Preparation method and application of small molecule oat beta-glucan

Abstract

The application discloses a preparation method of small-molecule oat beta-glucan and application thereof, and the preparation method comprises the following steps: inoculating lactobacillus into a fermentation substrate composed of oat bran and water, carrying out fermentation culture, and sterilizing, and the preparation is completed; wherein the lactobacillus is composed of lactobacillus helveticus and lactobacillus plantarum, and the viable bacterial count ratio of the lactobacillus helveticus to the lactobacillus plantarum is 1:(0.5-2). The application utilizes lactic acid bacteria fermentation to improve the utilization rate of oat bran, reduces the loss of active ingredients, has the advantages of low cost, small energy consumption, environmental friendliness and the like, and can be directly used as an additive for preparing a mask, essence or toner; the product prepared has better DPPH free radical scavenging capacity, total antioxidant capacity, cytotoxicity, total sugar content, anti-inflammatory performance and molecular weight than a product prepared by a traditional method, and has a good market application prospect.

Description

Technical Field

[0001] This application belongs to the field of cosmetic technology, and in particular relates to a method for preparing small molecule oat β-glucan and its application. Background Technology

[0002] With technological advancements, consumers have increasingly higher demands for cosmetics. They not only expect products to have targeted, scientifically validated, and multi-functional benefits, but also emphasize the use of natural ingredients and transparent ingredient sourcing. Leveraging technology, the cosmetics industry is using biotechnology, digital technology, and other means to achieve personalized product customization, efficacy verification, and environmentally sustainable development, thereby meeting consumers' needs for high-quality, safe, and effective cosmetics.

[0003] There are various methods for producing oat β-glucan, including traditional extraction, biotechnology, physical and chemical methods, with water extraction being the most common. However, water extraction may encounter problems such as lower extraction rates and purity, more complex processes, higher costs, and potential damage to the bioactivity of the glucan.

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

[0005] The technical problem this application aims to solve is to overcome the high cost and low extraction rate of existing oat β-glucan extraction methods, and to provide a method for preparing small-molecule oat β-glucan and its application. This application solves the above-mentioned technical problem by using lactic acid bacteria fermentation to extract active ingredients from oat bran, which not only reduces the loss of active ingredients but also better enhances their skincare effects. The small-molecule oat β-glucan prepared by this application can be used directly in cosmetics or added to basic cosmetic formulations, exhibiting high safety and good antioxidant, anti-inflammatory, and skin-nourishing effects.

[0006] This application provides a method for preparing small molecule oat β-glucan, which includes the following steps: inoculating lactobacillus into a fermentation substrate composed of oat bran and water, fermenting and culturing, and then sterilizing.

[0007] The lactobacillus is composed of Lactobacillus helveticus and Lactobacillus plantarum, and the ratio of viable bacteria of Lactobacillus helveticus to Lactobacillus plantarum is 1:(0.5-2).

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

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

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

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

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

[0013] When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization temperature can be a temperature commonly used in this type of operation in the art, preferably 110-125°C, and more preferably 115-121°C.

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

[0015] When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization temperature is preferably 15-35 min, more preferably 15-25 min, such as 20 min, which is common practice in the art.

[0016] The sterilization process of the fermentation substrate may further include a cooling process, preferably cooling to room temperature.

[0017] In some embodiments, the *Lactobacillus helveticus* includes *Lactobacillus helveticus* deposited at the China Industrial Microbial Culture Collection Center with accession number CICC 20243.

[0018] In some embodiments, the *Lactiplantibacillus plantarum* includes *Lactiplantibacillus plantarum* BSJM23L004, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, on April 16, 2024, with accession number CGMCC No. 30350.

[0019] In some embodiments, the *Lactobacillus helveticus* is added in the form of a *Lactobacillus helveticus* bacterial suspension, and the viable count of the *Lactobacillus helveticus* is 10-1. 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 suspension, and the viable count of the *Lactobacillus plantarum* is 10-1. 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 may be anaerobic fermentation.

[0022] In some embodiments, the fermentation culture may be carried out in a thermostat in accordance with conventional practices in the art.

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

[0024] In some embodiments, the fermentation culture time may 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 sterilization, the sterilization temperature can be 110–125°C, preferably 115–121°C. When the high-temperature sterilization method is used for sterilization, the sterilization pressure can be 0.1–0.14 MPa, preferably 0.12–0.13 MPa. When the high-temperature sterilization method is used for sterilization, the sterilization time can be 15–35 min, preferably 15–25 min, and more preferably 20 min.

[0027] In some embodiments, the sterilization process may further include centrifugation and collection of the supernatant.

[0028] The centrifugation speed can be 3000-9000 rpm, preferably 4000-6000 rpm, and more preferably 4800 rpm.

[0029] The radius of the centrifuge is 8-15 cm, preferably 10 cm.

[0030] The centrifugation time can be 10-40 min, preferably 20-40 min, and more preferably 30 min.

[0031] The centrifugation process may further include at least one of the following operations: decolorizing the supernatant, secondary sterilizing it, and mixing it with a preservative.

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

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

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

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

[0036] The secondary sterilization method can be conventional in the art, generally a high-temperature sterilization method. When the secondary sterilization is performed using the high-temperature sterilization method, the sterilization temperature can be a temperature conventional for this type of operation in the art, preferably 115–125°C, more preferably 118–121°C. When the secondary sterilization is performed using the high-temperature sterilization method, the sterilization pressure can be conventional in the art, preferably 0.1–0.14 MPa, preferably 0.12–0.13 MPa. When the secondary sterilization is performed using the high-temperature sterilization method, the sterilization temperature can be conventional in the art, preferably 15–25 min, for example 20 min.

[0037] The preservative may include p-hydroxyacetophenone and 1,2-hexanediol. When the preservative includes p-hydroxyacetophenone, the p-hydroxyacetophenone accounts for 0.1% to 1% of the supernatant by mass. When the preservative includes 1,2-hexanediol, the 1,2-hexanediol accounts for 0.5% to 2% of the supernatant by mass.

[0038] During the mixing process with the preservative, the mixing temperature can be 50-80°C, preferably 60-80°C, for example 75°C.

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

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

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

[0042] Preferably, the small molecule oat β-glucan is used as the anti-inflammatory active ingredient in the topical skin agent; more preferably, the anti-inflammatory active ingredient is an anti-inflammatory active ingredient that reduces IL-8 and IL-6 inflammatory factors.

[0043] This application also provides a topical skin agent comprising the small molecule oat β-glucan as described above.

[0044] In some embodiments, the topical skin agent may further include 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 agents used may be, in accordance with the conventions of the art, including but not limited to, face masks, serums, or toners.

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

[0047] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this application.

[0048] All reagents and raw materials used in this application are commercially available.

[0049] The positive and progressive effects of this application are as follows: the small molecule oat β-glucan prepared by this application has ideal antioxidant properties, can reduce the loss of active ingredients in oat bran, improve extraction efficiency, and has high utilization of oat bran. The preparation process is simple, the fermentation conditions are mild, the energy consumption is low, the cost is low, and the waste of oat bran resources is effectively reduced. Attached Figure Description

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

[0051] in:

[0052] Figure 1 This is a comparison chart of the DPPH free radical scavenging abilities of Examples 1-5 and Comparative Examples 1-5;

[0053] Figure 2 This is a comparison chart of the total antioxidant capacity of Examples 1-5 and Comparative Examples 1-5;

[0054] Figure 3 This is a comparison chart of the toxicity test results of Examples 1-5 and Comparative Examples 1-5 on human immortalized epidermal cells;

[0055] Figure 4 This is a comparison chart of the total sugar content of Examples 1-5 and Comparative Examples 1-5;

[0056] Figure 5 The effects 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 The effects 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 The effects of Examples 1-5 and Comparative Examples 1-5 on the relative mRNA expression levels of IL-8 in human immortalized epidermal cells;

[0059] Figure 8 The effects of Examples 1-5 and Comparative Examples 1-5 on the relative expression levels of IL-6 mRNA in human immortalized epidermal cells were investigated.

[0060] Figure 9 This is a comparison chart of the molecular weights of Examples 1-5 and Comparative Examples 1-5. Detailed Implementation

[0061] The present application is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the present application. Experimental methods not specifically described in the following embodiments are performed according to conventional methods and conditions, or as selected in accordance with the product instructions.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0063] The heat-resistant amylases used in the following examples and comparative examples were obtained from commercially available sources.

[0064] The oat bran used in the following examples and comparative examples was sourced from commercially available sources.

[0065] The Lactiplantibacillus plantarum BSJM23L004 used in the following examples and comparative examples is classified as Lactiplantibacillus plantarum. Its depository institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The deposit date is April 16, 2024, and the accession number is CGMCC No. 30350.

[0066] The Lactobacillus helveticus used in the following examples and comparative examples was purchased from the China Industrial Microbial Culture Collection Center, with accession number CICC 20243.

[0067] The Lactobacillus helveticus in the following comparative examples was purchased from the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 19090.

[0068] The species *Schizophyllum commune* in the following comparative example is classified and named as follows: *Schizophyllum commune*, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, on March 9, 2023, with accession number CGMCC NO.40388.

[0069] The Lacticaseibacillus paracasei in the following comparative examples was purchased from the China Industrial Microbial Culture Collection Center, with accession number CICC 20241.

[0070] The 0.25% (EDTA-containing) trypsin in the following examples and comparative examples was manufactured by GIBCO Corporation, USA; the MEM culture medium was manufactured by GIBCO Corporation, USA.

[0071] The bispecific antibody in the following efficacy examples was manufactured by Corning Incorporated, Inc. (USA); the CCK-8 was manufactured by Beijing Bairddi Biotechnology Co., Ltd.; and the fetal bovine serum was manufactured by GIBCO, Inc. (USA).

[0072] The phosphate buffer used in the following effect examples was manufactured by Beijing Bairui Biotechnology Co., Ltd.

[0073] The WJ-80A-Ⅱ CO2 constant temperature incubator used in the following effect examples is manufactured by Shanghai Shengke Instrument Equipment Co., Ltd.; the Sunrise microplate reader is manufactured by Diken Trading Co., Ltd.

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

[0075] Example 1

[0076] Commercially available oat bran was pulverized and sieved to a particle size of 100 mesh. 3g of oat bran powder and 300mL of deionized water were mixed evenly, and then heat-resistant amylase was added. The mixture was stirred and hydrolyzed at 70℃ for 30min. The ratio of heat-resistant amylase added was 1mL / L, and the stirring speed was 300r / min. After the hydrolysis was completed, the mixture was mixed evenly and sterilized in an autoclave at 121℃ for 20min. 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, with the viable cell count of Lactobacillus helveticus CICC20243 in both cultures being 10⁻⁶. 7 The inoculum size was 7.5 mL, and the viable count of *Lactobacillus plantarum* culture was 10 CFU / mL. 7 The inoculum was 7.5 mL at CFU / mL and shaken well. It was then incubated at 43℃ for 15 h. After fermentation, the fermentation broth was sterilized at 121℃ for 20 min in an autoclave. After sterilization, the fermentation broth was cooled to room temperature and centrifuged at 4800 r / min for 30 min.

[0078] After centrifugation, the supernatant was collected and decolorized using activated carbon as the decolorizing agent. The mass-volume ratio of activated carbon to supernatant was 20 g / L. The decolorization stirring rate was 300 r / min, and the decolorization time was 60 min. After decolorization, the mixture was first filtered through 12 μm filter paper and then second filtered through 0.45 μm filter paper to remove the decolorizing agent. After decolorization, the mixture was sterilized for 20 min at 121℃ and 0.13 MPa. After sterilization, a preservative was added to the fermentation broth. During the mixing process with the preservative, the mixing temperature was 75℃. Based on the mass of the original fermentation broth, 0.5% p-hydroxyacetophenone and 0.8% 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 hours, while all other conditions are the same as in Example 1. The specific operation is as follows:

[0081] Commercially available oat bran was pulverized and sieved to a particle size of 100 mesh. 3g of oat bran powder and 300mL of deionized water were mixed evenly, and then heat-resistant amylase was added. The mixture was stirred and hydrolyzed at 70℃ for 30min. The ratio of heat-resistant amylase was 1mL / L, and the stirring speed was 300r / min. After the hydrolysis was completed, the mixture was mixed evenly and sterilized in an autoclave at 121℃ for 20min. After sterilization, the mixture was cooled to room temperature to obtain the initial fermentation system.

[0082] Lactobacillus helveticus and Lactobacillus plantarum were inoculated into the initial fermentation system for fermentation, with the viable cell count of Lactobacillus helveticus CICC20243 in both cultures being 10⁻⁶. 7 The inoculum size was 7.5 mL, and the viable count of *Lactobacillus plantarum* culture was 10 CFU / mL. 7 The inoculum was prepared at CFU / mL and 7.5 mL. After shaking well, the mixture was incubated at 43℃ for 24 h. After fermentation, the fermentation broth was sterilized at 121℃ for 20 min in an autoclave. After sterilization, the fermentation broth was cooled to room temperature and centrifuged at 4800 r / min for 30 min.

[0083] After centrifugation, the supernatant was collected and decolorized using activated carbon as the decolorizing agent. The mass-volume ratio of activated carbon to supernatant was 20 g / L. The decolorization stirring rate was 300 r / min, and the decolorization time was 60 min. After decolorization, the mixture was first filtered through 12 μm filter paper and then second filtered through 0.45 μm filter paper to remove the decolorizing agent. After decolorization, the mixture was sterilized for 20 min at 121℃ and 0.13 MPa. After sterilization, a preservative was added to the fermentation broth. During the mixing process with the preservative, the mixing temperature was 75℃. Based on the mass of the original fermentation broth, 0.5% p-hydroxyacetophenone and 0.8% 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 amount of oat bran used is 5g, and all other conditions are the same as in Example 1. The specific operation is as follows:

[0086] Commercially available oat bran was pulverized and sieved to a particle size of 100 mesh. 5g of oat bran and 300mL of deionized water were mixed evenly, and then heat-resistant amylase was added. The mixture was stirred and hydrolyzed at 70℃ for 30min. The ratio of heat-resistant amylase was 1mL / L, and the stirring speed was 300r / min. After the hydrolysis was completed, the mixture was mixed evenly and sterilized in an autoclave at 121℃ for 20min. After sterilization, the mixture 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, with the viable cell count of Lactobacillus helveticus CICC20243 in both cultures being 10⁻⁶. 7 The inoculum size was 7.5 mL, and the viable count of *Lactobacillus plantarum* culture was 10 CFU / mL. 7 The inoculum was prepared at CFU / mL and 7.5 mL. After shaking well, the mixture was incubated at 43℃ for 15 h. After fermentation, the fermentation broth was sterilized at 121℃ for 20 min in an autoclave. After sterilization, the fermentation broth was cooled to room temperature and centrifuged at 4800 r / min for 30 min.

[0088] After centrifugation, the supernatant was collected and decolorized using activated carbon as the decolorizing agent. The mass-volume ratio of activated carbon to supernatant was 20 g / L. The decolorization stirring rate was 300 r / min, and the decolorization time was 60 min. After decolorization, the mixture was first filtered through 12 μm filter paper and then second filtered through 0.45 μm filter paper to remove the decolorizing agent. After decolorization, the mixture was sterilized for 20 min at 121℃ and 0.13 MPa. After sterilization, a preservative was added to the fermentation broth. During the mixing process with the preservative, the mixing temperature was 75℃. Based on the mass of the original fermentation broth, 0.5% p-hydroxyacetophenone and 0.8% 1,2-hexanediol were added and mixed evenly to obtain small molecule oat β-glucan.

[0089] Example 4

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

[0091] Commercially available oat bran was pulverized and sieved to a particle size of 100 mesh. 300 mL of deionized water and 3 g of oat bran were mixed evenly, and then heat-resistant amylase was added. The mixture was stirred and hydrolyzed at 70°C for 30 min. The ratio of heat-resistant amylase was 1 mL / L, and the stirring speed was 300 r / min. After the hydrolysis was completed, the mixture was mixed evenly and sterilized in an autoclave at 121°C for 20 min. After sterilization, the mixture was cooled to room temperature to obtain the initial fermentation system.

[0092] Lactobacillus helveticus and Lactobacillus plantarum were inoculated into the initial fermentation system for fermentation, with the viable cell count of Lactobacillus helveticus CICC20243 in both cultures being 10⁻⁶. 7 The inoculum size was 7.5 mL, and the viable count of *Lactobacillus plantarum* culture was 10 CFU / mL. 7 The inoculum was prepared at CFU / mL and 15 mL. After shaking well, the mixture was incubated at 43°C for 15 h. After fermentation, the fermentation broth was sterilized at 121°C for 20 min in an autoclave. After sterilization, the fermentation broth was cooled to room temperature and centrifuged at 4800 r / min for 30 min.

[0093] After centrifugation, the supernatant was collected and decolorized using activated carbon as the decolorizing agent. The mass-volume ratio of activated carbon to supernatant was 20 g / L. The decolorization stirring rate was 300 r / min, and the decolorization time was 60 min. After decolorization, the mixture was first filtered through 12 μm filter paper and then second filtered through 0.45 μm filter paper to remove the decolorizing agent. After decolorization, the mixture was sterilized for 20 min at 121℃ and 0.13 MPa. After sterilization, a preservative was added to the fermentation broth. During the mixing process with the preservative, the mixing temperature was 75℃. Based on the mass of the original fermentation broth, 0.5% p-hydroxyacetophenone and 0.8% 1,2-hexanediol were added and mixed evenly to obtain small molecule oat β-glucan.

[0094] Example 5

[0095] Compared with Example 1, the only difference is that the amount of amylase added is 1.2 mL / L, the enzymatic hydrolysis temperature is 60℃, and the enzymatic hydrolysis time is 35 min. All other conditions are the same as in Example 1. The specific operation is as follows:

[0096] Commercially available oat bran was pulverized and sieved to a particle size of 100 mesh. 3g of oat bran and 300mL of deionized water were mixed evenly, and then heat-resistant amylase was added. The mixture was stirred and hydrolyzed at 60℃ for 35min. The ratio of heat-resistant amylase added was 1.2mL / L, and the stirring speed was 300r / min. After the hydrolysis was completed, the mixture was mixed evenly and sterilized in an autoclave at 121℃ for 20min. After sterilization, it was cooled to room temperature to obtain the initial fermentation system.

[0097] Lactobacillus helveticus and Lactobacillus plantarum were inoculated into the initial fermentation system for fermentation, with the viable cell count of Lactobacillus helveticus CICC20243 in both cultures being 10⁻⁶. 7 The inoculum size was 7.5 mL, and the viable count of *Lactobacillus plantarum* culture was 10 CFU / mL. 7The inoculum was prepared at CFU / mL and 7.5 mL. After shaking well, the mixture was incubated at 43℃ for 15 h. After fermentation, the fermentation broth was sterilized at 121℃ for 20 min in an autoclave. After sterilization, the fermentation broth was cooled to room temperature and centrifuged at 4800 r / min for 30 min.

[0098] After centrifugation, the supernatant was collected and decolorized using activated carbon as the decolorizing agent. The mass-volume ratio of activated carbon to supernatant was 20 g / L. The decolorization stirring rate was 300 r / min, and the decolorization time was 60 min. After decolorization, the mixture was first filtered through 12 μm filter paper and then second filtered through 0.45 μm filter paper to remove the decolorizing agent. After decolorization, the mixture was sterilized for 20 min at 121℃ and 0.13 MPa. After sterilization, a preservative was added to the fermentation broth. During the mixing process with the preservative, the mixing temperature was 75℃. Based on the mass of the original fermentation broth, 0.5% p-hydroxyacetophenone and 0.8% 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 only difference is that no inoculation fermentation is performed, only extraction is performed. All other conditions are the same as in Example 1. The specific operation is as follows:

[0101] Commercially available oat bran was pulverized and sieved to a particle size of 100 mesh. 3g of oat bran powder was mixed with 300mL of deionized water, and then a thermoresistant amylase was added. The mixture was hydrolyzed at 70℃ for 30 minutes with stirring. The thermoresistant amylase concentration was 1mL / L, and the stirring speed was 300 rpm. After hydrolysis, the pH was adjusted to 7, and the mixture was extracted in a 70℃ water bath with stirring at 300 rpm for 120 minutes. The extract was then centrifuged at 4800 rpm for 30 minutes. The supernatant was collected after centrifugation and then decolorized with stirring at 300 rpm. The supernatant was decolorized in activated carbon for 60 minutes, with a total volume of activated carbon and supernatant of 20 g / L. After decolorization, the supernatant was first filtered through 12 μm filter paper and then second filtered through 0.45 μm filter paper to remove the decolorizing agent. After decolorization, the supernatant was sterilized for 20 minutes at 121°C and 0.12 MPa. After sterilization, a preservative was added to the fermentation broth, and the mixing temperature was 75°C. Based on the mass of the fermentation broth, 0.5% p-hydroxyacetophenone and 0.8% 1,2-hexanediol were added and mixed thoroughly.

[0102] Comparative Example 2

[0103] Compared with Example 1, the only difference is the inoculation with Lactobacillus helveticus CGMCC No. 19090; all other conditions are the same as in Example 1. The specific operation is as follows:

[0104] Commercially available oat bran was pulverized and sieved to a particle size of 100 mesh. 3g of oat bran powder and 300mL of deionized water were mixed evenly, and then heat-resistant amylase was added. The mixture was stirred and hydrolyzed at 70℃ for 30min. The ratio of heat-resistant amylase was 1mL / L, and the stirring speed was 300r / min. After the hydrolysis was completed, the mixture was mixed evenly and sterilized in an autoclave at 121℃ for 20min. After sterilization, the mixture was cooled to room temperature to obtain the initial fermentation system.

[0105] The initial fermentation system was inoculated with *Lactobacillus helveticus* CGMCC No. 19090 for fermentation, with a viable count of 10⁻⁶ for all *Lactobacillus helveticus* CGMCC No. 19090. 7 The inoculum was prepared at CFU / mL and 15mL. After shaking well, the mixture was incubated at 43℃ for 15 hours. After fermentation, the fermentation broth was sterilized at 121℃ for 20 minutes in an autoclave. After sterilization, the fermentation broth was cooled to room temperature and centrifuged at 4800 rpm for 30 minutes.

[0106] After centrifugation, the supernatant was collected and decolorized using activated carbon as the decolorizing agent. The mass-volume ratio of activated carbon to supernatant was 20 g / L. The decolorization stirring rate was 300 r / min, and the decolorization time was 60 min. After decolorization, the mixture was first filtered through 12 μm filter paper and then second filtered through 0.45 μm filter paper to remove the decolorizing agent. After decolorization, the mixture was sterilized for 20 min at 121℃ and 0.13 MPa. After sterilization, a preservative was added to the fermentation broth. During the mixing process with the preservative, the mixing temperature was 75℃. Based on the mass of the original fermentation broth, 0.5% p-hydroxyacetophenone and 0.8% 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 only difference is the addition of a carbon source and a nitrogen source to the fermentation substrate; all other conditions are the same as in Example 1.

[0109] Commercially available oat bran was pulverized and sieved to a particle size of 100 mesh. 3g of oat bran powder and 300mL of deionized water were mixed evenly, and then heat-resistant amylase was added. The mixture was stirred and hydrolyzed at 70℃ for 30min. The ratio of heat-resistant amylase was 1mL / L, and the stirring speed was 300r / min. After hydrolysis, 1.5g of soybean peptide and 1.0g of glucose were added and mixed evenly. The mixture was then sterilized in an autoclave at 121℃ for 20min. 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, with the viable cell count of Lactobacillus helveticus CICC20243 in both cultures being 10⁻⁶. 7 The inoculum size was 7.5 mL, and the viable count of *Lactobacillus plantarum* culture was 10 CFU / mL. 7 The inoculum was prepared at CFU / mL and 7.5 mL. After shaking well, the mixture was incubated at 43℃ for 15 h. After fermentation, the fermentation broth was sterilized at 121℃ for 20 min in an autoclave. After sterilization, the fermentation broth was cooled to room temperature and centrifuged at 4800 r / min for 30 min.

[0111] After centrifugation, the supernatant was collected and decolorized using activated carbon as the decolorizing agent. The mass-volume ratio of activated carbon to supernatant was 20 g / L. The decolorization stirring rate was 300 r / min, and the decolorization time was 60 min. After decolorization, the mixture was first filtered through 12 μm filter paper and then second filtered through 0.45 μm filter paper to remove the decolorizing agent. After decolorization, the mixture was sterilized for 20 min at 121℃ and 0.13 MPa. After sterilization, a preservative was added to the fermentation broth. During the mixing process with the preservative, the mixing temperature was 75℃. Based on the mass of the original fermentation broth, 0.5% p-hydroxyacetophenone and 0.8% 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 only difference is the inoculation with Schizophyllum commune (CGMCC NO.40388) and Saccharomyces cerevisiae (CGMCC NO.17452), while all other conditions are the same as in Example 1.

[0114] Commercially available oat bran was pulverized and sieved to a particle size of 100 mesh. 3g of oat bran powder and 300mL of deionized water were mixed evenly, and then heat-resistant amylase was added. The mixture was stirred and hydrolyzed at 70℃ for 30min. The ratio of heat-resistant amylase added was 1mL / L, and the stirring speed was 300r / min. After the hydrolysis was completed, the mixture was mixed evenly and sterilized in an autoclave at 121℃ for 20min. After sterilization, it was cooled to room temperature to obtain the initial fermentation system.

[0115] Schizophyllum commune and yeast were inoculated into the initial fermentation system for fermentation. The inoculation amounts of Schizophyllum commune and yeast were 7.5 mL each. After mixing, the mixture was incubated statically at 43℃ for 15 h. After fermentation, the resulting fermentation broth was sterilized in an autoclave at 121℃ for 20 min. After sterilization, the fermentation broth was cooled to room temperature and centrifuged at 4800 r / min for 30 min.

[0116] After centrifugation, the supernatant was collected and decolorized using activated carbon as the decolorizing agent. The mass-volume ratio of activated carbon to supernatant was 20 g / L. The decolorization stirring rate was 300 r / min, and the decolorization time was 60 min. After decolorization, the mixture was first filtered through 12 μm filter paper and then second filtered through 0.45 μm filter paper to remove the decolorizing agent. After decolorization, the mixture was sterilized for 20 min at 121℃ and 0.13 MPa. After sterilization, a preservative was added to the fermentation broth. During the mixing process with the preservative, the mixing temperature was 75℃. Based on the mass of the original fermentation broth, 0.5% p-hydroxyacetophenone and 0.8% 1,2-hexanediol were added and mixed evenly to obtain small molecule oat β-glucan.

[0117] Comparative Example 5

[0118] Compared with Example 1, the only difference is that the inoculation species is Lactobacillus casei (CICC 20241), and all other conditions are the same as in Example 1.

[0119] Commercially available oat bran was pulverized and sieved to a particle size of 100 mesh. 3g of oat bran powder and 300mL of deionized water were mixed evenly, and then heat-resistant amylase was added. The mixture was stirred and hydrolyzed at 70℃ for 30min. The ratio of heat-resistant amylase added was 1mL / L, and the stirring speed was 300r / min. After the hydrolysis was completed, the mixture was mixed evenly and sterilized in an autoclave at 121℃ for 20min. After sterilization, it was cooled to room temperature to obtain the initial fermentation system.

[0120] Lactobacillus casei was inoculated into the initial fermentation system for fermentation, with a viable count of 10⁻⁶ for each type of Lactobacillus casei. 7The inoculum was prepared at CFU / mL and 7.5 mL. After shaking well, the mixture was incubated at 43℃ for 15 h. After fermentation, the fermentation broth was sterilized at 121℃ for 20 min in an autoclave. After sterilization, the fermentation broth was cooled to room temperature and centrifuged at 4800 r / min for 30 min.

[0121] After centrifugation, the supernatant was collected and decolorized using activated carbon as the decolorizing agent. The mass-volume ratio of activated carbon to supernatant was 20 g / L. The decolorization stirring rate was 300 r / min, and the decolorization time was 60 min. After decolorization, the mixture was first filtered through 12 μm filter paper and then second filtered through 0.45 μm filter paper to remove the decolorizing agent. After decolorization, the mixture was sterilized for 20 min at 121℃ and 0.13 MPa. After sterilization, a preservative was added to the fermentation broth. During the mixing process with the preservative, the mixing temperature was 75℃. Based on the mass of the original fermentation broth, 0.5% p-hydroxyacetophenone and 0.8% 1,2-hexanediol were added and mixed evenly to obtain small molecule oat β-glucan.

[0122] Example 1: DPPH free radical scavenging experiment

[0123] DPPH is a stable nitrogen-centered free radical that appears purple in organic solvents and exhibits maximum absorption at 517 nm. In DPPH experiments, when antioxidants are added to a DPPH solution, they react with the DPPH free radical, capturing its unpaired electrons and causing it to lose its free radical properties, changing from purple to colorless. By comparing the changes in absorbance before and after the reaction, the antioxidant capacity of a sample can be quantitatively assessed. This method is simple, rapid, and widely used in the food, pharmaceutical, and cosmetic industries to evaluate the antioxidant performance of products. Therefore, the scavenging effect of a sample on DPPH free radicals can be evaluated by measuring changes in absorbance.

[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 2 × 10 -4 Mix the mol / L DPPH solution thoroughly (tube A1);

[0126] (2) Take an equal volume (1 mL) of anhydrous ethanol (the solvent for the analyte) and 2 × 10⁻⁶ mol / L. -4 Mix the mol / L DPPH solution thoroughly (tube A2);

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

[0128] (4) After reacting in the dark for 30 minutes, measure the absorbance values ​​of tubes A1, A2 and A3 at 517 nm; the clearance rate is calculated as follows: clearance rate = [(A2+A3)-A1] / A2×100%.

[0129] This experiment tested the DPPH free radical scavenging assay 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 (significant difference), 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 free 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 composition of fermentation strains and fermentation substrates have a significant impact on the antioxidant performance of the final product.

[0134] Example 2: Determination of total antioxidant capacity

[0135] The total antioxidant capacity of the products prepared in the examples and comparative examples was tested using the Total Antioxidant Capacity Assay Kit (FRAP method) manufactured by Beyotime Biotechnology Co., Ltd. ns p > 0.05, no significant difference; * p<0.05, ** p<0.01, *** p<0.001 (significant difference), 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 showed that the total antioxidant capacity of the products prepared in Examples 1-5 was 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 fermentation strains and fermentation substrates have a significant impact on the total antioxidant capacity of the final product.

[0139] Example 3: Human Immortalized Epidermal Cell Toxicity Experiment

[0140] This experiment used immortalized human epidermal cells from the Chinese Scientific Cell Bank to verify the cytotoxicity of the products prepared in the above examples and comparative examples.

[0141] 1. Experimental steps:

[0142] The products obtained in Examples 1-5 and Comparative Examples 1-5 were respectively prepared into experimental test solutions with a volume percentage of 1.25% using serum-free MEM medium. Human immortalized epidermal cells were cultured in a medium containing 10% fetal bovine serum and 1% penicillin-antibody (1×10⁻⁶) 5 Cells were cultured in MEM medium containing 100 mg / L penicillin and 100 mg / L streptomycin. Cells were grown in a 37°C, 5% CO2 saturated humidity incubator. When cell confluence reached 85% or higher, logarithmic growth phase cells were digested with 0.05% trypsin, and the digestion was terminated with serum-containing MEM. Cells were counted using a cell counting chamber, and the cell suspension concentration was adjusted to 7 × 10⁻⁶ cells / mL. 4 Cells were seeded at a rate of 100 μL per well in a 96-well plate 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-buffered saline (PBS). In each experimental group, 100 μL of the pre-filtered and sterilized test solution of different concentrations was added to each well, with six replicates per test solution. The control group contained cells and was treated with serum-free MEM medium; the blank control group contained no cells and was treated with 100 μL of PBS. The cells were then incubated at 37°C and 5% CO2 for 24 h. The old culture medium was discarded, and the cells were washed twice with phosphate-buffered saline (PBS). Then, 100 μL of serum-free MEM medium and 10 μL of CCK-8 solution were added to each well, and the cells were incubated for another 3 h. The absorbance was measured at 450 nm, and the cell viability of each group was calculated. ns p > 0.05, no significant difference; * p<0.05, ** p<0.01, *** p<0.001 (significant difference), results are shown in Table 3 and Figure 3 .

[0143] The formula for calculating cell viability is as follows:

[0144] Cell viability (%) = (A experimental group - A blank control group) / (A control group - A blank 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 showed that the HaCaT cell survival rate of the products prepared in Examples 1-5 was higher than that of the products prepared in Comparative Examples 1-5. This indicates that the products prepared in the examples of this application have a significant proliferative effect on HaCaT cells, and the composition of the fermentation strain and fermentation substrate have a significant impact on the proliferative effect of HaCaT cells in the final product.

[0148] 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 manufactured by Beijing Solarbio Technology Co., Ltd. ns p > 0.05, no significant difference; * p<0.05, ** p<0.01, *** p<0.001 (significant difference), 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] From Table 4 and Figure 4 The results showed that the total sugar content of the products prepared in Examples 1-5 was higher than that of the products prepared in Comparative Examples 1-5, indicating that the composition of the fermentation strain and the fermentation substrate had a significant impact on the total sugar content of the final product.

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

[0154] Example 5: Measurement of the inflammatory factor IL-8 (repairing cells)

[0155] Sample preparation: The products prepared in Examples 1-5 and Comparative Examples 1-5 were used to prepare an experimental group test solution with a volume fraction of 2% using oat (AVENA SATIVA) β-glucan in serum-free MEM medium.

[0156] HaCaT cells were seeded at a density of 250,000 cells / mL into 6-well cell culture plates, with 2 mL of cell suspension added to each well. After 12 h of culture, the supernatant was discarded. For the control group, 2 mL of MEM solution was added. For both the experimental and model groups, cells were induced with 800 μg / mL LPS for 6 h, and the supernatant was discarded. For the experimental group, 2 mL of sample solution was added, and for the model group, 2 mL of MEM solution was added. Cells were treated for 24 h. The supernatant was collected, and 200 μL of cell lysis buffer was added to each well. Cells were centrifuged at 10000 rpm at 4°C for 10 min, and the supernatant was collected. 20 μL of the cell lysis buffer was used to detect the total protein content in the sample using a BCA assay kit. The inflammatory factors were measured according to the ELISA kit instructions, with OD values ​​measured at 450 nm. Based on the OD values, the IL-8 release level was calculated.

[0157] BCA reagent kit operation steps:

[0158] 1. Sample pretreatment: Prepare lysis buffer according to the amount to be used, with a ratio of RIPA:PMSF of 100:1. Remove the culture medium, wash once with PBS, add 150-250 μL of lysis buffer per well of a 6-well plate, and pipette a few times to ensure that the lysis buffer is in full contact with the cells.

[0159] 2. Post-processing: Centrifuge the lysed sample at 10,000 rpm for 10 min, collect the supernatant, and then proceed with the subsequent protein concentration determination;

[0160] 3. According to the ratio of reagent (A) BCA solution to reagent (B) Cu 2+ The BCA working solution was prepared at a ratio of 50:1.

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

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

[0163] 6. Detect absorbance at 562 nm;

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

[0165] ELISA kit detection steps:

[0166] 1. Reagent preparation

[0167] 1) Reagent warming: First, warm the reagent kit to room temperature 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 crystals are dissolved.

[0168] 2) Preparation of washing solution: Calculate the volume of the diluted washing solution to be used in advance, and then dilute the 20-fold concentrated washing solution with deionized water to make a 1-fold application solution. Store any unused concentrated washing solution at 4°C.

[0169] 3) Sample gradient dilution: Add 1 mL of standard / sample diluent (SR1) to the lyophilized standard, let stand for 15 minutes until completely dissolved, then gently mix (concentration 1000 pg / mL). Then add 500 μL of standard / sample diluent (SR1) to each of the remaining 6 tubes, and perform 2-fold dilutions at the following concentrations: 500, 250, 125, 62.5, 31.25, 15.62, and 0 pg / mL. 1000 pg / mL is the highest concentration of the standard curve, and the standard / sample diluent (SR1) is used as the zero point of the standard curve. Any unused reconstituted standard stock solution (concentration 1000 pg / mL) should be discarded or aliquoted as needed and stored at -80°C.

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

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

[0172] 6) Washing method: Shake off all the liquid in the wells of the microplate, pat dry on absorbent paper, add 300 μL of washing buffer per well in a wash bottle, let stand for 30 seconds, shake off all the liquid in the wells of the microplate, and pat dry on absorbent paper.

[0173] 2. Testing Procedures

[0174] 1) 30 minutes before the experiment, take out the reagent kit, allow it to return to room temperature, wash the plate three times and shake dry before adding the standards / samples;

[0175] 2) Add 100 μL of standard / sample to the reaction wells, seal the plate and incubate at 37°C for 90 min, then tap the plate and wash it 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 min, then tap 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 min, then tap the plate and wash it 5 times.

[0178] 5) Add 50 μL of chromogenic substrate to the reaction wells, seal the plate, and incubate at 37°C in the dark for 15 min.

[0179] 6) Add 50 μL of stop solution and immediately measure the OD value at 450 nm wavelength using a microplate reader (within 5 min) (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 1-5 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 anti-inflammatory effects, and the composition of fermentation strains and fermentation substrates have a significant impact on the anti-inflammatory properties of the final product.

[0184] Example 6: Measurement of the inflammatory factor IL-6 (repairing cells)

[0185] ELISA kit detection steps:

[0186] 1. Reagent preparation

[0187] 1) Reagent warming: First, warm the reagent kit to room temperature 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 crystals are dissolved.

[0188] 2) Preparation of washing solution: Calculate the volume of the diluted washing solution to be used in advance, and then dilute the 20-fold concentrated washing solution with deionized water to make a 1-fold application solution. Store any unused concentrated washing solution at 4°C.

[0189] 3) Sample gradient dilution: Add 1 mL of standard / sample diluent (SR1) to the lyophilized standard, let stand for 15 minutes until completely dissolved, then gently mix (concentration 1000 pg / mL). Then add 500 μL of standard / sample diluent (SR1) to each of the remaining 6 tubes, and perform 2-fold dilutions at the following concentrations: 500, 250, 125, 62.5, 31.25, 15.62, and 0 pg / mL. 1000 pg / mL is the highest concentration of the standard curve, and the standard / sample diluent (SR1) is used as the zero point of the standard curve. Any unused reconstituted standard stock solution (concentration 1000 pg / mL) should be discarded or aliquoted as needed and stored at -80°C.

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

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

[0192] 6) Washing method: Shake off all the liquid in the wells of the microplate, pat dry on absorbent paper, add 300 μL of washing buffer per well in a wash bottle, let stand for 30 seconds, shake off all the liquid in the wells of the microplate, and pat dry on absorbent paper.

[0193] 2. Testing Procedures

[0194] 1) 30 minutes before the experiment, take out the reagent kit, allow it to return to room temperature, wash the plate three times and shake dry before adding the standards / samples;

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

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

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

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

[0199] 6) Add 50 μL of stop solution and immediately measure the OD value at 450 nm wavelength using a microplate reader (within 5 min) (nsp > 0.05, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, significant difference). The results are shown in Table 6. 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] From Table 6 and Figure 6 The results show that the IL-6 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 anti-inflammatory effects, and the composition of fermentation strains and fermentation substrates have a significant impact on the anti-inflammatory properties of the final product.

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

[0204] RNA was extracted from HaCaT cells using the TriQuick total RNA extraction reagent according to the manufacturer's instructions. First-strand cDNA synthesis was performed using the FastQuant cDNA kit. qPCR MasterMix and qRT-PCR were used to detect the cDNA obtained from further reverse transcription. Specific primer sequences are shown in Tables 7-8 below. Figures 7-8 As shown ( ns p > 0.05, no significant difference; * p<0.05, ** p<0.01, *** p<0.001, indicating a significant difference.

[0205] qRT-PCR primer sequence listing

[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] From Table 7 and Figure 7 The results showed that the relative expression level of IL-8 mRNA in the products prepared in Examples 1-5 was lower than that in the products prepared in Comparative Examples 1-5. This indicates that the products prepared in the examples of this application have ideal anti-inflammatory effects, and the composition of the fermentation strain and fermentation substrate has a significant impact on the anti-inflammatory properties of the final product.

[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] From Table 8 and Figure 8 The results showed that the relative expression level of IL-6 mRNA in the products prepared in Examples 1-5 was lower than that in the products prepared in Comparative Examples 1-5. This indicates that the products prepared in the examples of this application have ideal anti-inflammatory effects, and the composition of the fermentation strain and fermentation substrate has a significant impact on the anti-inflammatory properties of the final product.

[0213] Example 8: Molecular weight of oat beta-glucan

[0214] Proteins were removed from the samples of Examples 1-5 and Comparative Examples 1-5, and oat β-glucan was precipitated by alcohol precipitation. The crude product was obtained by centrifugation and drying. An oat β-glucan solution was prepared by placing an Ubbelohde viscometer in a constant temperature water bath at 25°C. First, the time it took for distilled water to flow through the capillary was measured as a blank control (t0). Then, the time it took for each sample to flow through the capillary (t) was measured. The relative viscosity (ηr = t / t0) was calculated, and the specific viscosity (ηsp) was calculated. A standard curve was plotted 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, indicating a significant difference.

[0215] Table 9

[0216]

[0217]

[0218] From Table 9 and Figure 9 The results showed that the molecular weight of the products prepared in Examples 1-5 was lower 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 a smaller molecular weight and are easier to be absorbed by the skin. Moreover, the composition of the fermentation strain and the fermentation substrate have a significant impact on the molecular weight of the final product.

[0219] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof in this application are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

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

Claims

1. A method for preparing small molecule oat β-glucan, characterized in that, It includes the following steps: inoculating lactobacillus into a fermentation substrate composed of oat bran and water, fermenting and culturing, and then sterilizing; The lactobacillus is composed of Lactobacillus helveticus and Lactobacillus plantarum, and the ratio of viable bacteria of Lactobacillus helveticus to Lactobacillus plantarum is 1:(0.5~2). The plant lactobacillus is *Lactobacillus plantarum* (… Lactiplantibacillus plantarum BSJM23L004, the plant lactobacillus ( Lactiplantibacillus plantarum BSJM23L004, deposited at the China General Microbiological Culture Collection Center (CGMCC), No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China, on April 16, 2024, with accession number CGMCC No. 30350. The Lactobacillus helveticus mentioned is Lactobacillus helveticus deposited at the China Industrial Microbial Culture Collection Center, with accession number CICC 20243; The fermentation culture temperature was 43℃; The fermentation culture time is 15h~24h; The sterilization process includes cooling and centrifugation, followed by collection of the supernatant.

2. The method for preparing small molecule oat β-glucan as described in claim 1, characterized in that, The oat bran also includes oat bran powder obtained by crushing and sieving; the oat bran also includes an enzymatic hydrolysis process before use; the fermentation substrate also includes a sterilization process before use.

3. The method for preparing small molecule oat β-glucan as described in claim 2, characterized in that, The method for preparing the small molecule oat β-glucan satisfies at least one of the following conditions: The pulverized and sieved material has a mesh size of 80-150 mesh; The enzymatic hydrolysis temperature is 50~90℃; The enzymatic hydrolysis time is 20-40 minutes; The mass-to-volume ratio of the enzyme to the water is 0.5~2 mL / L; The enzymes used in the enzymatic hydrolysis include thermoresistant amylase; The sterilization method is high-temperature sterilization.

4. The method for preparing small molecule oat β-glucan as described in claim 3, characterized in that, The method for preparing the small molecule oat β-glucan satisfies at least one of the following conditions: The pulverized and sieved material has a mesh size of 90-120. The enzymatic hydrolysis temperature is 60~70℃; The enzymatic hydrolysis time is 25-35 minutes; The mass-to-volume ratio of the enzyme to the water is 0.8~1.5 mL / L; When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization temperature is 110~125℃; the sterilization pressure is 0.1~0.14MPa; and the sterilization time is 15~35min.

5. The method for preparing small molecule oat β-glucan as described in claim 4, characterized in that, When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization temperature is 115~121℃; the sterilization pressure is 0.12~0.13MPa; and the sterilization time is 15~25min.

6. The method for preparing small molecule oat β-glucan as described in claim 1, characterized in that, The method for preparing the small molecule oat β-glucan satisfies at least one of the following conditions: The *Lactobacillus helveticus* was added in the form of a *Lactobacillus helveticus* bacterial suspension, and the viable count of the *Lactobacillus helveticus* was 10-1. 5 ~10 10 CFU / mL; The *Lactobacillus plantarum* was added in the form of a *Lactobacillus plantarum* bacterial suspension, and the viable count of the *Lactobacillus plantarum* was 10-1. 5 ~10 10 CFU / mL.

7. The method for preparing small molecule oat β-glucan as described in claim 6, characterized in that, The method for preparing the small molecule oat β-glucan satisfies at least one of the following conditions: The viable count of the *Lactobacillus helveticus* was 10. 6 ~10 8 CFU / mL; The viable count of the *Lactobacillus plantarum* was 10. 6 ~10 8 CFU / mL.

8. The method for preparing small molecule oat β-glucan as described in claim 7, characterized in that, The method for preparing the small molecule oat β-glucan satisfies at least one of the following conditions: The viable count of the *Lactobacillus helveticus* was 10. 7 CFU / mL; The viable count of the *Lactobacillus plantarum* was 10. 7 CFU / mL.

9. The method for preparing small molecule oat β-glucan as described in claim 1, characterized in that, The method for preparing the small molecule oat β-glucan satisfies at least one of the following conditions: The fermentation culture is an anaerobic fermentation; The fermentation culture was carried out in a constant temperature incubator; The mass ratio of the oat bran to the water is 1:(20~200).

10. The method for preparing small molecule oat β-glucan as described in claim 9, characterized in that... The mass ratio of the oat bran to the water is 1:(50~150).

11. The method for preparing small molecule oat β-glucan as described in claim 1, characterized in that, The preparation method of the small molecule oat β-glucan satisfies at least one of the following conditions: the cooling is cooling to room temperature; The centrifuge speed is 3000~9000 rpm; The radius of the centrifuge is 8~15cm; The centrifugation time is 10-40 minutes.

12. The method for preparing small molecule oat β-glucan as described in claim 11, characterized in that, The method for preparing the small molecule oat β-glucan satisfies at least one of the following conditions: The centrifuge speed is 4000~6000 rpm; The radius of the centrifuge is 10 cm; The centrifugation time is 20-40 minutes.

13. The method for preparing small molecule oat β-glucan as described in claim 1, characterized in that, The method for preparing the small molecule oat β-glucan satisfies at least one of the following conditions: The centrifuge speed is 4800 rpm; The centrifugation time was 30 minutes.

14. The method for preparing small molecule oat β-glucan as described in claim 1, characterized in that, The centrifugation process is followed by at least one of the following operations: decolorization, secondary sterilization, and mixing with a preservative.

15. The method for preparing small molecule oat β-glucan as described in claim 14, characterized in that, The method for preparing the small molecule oat β-glucan satisfies at least one of the following conditions: The decolorizing agent used in the decolorization includes one or more of activated carbon, bentonite, and diatomaceous earth; The mass-to-volume ratio of the decolorizing agent to the supernatant is 10~30g / L; The stirring rate for decolorization is 200~500 r / min; The decolorization time is 50-70 minutes; The secondary sterilization method is high-temperature sterilization. During the mixing process with the preservative, the mixing temperature is 50–80°C; During the mixing process with the preservative, the preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol.

16. The method for preparing small molecule oat β-glucan as described in claim 15, characterized in that, The method for preparing the small molecule oat β-glucan satisfies at least one of the following conditions: The decolorizing agent used in the decolorization process is activated carbon; The mass-to-volume ratio of the decolorizing agent to the supernatant is 15-25 g / L; The stirring rate for decolorization is 300~400 r / min; The decolorization time is preferably 55-65 minutes; When the high-temperature sterilization method is used for the secondary sterilization, the temperature of the secondary sterilization is 115~125℃; the time of the secondary sterilization is 15~25min; and the pressure of the secondary sterilization is 0.1~0.14MPa. During the mixing process with the preservative, the mixing temperature is 60~80℃; When the preservative includes p-hydroxyacetophenone and 1,2-hexanediol, the p-hydroxyacetophenone accounts for 0.1% to 1% of the mass of the supernatant obtained after centrifugation, and the 1,2-hexanediol accounts for 0.5% to 2% of the mass of the supernatant obtained after centrifugation.

17. The method for preparing small molecule oat β-glucan as described in claim 16, characterized in that, The method for preparing the small molecule oat β-glucan satisfies at least one of the following conditions: When the high-temperature sterilization method is used for the secondary sterilization, the temperature of the secondary sterilization is 118~121℃; the time of the secondary sterilization is 20min; and the pressure of the secondary sterilization is 0.12~0.13MPa. During the mixing process with the preservative, the mixing temperature is 75°C; The p-hydroxyacetophenone accounts for 0.5% of the mass of the supernatant obtained after centrifugation, and the 1,2-hexanediol accounts for 0.8% of the mass of the supernatant obtained after centrifugation.

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