacity detection kit (FRAP method) produced by Beyotime Biotechnology Co., Ltd. to test the total antioxidant capacity of the products prepared in the examples and comparative examples ( ns p > 0.05, no significant difference; * p < 0.05, ** p < 0.01, *** p < 0.001, there is a significant difference), and the results are shown in Table 2 and Figure 2 .

[0136] Table 2

[0137] Total antioxidant capacity (mM) Example 1 1.74±0.08 Example 2 1.66±0.08 Example 3 1.97±0.04 Example 4 1.64±0.05 Example 5 1.55±0.08 Comparative Example 1 1.02±0.08 Comparative Example 2 0.86±0.03 Comparative Example 3 0.93±0.08 Comparative Example 4 0.77±0.04 Comparative Example 5 0.85±0.08

[0138] From Table 2 and Figure 2 the results show that the total antioxidant capacity of the products prepared in Examples 1-5 is better than that of the products prepared in Comparative Examples 1-5. It can be seen that the products prepared in the examples of this application have ideal antioxidant effects, and the components of the fermentation strains and fermentation substrates have great effects on the total antioxidant capacity of the final products.

[0139] Effect Example 3: Human immortalized epidermal cell cytotoxicity experiment

[0140] In this experiment, human immortalized epidermal cells from the Chinese Academy of Sciences Cell Bank were used to verify the cytotoxicity of the products prepared in the above-mentioned examples and comparative examples.

[0141] 1. Experimental steps:

[0142] The products prepared in Examples 1-5 and Comparative Examples 1-5 were respectively configured into experimental group test solutions with a volume percentage of 1.25% using serum-free MEM medium. Human immortalized epidermal cells were cultured in MEM medium containing 10% fetal bovine serum and 1% double antibody (1×10 5 U / L penicillin, 100 mg / L streptomycin). The cells grew in an incubator at 37°C and 5% CO2 saturated humidity. When the cell confluence reached more than 85%, the cells in the logarithmic growth phase were digested with 0.05% trypsin, and the digestion reaction was terminated with serum-containing MEM. The cells were counted with a cell counting plate, and the cell suspension concentration was adjusted to 7×10 4 cells / mL. The cell suspension was inoculated into a 96-well plate at a ratio of 100 μL per well and incubated at 37°C and 5% CO2 for 12 h. The old culture medium was removed, and the cells were washed twice with phosphate buffer solution. In the experimental group, 100 μL of the above-prepared filtered and sterilized test solutions with different concentrations were added to each well, and 6 replicates were made for each test solution; the control group contained cells and serum-free MEM medium was added; the blank control group had no cells and 100 μL of PBS was added. Then it was incubated at 37°C and 5% CO2 for 24 h. The old culture medium was aspirated, and the cells were washed twice with phosphate buffer solution. Then 100 μL of serum-free MEM medium and 10 μL of CCK-8 solution were added to each well, and it was incubated for another 3 h. The absorbance value was measured at a wavelength of 450 nm, and the cell survival rate of each group was calculated ([ ns p > 0.05, no significant difference; * p < 0.05, ** p < 0.01, *** p < 0.001, there is a significant difference), and the results are shown in Table 3 and Figure 3 .

[0143] The calculation formula for the cell survival rate is as follows:

[0144] Cell survival rate (%) = (Aexperimental group - Ablank control group) / (Acontrol group - Ablank control group) × 100%.

[0145] Table 3

[0146] Cell viability (%) Example 1 112.93±5.38 Example 2 115.40±1.93 Example 3 112.67±6.32 Example 4 110.33±2.29 Example 5 106.69±1.58 Comparative Example 1 99.53±1.03 Comparative Example 2 93.16±5.66 Comparative Example 3 89.77±1.03 Comparative Example 4 96.54±1.76 Comparative Example 5 88.86±5.65

[0147] From Table 3 and Figure 3The results show that the survival rates of the products prepared in Examples 1-5 for HaCaT cells are higher than those of the products prepared in Comparative Examples 1-5 for HaCaT cells. It can be seen that the products prepared in the examples of this application have a significant proliferative effect on HaCaT cells, and the components of the fermentation strains and fermentation substrates both have a great influence on the proliferative effect of the final products on HaCaT cells.

[0148] Effect Example 4: Determination of total sugar content

[0149] The total sugar content in the products prepared in the examples and comparative examples was tested using a total sugar content detection kit produced by Beijing Solarbio Science & Technology Co., Ltd. ( ns p > 0.05, no significant difference; * p < 0.05, ** p < 0.01, *** p < 0.001, there is a significant difference), and the results are shown in Table 4 and Figure 4 .

[0150] Table 4

[0151] Total sugar content (mg / mL) Example 1 1.83±0.05 Example 2 1.83±0.05 Example 3 2.00±0.05 Example 4 1.97±0.05 Example 5 1.89±0.03 Comparative Example 1 1.61±0.03 Comparative Example 2 1.62±0.11 Comparative Example 3 1.62±0.03 Comparative Example 4 1.54±0.01 Comparative Example 5 1.65±0.02

[0152] As shown in Table 4 and Figure 4 The results show that the total sugar content of the products prepared in Examples 1-5 is higher than that of the products prepared in Comparative Examples 1-5. It can be seen that the components of the fermentation strains and fermentation substrates both have a great influence on the total sugar content of the final products.

[0153] The results show that the total sugar content of Examples 1-5 is higher than that of Comparative Examples 1-5;

[0154] Effect Example 5: Determination of inflammatory factor IL-8 (repair cells)

[0155] Preparation of the sample to be tested: The products prepared in Examples 1-5 and Comparative Examples 1-5 above were used to prepare a test solution to be tested with a volume fraction of 2% of Avena sativa β-glucan in serum-free MEM medium.

[0156] HaCaT cells were taken and the cell suspension was inoculated on a 6-well cell culture plate at a density of 250,000 cells / mL. 2 mL of the cell suspension was added to each well and cultured for 12 h, and then the supernatant was aspirated. 2 mL of MEM solution was added to the blank group; in the experimental group and the model group, cells were damaged with 800 μg / mL LPS for 6 hours, and then the supernatant was aspirated. 2 mL of the sample to be tested was added to the experimental group, and 2 mL of MEM solution was added to the model group. The cells were treated for 24 h. The supernatant was collected, 200 μL of cell lysate was added to each well to treat the cells, and centrifuged at 10,000 r / min at 4 °C for 10 min. The supernatant was taken to obtain the cell lysate supernatant. 20 μL of the cell lysate was used to detect the total protein content in the sample with a BCA kit; the determination of inflammatory factors was carried out according to the instructions of the ELISA kit. The OD values were measured at 450 nm, and according to the OD values, the release level of IL-8 was calculated.

[0157] Operating steps of the BCA kit:

[0158] 1. Sample pretreatment: According to the usage amount, prepare the lysate according to the ratio of RIPA:PMSF of 100:1. Remove the culture medium, wash it once with PBS, and add 150 - 250 μL of lysate according to the cell amount in each well of the 6-well plate. Pipette several times to make the lysate fully contact with the cells;

[0159] 2. Post-treatment: Centrifuge the lysed sample at 10,000 rpm for 10 min, take the supernatant, and then the subsequent protein concentration determination can be carried out;

[0160] 3. Prepare the BCA working solution according to the ratio of reagent (A) BCA solution:reagent (B) Cu 2+ solution of 50:1;

[0161] 4. Add 20 μL of the protein sample to be tested into a 96-well plate;

[0162] 5. Add 200 μL of the prepared BCA working solution to each well and place it at 37 °C for 20 - 30 min.

[0163] 6. Detect the absorbance at 562 nm;

[0164] 7. Calculate the protein concentration of the sample according to the standard curve and dilution factor.

[0165] Detection steps of the ELISA kit:

[0166] 1. Reagent preparation

[0167] 1) Reagent warming: First, warm the kit 30 min before the experiment. Place the samples to be tested at room temperature. If crystals appear in the concentrated washing solution, place it in a 37 °C water bath until all the crystals dissolve.

[0168] 2) Preparation of washing solution: Calculate in advance the volume of the diluted washing solution to be used. Then dilute the 20-fold concentrated washing solution with deionized water to a 1-fold application solution. The unused concentrated washing solution should be stored at 4°C.

[0169] 3) Serial dilution of samples: Add 1 mL of standard / sample diluent (SR1) to the lyophilized standard. Let it stand for 15 minutes, and after it is completely dissolved, gently mix well (concentration 1000 pg / mL). Then add 500 μL of standard / sample diluent (SR1) to each of the remaining 6 tubes and perform a 2-fold dilution according to the following concentrations: 500, 250, 125, 62.5, 31.25, 15.62, 0 pg / mL. 1000 pg / mL is the highest concentration of the standard curve, and the standard / sample diluent (SR1) serves as the zero point of the standard curve. The unused reconstituted standard stock solution (concentration 1000 pg / mL) should be discarded or aliquoted according to the single-use requirement and stored in a -80°C refrigerator.

[0170] 4) Biotinylated antibody working solution: Calculate in advance the amount required for the experiment. Dilute the 100-fold antibody concentrate with the detection diluent (SR2) to a 1-fold application working solution (mix well before dilution) and add it to the reaction wells within 30 minutes.

[0171] 5) Enzyme conjugate working solution: Prepare according to the amount required for each experiment. Dilute the 40-fold concentrated enzyme conjugate with the enzyme conjugate diluent (SR3) to a 1-fold application working solution (centrifuge before dilution) and use it within 30 minutes.

[0172] 6) Washing method: Discard the liquid in the enzyme-labeled plate wells by flicking, pat dry on absorbent paper, add 300 μL of washing solution per well with a wash bottle, let it stand for 30 s, then discard the liquid in the enzyme-labeled plate wells by flicking and pat dry on absorbent paper.

[0173] 2. Detection procedure

[0174] 1) 30 minutes before the experiment, take out the kit and let it return to room temperature. Before adding the standard / sample, wash the plate three times and flick dry.

[0175] 2) Add 100 μL of standard / sample to the reaction wells, seal the plate and incubate at 37°C for 90 minutes, tap the plate and wash the plate 4 times.

[0176] 3) Add 100 μL of biotinylated antibody working solution to the reaction wells, seal the plate and incubate at 37°C for 60 minutes, tap the plate and wash the plate 4 times.

[0177] 4) Add 100 μL of enzyme conjugate working solution to the reaction wells, seal the plate and incubate at 37°C for 30 minutes, tap the plate and wash the plate 5 times.

[0178] 5) Add 50 μL of color development substrate to the reaction wells, seal the plate and incubate in a 37°C incubator in the dark for 15 min;

[0179] 6) Add 50 μL of stop solution and immediately measure the OD value (within 5 min) at a wavelength of 450 nm using an enzyme reader (nsp>0.05, no significant difference; *p<0.05, **p<0.01, ***p<0.001, significant difference). The results are shown in Table 5 and Figure 5 .

[0180] Table 5

[0181]

[0182]

[0183] From Table 5 and Figure 5 The results show that the IL-8 scavenging ability of the products prepared in Examples 1 to 5 is better than that of the products prepared in Comparative Examples 1 to 5. It can be seen that the products prepared in the examples of the present application have ideal anti-inflammatory effects, and the components of the fermentation bacteria and the fermentation substrate have a great influence on the anti-inflammatory properties of the final product.

[0184] Effect Example 6: Determination of Inflammatory Factor IL-6 (Repair Cells)

[0185] ELISA kit detection steps:

[0186] 1. Reagent Preparation

[0187] 1) Reagent warming: First, warm the reagent kit 30 minutes before the experiment. Place the sample to be tested at room temperature. If crystals appear in the concentrated washing solution, place it in a 37°C water bath until all the crystals are dissolved.

[0188] 2) Prepare the washing solution: Calculate the volume of the diluted washing solution in advance, then dilute the 20-fold concentrated washing solution into 1-fold application solution with deionized water. Store the unused concentrated washing solution at 4°C.

[0189] 3) Sample serial dilution: Add 1 mL of Standard / Sample Diluent (SR1) to the lyophilized standard, let it stand for 15 minutes, and gently mix after it is completely dissolved (concentration 1000 pg / mL). Then add 500 μL of Standard / Sample Diluent (SR1) to each of the remaining 6 tubes and perform a 2-fold dilution according to the following concentrations: 500, 250, 125, 62.5, 31.25, 15.62, 0 pg / mL. 1000 pg / mL is the highest concentration of the standard curve, and the Standard / Sample Diluent (SR1) serves as the zero point of the standard curve. Any unused reconstituted standard stock solution (concentration 1000 pg / mL) should be discarded or aliquoted according to the single-use requirement and stored in a -80 °C refrigerator.

[0190] 4) Biotinylated antibody working solution: Calculate the required amount for the experiment in advance, dilute the 100-fold antibody concentrate to a 1-fold working solution with Detection Diluent (SR2) (mix well before dilution), and add it to the reaction wells within 30 minutes.

[0191] 5) Enzyme conjugate working solution: Prepare according to the required amount for each experiment, dilute the 40-fold concentrated enzyme conjugate to a 1-fold working solution with Enzyme Conjugate Diluent (SR3) (centrifuge before dilution), and use it within 30 minutes.

[0192] 6) Washing method: Discard the liquid in the enzyme-labeled plate wells by flicking, pat dry on absorbent paper, add 300 μL of washing solution per well with a wash bottle, let it stand for 30 s, then discard the liquid in the enzyme-labeled plate wells by flicking and pat dry on absorbent paper.

[0193] 2. Detection procedure

[0194] 1) 30 minutes before the experiment, take out the kit and let it return to room temperature. Before adding the standard / sample, wash the plate three times and flick dry.

[0195] 2) Add 100 μL of standard / sample to the reaction wells, seal the plate, incubate at 37 °C for 90 minutes, tap the plate, and wash the plate 4 times.

[0196] 3) Add 100 μL of biotinylated antibody working solution to the reaction wells, seal the plate, incubate at 37 °C for 60 minutes, tap the plate, and wash the plate 4 times.

[0197] 4) Add 100 μL of enzyme conjugate working solution to the reaction wells, seal the plate, incubate at 37 °C for 30 minutes, tap the plate, and wash the plate 5 times.

[0198] 5) Add 50 μL of chromogenic substrate to the reaction wells, seal the plate, and incubate in the 37 °C incubator in the dark for 15 minutes.

[0199] 6) Add 50 μL of the termination solution, and immediately measure the OD value at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader (within 5 minutes) (nsp > 0.05, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, significant difference). The results are shown in Table 6 and Figure 6 .

[0200] Table 6

[0201] IL-6 content (pg / mg) Example 1 1306.73±10.58 Example 2 1276.10±30.81 Example 3 1334.4±4.71 Example 4 1321.81±37.32 Example 5 1410.93±3.86 Comparative Example 1 1663.61±57.85 Comparative Example 2 1668.19±59.04 Comparative Example 3 1648.46±17.34 Comparative Example 4 1744.17±13.08 Comparative Example 5 1773.27±123.04

[0202] As shown in Table 6 and Figure 6 the results indicate that the ability of the products prepared in Examples 1 - 5 to scavenge IL-6 is superior to that of the products prepared in Comparative Examples 1 - 5. It can be seen that the products prepared in the examples of this application have an ideal anti-inflammatory effect, and the components of the fermentation strains and fermentation substrates both have a great influence on the anti-inflammatory performance of the final products.

[0203] Effect Example 7: Relative mRNA expression levels of IL-6 and IL-8

[0204] Use TriQuick total RNA extraction reagent to extract RNA from HaCaT cells according to the instructions. Use FastQuant cDNA kit for the first-strand cDNA synthesis reaction. Use Fast Super qPCR MasterMix and qRT-PCR to detect the cDNA obtained by further reverse transcription. The specific primer sequences are shown in Tables 7 - 8 and Figure 7 - 8 as follows ([[]] ns p > 0.05, no significant difference; * p < 0.05, ** p < 0.01, *** p < 0.001, significant difference).

[0205] qRT-PCR primer sequence table

[0206]

[0207] Table 7

[0208] Relative expression level of IL-8 mRNA Example 1 1.17±0.07 Example 2 1.25±0.15 Example 3 1.15±0.06 Example 4 1.31±0.09 Example 5 1.45±0.04 Comparative Example 1 1.92±0.09 Comparative Example 2 2.07±0.18 Comparative Example 3 1.98±0.15 Comparative Example 4 1.97±0.13 Comparative Example 5 1.85±0.06

[0209] As shown in Table 7 and Figure 7 the results indicate that the relative mRNA expression level of IL-8 in the products prepared in Examples 1 - 5 is lower than that in the products prepared in Comparative Examples 1 - 5. It can be seen that the products prepared in the examples of this application have an ideal anti-inflammatory effect, and the components of the fermentation strains and fermentation substrates both have a great influence on the anti-inflammatory performance of the final products.

[0210] Table 8

[0211] Relative expression level of IL-6 mRNA Example 1 1.1±0.07 Example 2 1.25±0.15 Example 3 1.04±0.06 Example 4 1.06±0.09 Example 5 1.34±0.04 Comparative Example 1 1.77±0.09 Comparative Example 2 1.6±0.18 Comparative Example 3 1.95±0.15 Comparative Example 4 1.94±0.13 Comparative Example 5 1.66±0.06

[0212] As shown in Table 8 and Figure 8 the results indicate that the relative mRNA expression levels of IL-6 in the products prepared in Examples 1 to 5 are lower than those in the products prepared in Comparative Examples 1 to 5. It can be seen that the products prepared in the examples of this application have ideal anti-inflammatory effects, and the components of the fermentation strains and fermentation substrates both have great influences on the anti-inflammatory properties of the final products.

[0213] Effect Example 8: Molecular weight of oat glucan

[0214] Remove proteins from the samples of Examples 1 to 5 and Comparative Examples 1 to 5, precipitate oat β-glucan by alcohol precipitation, and centrifuge and dry to obtain the crude product. Prepare an oat β-glucan solution, place the Ubbelohde viscometer in a constant temperature water bath at 25°C, first measure the time for distilled water to flow through the capillary as the blank control (t0), then measure the time for each sample to flow through the capillary (t), calculate the relative viscosity (ηr = t / t0), calculate the specific viscosity (ηsp), and make a standard curve to estimate the molecular weight of oat β-glucan ( ns p > 0.05, no significant difference; * p < 0.05, ** p < 0.01, *** p < 0.001, there is a significant difference).

[0215] Table 9

[0216]

[0217]

[0218] As shown in Table 9 and Figure 9 the results indicate that the molecular weights of the products prepared in Examples 1 to 5 are lower than those of the products prepared in Comparative Examples 1 to 5. It can be seen that the products prepared in the examples of this application have smaller molecular weights and are more easily absorbed by the skin, and the components of the fermentation strains and fermentation substrates both have great influences on the molecular weights of the final products.

[0219] Finally, it should also be noted that in this application, the terms "comprise", "include" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0220] Although the present application has been disclosed above through the description of specific embodiments of the present application, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present application within the spirit and scope of the appended solutions. These modifications, improvements or equivalents should also be considered to be included within the scope claimed by the present application.

Claims

1. A method for preparing small molecule oat β-glucan, characterized in that: The method comprises the following steps: inoculating lactobacillus into a fermentation substrate composed of oat bran and water, culturing by fermentation, and sterilizing; The lactobacillus is composed of Lactobacillus helveticus and Lactobacillus plantarum, and the ratio of the number of live bacteria of the Lactobacillus helveticus to the Lactobacillus plantarum is 1:(0.5-2).

2. The method for preparing small molecule oat β-glucan according to claim 1, characterized in that: The oat bran also includes oat bran powder obtained by crushing and sieving; Preferably, the oat bran further undergoes enzymatic hydrolysis before use; Preferably, the fermentation substrate also includes a sterilization operation before use.

3. The method for preparing small molecule oat β-glucan according to claim 2, characterized in that: The preparation method of the small molecule oat β-glucan meets at least one of the following conditions: The mesh size of the crushed and sieved material is 80 to 150 meshes, preferably 90 to 120 meshes; The enzymatic hydrolysis temperature is 50-90°C, preferably 60-70°C; The enzymatic hydrolysis time is 20 to 40 minutes, preferably 25 to 35 minutes; The mass volume ratio of the enzyme to the water is 0.5-2 mL / L, preferably 0.8-1.5 mL / L; The enzyme used in the enzymolysis includes thermostable amylase; The sterilization is a high-temperature sterilization method. Preferably, when the fermentation substrate is sterilized by the high-temperature sterilization method, the sterilization temperature is 110-125°C, and more preferably 115-121°C; when the fermentation substrate is sterilized by the high-temperature sterilization method, the sterilization pressure is 0.1-0.14MPa, and preferably 0.12-0.13MPa; when the fermentation substrate is sterilized by the high-temperature sterilization method, the sterilization temperature is 15-35min, and preferably 15-25min, for example 20min.

4. The method for preparing small molecule oat β-glucan according to claim 1, characterized in that: The preparation method of the small molecule oat β-glucan meets at least one of the following conditions: The Lactobacillus helveticus includes Lactobacillus helveticus deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC20243; The plant lactobacillus includes plant lactobacillus (Lactiplantibacillus plantarum) BSJM23L004, the plant lactobacillus (Lactiplantibacillus plantarum) BSJM23L004, the depository of which is the General Microbiological Center of China National Microbiological Culture Collection Committee (CGMCC for short), the address of which is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, the deposit date is April 16, 2024, and the deposit number is CGMCC No.30350; The Lactobacillus helveticus is added in the form of a Lactobacillus helveticus bacterial liquid, and the number of live bacteria of the Lactobacillus helveticus is 10 5 ~10 10 CFU / mL, preferably 10 6 ~10 8 CFU / mL, e.g. 10 7 CFU / mL; The plant lactobacillus is added in the form of plant lactobacillus bacterial liquid, and the number of live bacteria of the plant lactobacillus is 10 5 ~10 10 CFU / mL, preferably 10 6 ~10 8 CFU / mL, e.g. 10 7 CFU / mL.

5. The method for preparing small molecule oat β-glucan according to claim 1, characterized in that: The preparation method of the small molecule oat β-glucan meets at least one of the following conditions: The fermentation culture is anaerobic fermentation; The fermentation culture is carried out in a constant temperature incubator; The fermentation culture temperature is 35-50°C, preferably 40-45°C, for example 43°C; The fermentation culture time is 12 to 48 hours, preferably 15 to 30 hours; The mass ratio of the oat bran to the water is 1:(20-200), preferably 1:(50-150).

6. The method for preparing small molecule oat β-glucan according to claim 1, characterized in that: After the sterilization operation, the process further includes cooling and centrifuging to collect the supernatant; Preferably, the cooling is cooling to room temperature; Preferably, the centrifugal speed is 3000-9000 rpm, preferably 4000-6000 rpm, for example 4800 rpm; Preferably, the radius of the centrifuge is 8 to 15 cm, for example 10 cm; Preferably, the centrifugation time is 10 to 40 min, more preferably 20 to 40 min, for example 30 min.

7. The method for preparing small molecule oat β-glucan according to claim 6, characterized in that: The centrifugal operation further includes at least one of decolorization, secondary sterilization, and mixing with a preservative; Preferably, the decolorizing agent used in the decolorization comprises one or more of activated carbon, bentonite and diatomaceous earth, more preferably activated carbon; Preferably, the mass volume ratio of the decolorizing agent to the supernatant is preferably 10 to 30 g / L, more preferably 15 to 25 g / L; Preferably, the stirring rate of the decolorization is 200 to 500 r / min, preferably 300 to 400 r / min; Preferably, the decolorization time is preferably 50 to 70 minutes, preferably 55 to 65 minutes; Preferably, the secondary sterilization method is a high temperature sterilization method; when the high temperature sterilization method is used for the secondary sterilization, the temperature of the secondary sterilization is 115-125°C, preferably 118-121°C; when the high temperature sterilization method is used for the secondary sterilization, the time of the secondary sterilization is 15-25min, preferably 20min; when the high temperature sterilization method is used for the secondary sterilization, the pressure of the secondary sterilization is 0.1-0.14MPa, preferably 0.12-0.13MPa; Preferably, during the mixing process with the preservative, the mixing temperature is 50-80°C, preferably 60-80°C, for example 75°C; Preferably, during the process of mixing with the preservative, the preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol; when the preservative includes p-hydroxyacetophenone and 1,2-hexanediol, the mass percentage of p-hydroxyacetophenone in the supernatant obtained after centrifugation is 0.1% to 1%, and the mass percentage of 1,2-hexanediol in the supernatant obtained after centrifugation is 0.5% to 2%; preferably, the mass percentage of p-hydroxyacetophenone in the supernatant obtained after centrifugation is 0.5%, and the mass percentage of 1,2-hexanediol in the supernatant obtained after centrifugation is 0.8%.

8. A small molecule oat β-glucan, characterized in that: The small molecule oat beta-glucan is prepared by the preparation method of the small molecule oat beta-glucan as claimed in any one of claims 1 to 7.

9. A use of the small molecule oat β-glucan as claimed in claim 8 directly as a product, as an additive or as a base in the preparation of a skin topical preparation; Preferably, the small molecule oat β-glucan is used as the antioxidant active ingredient in the skin topical preparation, and more preferably, the antioxidant active ingredient is an antioxidant active ingredient having DPPH free radical scavenging effect and FRAP total antioxidant capacity; Preferably, the small molecule oat β-glucan is used as the anti-inflammatory active ingredient in the skin external preparation, and more preferably, the anti-inflammatory active ingredient is an anti-inflammatory active ingredient that can reduce IL-8 and IL-6 inflammatory factors.

10. A skin external preparation, characterized in that: It includes the small molecule oat β-glucan as claimed in claim 8; Preferably, the skin topical preparation further comprises at least one of a moisturizing active ingredient, a whitening active ingredient, an anti-inflammatory active ingredient, an anti-allergic active ingredient and an antioxidant active ingredient; Preferably, the skin external preparation comprises a facial mask, an essence or a toner; Preferably, the small molecule oat β-glucan accounts for 5% to 99% by mass of the external skin preparation, more preferably 60% to 99% by mass.

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