Semi-continuous fermentation combined with functional sugar addition for oat fermentation process and products and applications

Abstract

The application discloses a kind of semi-continuous fermentation combined with functional sugar addition oat fermentation method, comprising the following steps: 1) oat homogenate preparation;2) oat homogenate enzymolysis;3) oat medium preparation;4) first batch fermentation stage;5) semi-continuous fermentation stage: prepare new oat medium according to the above steps 1) -3);When tank is released after step 4) first batch fermentation stage ends, 10% of the mass fraction of liquid loading is retained in the fermenter, and new oat medium is added to the fermenter, to ensure that the mass fraction ratio of the remaining fermentation liquid and new oat medium in the fermenter is 1:9;Maintain the fermentation parameters in the first batch fermentation stage, restart fermentation, and observe the number of strains to determine the fermentation state, and when the amount of bacteria reaches the same amount as step 4), it is recorded as the fermentation completion standard;Then repeat step 5);6) post-treatment.The application has the characteristics of being able to effectively improve production efficiency and shorten production cycle.

Description

Technical Field

[0001] This invention relates to the field of oat fermentation preparation, and in particular to an oat fermentation method, product, and application that combines semi-continuous fermentation with the addition of functional sugars. Background Technology

[0002] Impaired skin barrier function can lead to numerous skin health problems. On one hand, a weakened barrier accelerates transepidermal water loss and the loss of skin nutrients. On the other hand, a fragile epidermal barrier enhances the penetration of irritants or allergens, and nerve endings are not adequately protected. Deeper layers of skin are exposed to microorganisms and external stimuli, triggering localized inflammation and symptoms such as redness, itching, burning, and stinging. Maintaining healthy skin homeostasis requires not only protecting the skin barrier and repairing it from multiple angles, but also controlling inflammatory responses.

[0003] Oats are rich in prebiotics such as protein, peptides, lipids, and beta-glucan. Their fermentable sugars serve as an excellent carbon source for probiotic fermentation, while other microorganisms and minerals provide beneficial nutrients for the skin. Currently, colloidal oats are widely used and have long been employed to relieve dry, itchy skin. They contain a unique antioxidant and anti-inflammatory phytochemical called anthranilamide, which promotes healthy skin and the overall skin barrier. Oat beta-(1,3,1,4)glucan has a unique structure different from yeast cell wall beta-(1,3,1,6)glucan, resulting in better water solubility. Through yeast fermentation, oats not only combine the properties of both types of glucan but also convert polysaccharides into oligosaccharides during fermentation, enhancing absorption.

[0004] Invention patent CN 111265468 A discloses a composition for repairing the skin's dual barriers. This composition, through the synergistic combination of Bifida ferment lysate filtrate, oat ferment filtrate, and a skin-like lipid complex, mimics prebiotics, probiotics, and microbial metabolites in the skin's microbiome, effectively stabilizing and balancing the skin's microbiome barrier. Meanwhile, the skin-like lipid complex and oat kernel oil primarily repair the skin's brick-and-mortar structure and improve the lipid composition of the stratum corneum, thereby achieving the goal of repairing the skin's physical barrier. Through the synergistic effect of these two barrier-repairing components, both the skin's microbiome barrier and physical barrier (stratum corneum) are simultaneously repaired, helping the skin rebuild a structurally intact stratum corneum and establish a healthy and thriving microbial community ecosystem.

[0005] Invention patent CN 113648263 A discloses a compound fermented product containing a topical skin agent, its preparation method, and its application. The preparation method of the compound fermented product includes the following steps: inoculating a fermentation strain into a fermentation substrate, followed by fermentation culture and sterilization; wherein the fermentation substrate is oat bran, acerola cherry extract, and water; the fermentation strain is a mixture of lactic acid bacteria and yeast, or yeast alone. Invention patent CN 113616579 A discloses an oat bran fermented product containing a topical skin agent, its preparation method, and its application. The preparation method of the oat bran fermented product includes the following steps: inoculating a fermentation strain into a fermentation substrate, followed by fermentation culture and sterilization; wherein the fermentation substrate is oat bran and water; the fermentation strain is a mixture of lactic acid bacteria and yeast, or lactic acid bacteria alone. In both of these patents, when yeast is used for fermentation, the required fermentation culture time is 30–50 hours.

[0006] In the authorized invention patent CN112494403, "An Oat Fermentation Broth and Its Preparation Method and Application," bio-enzymatic hydrolysis technology and lactic acid bacteria are used to ferment oats, improving the utilization rate of active ingredients and facilitating large-scale industrial production. This oat fermentation broth can be used to prepare cosmetics, exhibiting good anti-aging, antioxidant, and skin-firming effects.

[0007] Chinese patent CN 113018239 A discloses a method for preparing an oat fermentation extract. The method includes the following steps: Step 1, using oats as a fermentation substrate, inoculating with Bifidobacterium for anaerobic fermentation to obtain a fermentation broth; Step 2, inoculating the fermentation broth with yeast for aerobic fermentation to obtain the oat fermentation extract. This invention provides a method that utilizes Bifidobacterium and yeast to sequentially ferment oat raw materials through anaerobic and aerobic processes, and optimizes the fermentation control process to prepare an oat extract rich in various active ingredients such as oat β-glucan, glutathione, amino acids, lactic acid, total phenols, saponins, and flavonoids. The total fermentation time is 90-100 hours.

[0008] However, current oat-based fermentation processes primarily employ batch fermentation for small-scale preparation or production. This method is characterized by a lack of external material exchange within the entire culture system; filtered sterile air or oxygen is only introduced as needed. This approach is well-suited for small-batch, multi-variety fermentation production, allowing for immediate halting if contamination or strategy changes occur. However, the process necessitates the repeated preparation of seed culture and cleaning of fermentation equipment and pipelines between batches, resulting in repetitive consumption of time, labor, and energy.

[0009] In the above-mentioned fermentation of oats, the fermentation time for a single batch is more than 30 hours. This long-term single-batch fermentation mode is prone to the accumulation of toxic metabolites, the bacterial concentration is not easy to maintain and is prone to decline, and there is a high risk of bacterial mutation.

[0010] During fermentation, in order to maintain the accumulation of fermentation products, it is often necessary to add defoamers and pH adjusters to control the pH. The introduction of these additives due to the long fermentation period makes the composition of fermentation products more complex and causes certain difficulties in the later processing.

[0011] Therefore, there is an urgent need to develop an oat fermentation method that can improve production efficiency and shorten the production cycle. Summary of the Invention

[0012] The purpose of this invention is to provide a semi-continuous fermentation method, product, and application of oat fermentation combined with the addition of functional sugars. This invention effectively improves production efficiency and shortens the production cycle.

[0013] The technical solution of this invention: a semi-continuous fermentation method for oats combined with the addition of functional sugars, comprising the following steps:

[0014] 1) Preparation of oat homogenate;

[0015] Weigh out whole oat flour and mix it thoroughly with 8-10 times its weight of pure water to obtain oat homogenate.

[0016] 2) Oat homogenate enzymatic hydrolysis;

[0017] Add 0.03-0.05% (w / w) of Ban 480L medium-temperature α-amylase to the oat homogenate and stir for 0.6-1.5 h at 60-75℃.

[0018] Then, add 0.01-0.02% of Neutase 0.8L and Maltogenase 2X L of maltose amylase to the oat homogenate, and stir at 55-67℃ for 0.8-1.5h to obtain the enzymatic hydrolysate. After centrifugation, remove the precipitate, and the resulting clear liquid is the oat homogenate enzymatic hydrolysate.

[0019] 3) Preparation of oat culture medium;

[0020] Take oat homogenate hydrolysate, add 0.5-1% fermentation nutrients and 3-8% functional sugars of oat homogenate hydrolysate by mass, put it into a fermentation tank, sterilize it and cool it to room temperature for later use. The sterilized liquid obtained is oat culture medium.

[0021] 4) First fermentation stage: Add 0.005-0.02% (w / w) of brewer's yeast to the oat culture medium and ferment at 26-32℃ for 10-17 hours, maintaining an aeration ratio of 0.5-1.5 vvm and dissolved oxygen (DO) of 5-60%. Simultaneously, observe the bacterial count during fermentation to determine the fermentation status. When the bacterial count reaches 0.5-9.5 × 10⁻⁶... 7 When the concentration of CFU / mL reaches a certain level, it is recorded as the fermentation completion standard; the liquid obtained after fermentation is completed is discharged from the discharge port of the fermenter;

[0022] 5) Semi-continuous fermentation stage:

[0023] Prepare new oat culture medium according to steps 1)-3) above; when the first fermentation stage is completed in step 4), retain 10% of the fermentation liquid by mass in the fermentation tank and add new oat culture medium to the fermentation tank to ensure that the mass fraction ratio of the remaining fermentation liquid in the fermentation tank to the new oat culture medium is 1:9.

[0024] Maintain the fermentation parameters from the first fermentation stage, restart fermentation, and observe the number of bacteria to determine the fermentation status. When the number of bacteria reaches the same amount as in step 4), it is recorded as the fermentation completion standard. Then repeat step 5) to achieve a semi-continuous fermentation process production mode.

[0025] 6) Post-processing

[0026] After fermentation, the released liquid is filtered through an 80-mesh screen to remove solid impurities, and then homogenized under high pressure at 800–1,500 bar and 4°C. It is then centrifuged at 3,000–8,000 rpm and 4°C for 20 minutes. The resulting supernatant is the oat fermentation product.

[0027] In the aforementioned semi-continuous fermentation method of oat fermentation combined with the addition of functional sugars, the whole oat flour is whole wheat flour without de-braised, with a particle size of 50-100 mesh.

[0028] In the aforementioned semi-continuous fermentation method for oat fermentation combined with the addition of functional sugars, the obtained enzymatic hydrolysate is centrifuged at 25°C and 8000-10,000 rpm for 10-20 minutes to remove the precipitate, and the resulting clear liquid is the oat homogenate enzymatic hydrolysate.

[0029] In the aforementioned semi-continuous fermentation method for oat fermentation combined with the addition of functional sugars, the fermentation nutrients include ammonium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, glucose, and yeast extract.

[0030] In the aforementioned semi-continuous fermentation method for oat fermentation combined with the addition of functional sugars, the functional sugars are one or a combination of several of D-mannose, isomaltooligosaccharide, L-arabinose, D-trehalose, galactooligosaccharide, and fructooligosaccharide.

[0031] In the aforementioned semi-continuous fermentation method for oat fermentation combined with the addition of functional sugars, the sterilization temperature in step 3) of oat culture medium preparation is 121℃ and the time is 20min.

[0032] An oat fermentation product is prepared by the aforementioned oat fermentation method combining semi-continuous fermentation with the addition of functional sugars.

[0033] The oat fermentation products prepared by the aforementioned semi-continuous fermentation combined with functional sugar addition method can be used in the preparation of soothing toners, skin care essences, skin care gels, skin care lotions, skin care creams, makeup and / or facial cleansers and personal care products.

[0034] An emulsion comprising 0.5%-20% by mass of fermented oats; said fermented oats are prepared by the aforementioned semi-continuous fermentation method combined with the addition of functional sugars.

[0035] Compared with existing technologies, this invention utilizes semi-continuous fermentation technology combined with the addition of functional sugars during fermentation to enhance the stress resistance and vitality of fermentation strains. It maintains the strains in a stable and vigorous state during fermentation, thereby achieving the effects of rapidly accumulating fermentation strains, improving production efficiency, and shortening the production cycle.

[0036] This invention utilizes semi-continuous fermentation technology, which on the one hand avoids the waiting time associated with repetitive operations such as preparing inoculum, cleaning tanks, and sterilizing materials between batches; on the other hand, it significantly saves time required for multiple batches on the production line, thus improving production efficiency. Simultaneously, functional sugars are added during the fermentation process to protect the microbial cells and enhance their resilience to stress during long-term fermentation and product accumulation.

[0037] This invention shortens fermentation time and improves efficiency while simplifying the process. The entire fermentation process does not require the addition of other defoamers, pH adjusters, or other ingredients, ensuring that the fermented material does not need to undergo complex processes such as desalination and deodorization.

[0038] In summary, the present invention has the characteristics of effectively improving production efficiency and shortening the production cycle. Attached Figure Description

[0039] Figure 1 This is a diagram illustrating the fermentation process of process A in Example 1;

[0040] Figure 2 This is a diagram illustrating the fermentation process of process B in Example 1;

[0041] Figure 3 The growth curves of Saccharomyces cerevisiae on culture media with different functional sugars added (first fermentation);

[0042] Figure 4 The growth curves of Saccharomyces cerevisiae on culture media with different functional sugars (semi-continuous first time);

[0043] Figure 5 The growth curves of Saccharomyces cerevisiae on culture media with different functional sugars (semi-continuous second time);

[0044] Figure 6 The growth curves of Saccharomyces cerevisiae on culture media with different functional sugars (semi-continuous, 3rd iteration);

[0045] Figure 7 This is a diagram illustrating the fermentation process of process 3.1 in Example 3;

[0046] Figure 8 This is a diagram illustrating the fermentation process of process 3.2 in Example 3. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0048] Example. A semi-continuous fermentation method for oats combined with the addition of functional sugars, comprising the following steps:

[0049] 1) Preparation of oat homogenate;

[0050] Weigh out whole oat flour and mix it thoroughly with 8-10 times its weight of pure water to obtain oat homogenate.

[0051] 2) Oat homogenate enzymatic hydrolysis;

[0052] Add 0.03-0.05% (w / w) of Ban 480L medium-temperature α-amylase to the oat homogenate and stir for 0.6-1.5 h at 60-75℃.

[0053] Then, add 0.01-0.02% of Neutase 0.8L and Maltogenase 2X L of maltose amylase to the oat homogenate, and stir at 55-67℃ for 0.8-1.5h to obtain the enzymatic hydrolysate. After centrifugation, remove the precipitate, and the resulting clear liquid is the oat homogenate enzymatic hydrolysate.

[0054] 3) Preparation of oat culture medium;

[0055] Take oat homogenate hydrolysate, add 0.5-1% fermentation nutrients and 3-8% functional sugars of oat homogenate hydrolysate by mass, put it into a fermentation tank, sterilize it and cool it to room temperature for later use. The sterilized liquid obtained is oat culture medium.

[0056] 4) First fermentation stage: Add 0.005-0.02% (w / w) of brewer's yeast to the oat culture medium and ferment at 26-32℃ for 10-17 hours, maintaining an aeration ratio of 0.5-1.5 vvm and dissolved oxygen (DO) of 5-60%. Simultaneously, observe the bacterial count during fermentation to determine the fermentation status. When the bacterial count reaches 0.5-9.5 × 10⁻⁶... 7 When the concentration of CFU / mL reaches a certain level, it is recorded as the fermentation completion standard; the liquid obtained after fermentation is completed is discharged from the discharge port of the fermenter;

[0057] 5) Semi-continuous fermentation stage:

[0058] Prepare new oat culture medium according to steps 1)-3) above; when the first fermentation stage is completed in step 4), retain 10% of the fermentation liquid by mass in the fermentation tank and add new oat culture medium to the fermentation tank to ensure that the mass fraction ratio of the remaining fermentation liquid in the fermentation tank to the new oat culture medium is 1:9.

[0059] Maintain the fermentation parameters from the first fermentation stage, restart fermentation, and observe the number of bacteria to determine the fermentation status. When the number of bacteria reaches the same amount as in step 4), it is recorded as the fermentation completion standard. Then repeat step 5) to achieve a semi-continuous fermentation process production mode.

[0060] 6) Post-processing

[0061] After fermentation, the released liquid is filtered through an 80-mesh screen to remove solid impurities, and then homogenized under high pressure at 800–1,500 bar and 4°C. It is then centrifuged at 3,000–8,000 rpm and 4°C for 20 minutes. The resulting supernatant is the oat fermentation product.

[0062] Whole oat flour is made from whole wheat without the bran removed, with a particle size of 50-100 mesh.

[0063] After centrifuging the obtained enzymatic hydrolysate at 25℃ and 8000-10,000 rpm for 10-20 min, the precipitate was removed, and the resulting clear liquid was the oat homogenate enzymatic hydrolysate.

[0064] Fermented nutrients include ammonium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, glucose, and yeast extract.

[0065] The functional sugar is one or a combination of several of D-mannose, isomaltooligosaccharide, L-arabinose, D-trehalose, and galactooligosaccharide;

[0066] Step 3) The sterilization temperature in the oat culture medium preparation is 121℃ and the time is 20min.

[0067] An oat fermentation product is prepared by the aforementioned oat fermentation method combining semi-continuous fermentation with the addition of functional sugars.

[0068] Oat fermentation products prepared by a semi-continuous fermentation method combined with the addition of functional sugars can be used in the preparation of soothing toners, skin care essences, skin care gels, skin care lotions, skin care creams, makeup and / or facial cleansers.

[0069] An emulsion comprising 1%-1.5% by mass of fermented oats; said fermented oats are prepared by an oat fermentation method according to any one of claims 1-5, involving semi-continuous fermentation combined with the addition of functional sugars.

[0070] Example 1

[0071] Process A (Comparative Example): Batch fermentation process (4 batches): During the process, samples were taken at intervals of 0, 3, 4, 6, 7, 8, 10, 12, 13, and 14 hours to determine the viable cell count, pH, and Brix value.

[0072] Specific process:

[0073] 1) Weigh 250g of oat flour and mix thoroughly with 2.5L of pure water. Add 0.03% (w / w) of Ban 480L medium-temperature amylase and hydrolyze in a 73℃ water bath with stirring for 90 minutes. Continue to add 0.01% (w / w) of Neutase 0.8L neutral protease and Maltogenase 2XL maltose amylase and hydrolyze in a 60℃ water bath with stirring for 60 minutes. Centrifuge the resulting hydrolysate (10,000 rpm, 10 min), discard the precipitate, and keep the supernatant.

[0074] 2) Add 1% by mass of fermentation nutrients to 2.5L of enzymatic hydrolysis supernatant and put it into a fermentation tank. After autoclaving, cool it to room temperature for later use. This is called oat culture medium.

[0075] 3) Add 0.012% (w / w) of brewer's yeast to the oat culture medium, start fermentation, and set the fermentation parameters as follows: 32℃, aeration ratio of 0.8vvm, and DO control of 60%.

[0076] 4) After fermentation is complete, the fermentation liquid is filtered through an 80-mesh screen to remove solid impurities, then homogenized under high pressure at 800 bar and 4°C, and then centrifuged at 4,000 rpm for 20 minutes to obtain the oat fermentation product.

[0077] 5) Between fermentation batches, the tanks need to be cleaned, and the oat hydrolysate needs to be prepared, which takes 3-4 hours in total; the culture medium sterilization takes 2 hours. Therefore, in the batch fermentation process, at least a 5-6 hour interval is required before proceeding to the next production stage.

[0078] Process B (Implementation Example): Semi-continuous fermentation process (4 batches): During the process, samples were taken at certain intervals to determine the viable cell count, pH, and Brix value.

[0079] Specific process:

[0080] 1) The enzymatic hydrolysis of oat culture medium is the same as step 1 described in "Process A".

[0081] 2) Add 1% fermentation nutrients and 6% trehalose to 2.5L of enzymatic hydrolysis supernatant, and put it into a fermentation tank. After autoclaving, cool it to room temperature for later use. This is called oat culture medium. Add 0.012% brewer's yeast to the oat culture medium and set the fermentation parameters as follows: 32℃, aeration ratio of 0.8vvm, and DO control of 60%.

[0082] 3) When the fermentation is finished, leave 250mL of fermentation liquid in the tank and add 2.25L of sterilized oat culture medium (prepared as described in steps 1-2 of "Process A") to the fermentation tank to restart the fermentation. Observe the number of bacteria to determine the fermentation status. When the number of bacteria reaches the same amount, it is recorded as the fermentation completion standard.

[0083] 4) After fermentation is completed in step 3), retain 250 mL of fermentation liquid in the tank and add oat culture medium again. Repeat step 3 twice.

[0084] 5) After each batch of fermentation is completed, the fermentation liquid is filtered through an 80-mesh screen to remove solid impurities, then homogenized under high pressure at 800 bar and 4°C, and then centrifuged at 4,000 rpm for 20 minutes to obtain the oat fermentation product.

[0085] The results show:

[0086] In the batch fermentation process of process A, the brewing yeast is in the logarithmic growth phase for the first 6-12 hours of each batch of fermentation, with the number of viable cells increasing exponentially and the pH and Brix values ​​decreasing. All indicators tend to stabilize at 12-14 hours, and finally reach the fermentation endpoint as the number of cells increases.

[0087] In the semi-continuous fermentation process of process B, the brewing yeast enters the mid-log phase at about 10 hours and the stationary phase at about 13 hours in the first fermentation stage, which is similar to the indicators in the batch fermentation process. In the subsequent three semi-continuous fermentation stages, the brewing yeast enters the mid-log phase at about 4 hours and enters the stationary phase between 7 and 8 hours. Compared with the first fermentation, the growth of brewing yeast is significantly faster, and the time required to reach the same viable cell count is almost halved. Compared with the fermentation degree in process A, the time required for each batch and the total production time are both shortened.

[0088] Example 2

[0089] 3 batches of semi-continuous fermentation process

[0090] Specific process:

[0091] 1) Weigh 250g of oat flour and mix thoroughly with 2.5L of pure water. Add 0.05% (w / w) of Ban 480L medium-temperature amylase and hydrolyze in a 65℃ water bath with stirring for 40 minutes. Continue to add 0.015% (w / w) of Neutase 0.8L neutral protease and Maltogenase 2XL maltose amylase and hydrolyze in a 65℃ water bath with stirring for 90 minutes. Centrifuge the resulting hydrolysate (8,000 rpm, 20 min), discard the precipitate, and keep the supernatant.

[0092] 2) Add 0.8% fermentation nutrients and 3% trehalose to 2.5L of enzymatic hydrolysis supernatant, and put it into a fermentation tank. After autoclaving, cool it to room temperature for later use. This is called oat culture medium.

[0093] 3) Add 0.02% (w / w) of brewer's yeast to the oat culture medium, start fermentation, and set the fermentation parameters as follows: 28℃, aeration ratio of 1.5vvm, and DO control of 20%.

[0094] 4) When the fermentation is finished, leave 250mL of fermentation liquid in the tank and add 2.25L of sterilized oat culture medium into the fermentation tank to restart the fermentation.

[0095] 5) After each batch of fermentation is completed, the fermentation liquid is filtered through an 80-mesh screen to remove solid impurities, then homogenized under high pressure at 1250 bar and 4℃, and then centrifuged at 8,000 rpm for 20 min to obtain the oat fermentation product.

[0096] Example 3.

[0097] Process 3.1

[0098] 3 batches of semi-continuous fermentation process

[0099] 1) Weigh 1000g of oat flour and mix thoroughly with 8L of pure water. Add 0.04% (w / w) of Ban 480L medium-temperature amylase and hydrolyze in a 60℃ water bath with stirring for 80 minutes. Continue to add 0.02% (w / w) of Neutase 0.8L neutral protease and Maltogenase 2XL maltose amylase and hydrolyze in a 55℃ water bath with stirring for 50 minutes. Centrifuge the resulting hydrolysate (10,000 rpm, 15 min), discard the precipitate, and keep the supernatant.

[0100] 2) Add 0.5% fermentation nutrients and 8% arabinose to 8L of enzymatic hydrolysis supernatant and put it into a fermentation tank. After autoclaving, cool to room temperature for later use. This is called oat culture medium.

[0101] 3) Add 0.05% (w / w) of brewer's yeast to the oat culture medium, start fermentation, and set the fermentation parameters as follows: 26℃, aeration ratio of 0.5vvm, and DO control of 5%.

[0102] 4) When the fermentation is finished, leave 800mL of fermentation liquid in the tank and add 7.2L of sterilized oat culture medium into the fermentation tank to restart the fermentation.

[0103] 5) After each batch of fermentation is completed, the fermentation liquid is filtered through an 80-mesh screen to remove solid impurities, then homogenized under high pressure at 1500 bar and 4℃, and then centrifuged at 5,000 rpm for 20 min to obtain the oat fermentation product.

[0104] Process 3.2

[0105] The only difference from process 3.1 is the use of 8% mannose for comparison.

[0106] Experimental conclusions (such as...) Figure 7 and 8 As shown):

[0107] 1) Initial fermentation: In both groups, *Saccharomyces cerevisiae* entered the mid-log phase around 11 hours and the stationary phase around 14 hours. Comparatively, the addition of mannose promoted *Saccharomyces cerevisiae* proliferation more effectively, with the highest viable cell count reaching 1.31E+08 CFU / mL, slightly higher than the 8.26E+07 CFU / mL in the group with added arabinose. Both groups showed a highly synergistic trend in pH and Brix values, remaining relatively stable from 0-10 hours of fermentation and gradually decreasing slowly from 11-14 hours.

[0108] 2) Semi-continuous fermentation: The proliferation rate of brewer's yeast accelerated, entering the mid-log phase at around 5 hours and the stationary phase at around 8 hours. However, the highest viable cell count in the semi-continuous fermentation stage was slightly lower than that in the first fermentation stage. The viable cell count in the mannose-added experimental group was consistently slightly higher than that in the arabinose-added experimental group.

[0109] This proves that arabinose and mannose can also be used effectively.

[0110] Example 4. Preparation of emulsion from fermented oats.

[0111] Add phase A (A1-A6 in the table below) to a water pot, heat to 85℃, and stir until completely dissolved; accurately weigh phase B, heat to 85℃, and stir evenly; transfer phase A to an emulsification pot, then transfer phase B (B1-B4 in the table below), homogenize at 85℃ and 2500rpm for 6 minutes, and cool down; cool down to 50℃, add phase C (C1 in the table below) to the pot and stir evenly, and continue to cool down; cool down to below 37℃, add phase D (D1 in the table below), homogenize at 2500rpm for 2 minutes, stir evenly, and filter out the material after passing inspection at below 35℃.

[0112]

[0113] In vitro testing of soothing effects

[0114] 1. CCK-8 assay to detect the cytotoxic effects of oat fermentation products on RAW264.7 cells.

[0115] Samples were diluted with different concentration gradients to prepare different concentrations for each sample to be tested. The samples were then added to the cell seeding plate, and the following settings were configured:

[0116] Blank control: RAW264.7 cells + DMEM medium

[0117] Reagent blank control: DMEM medium,

[0118] The cell culture plates were then incubated in an incubator at 37°C and 5% CO2 for 24 hours.

[0119] After incubation, add 10 μl of CCK-8 solution (including reagent blank control wells) to each well of the plate, gently shake to mix, and then incubate at 37°C with 5% CO2 for 1-2 hours. After incubation, measure the absorbance of each well of the 96-well plate at 450 nm using a microplate reader.

[0120] Calculation formula: Cell viability (%) = (OD of test sample - OD of reagent blank) / (OD of blank group - OD of reagent blank) × 100%

[0121] 2. NO kit method for detecting the effects of oat fermentation product 1 (oat fermentation product obtained by process B in Example 1) and oat fermentation product 2 (oat fermentation product obtained in Example 2) on NO release from RAW264.7 cells.

[0122] Select sample concentrations with cytotoxicity survival rates greater than 90%, add the samples to the cell seeding plate, and simultaneously set:

[0123] Blank control: Complete culture medium + RAW264.7 cells; Positive control: Complete culture medium + RAW264.7 cells + 1 μg / mL LPS + 100 μg / mL dexamethasone

[0124] LPS group: Complete culture medium + RAW264.7 cells + 1 μg / mL LPS (final concentration)

[0125] After 24 hours of cell culture, 200 μL of culture medium was added to each well, followed by LPS solution (100 μg / mL) to bring the final concentration in the system to 1 μg / mL. After mixing, the cells were cultured for another 24 hours. After incubation, Griess Reagent solution from the NO detection kit was added sequentially, and the mixture was shaken and mixed. The absorbance of each well in the 96-well plate was measured at 540 nm using a microplate reader.

[0126] Calculation formula: NO release rate = 1 - [(OD sample group - OD blank group) / (OD LPS group - OD blank group)] × 100%.

[0127] 3. Statistical Analysis

[0128] All experiments were conducted in parallel design, with at least two replicates. Experimental data are expressed as mean ± standard deviation (Mean ± SD), and data analysis was performed using Excel statistical software.

[0129] Experimental data

[0130] 1) Effects of oat fermentation products on the cytotoxicity of RAW264.7 cells

[0131]

[0132]

[0133] 2) Effects of oat fermentation products on NO release from RAW264.7 cells

[0134]

[0135] 3) Experimental Results

[0136] 1. According to the CCK-8 results, oat fermentation products at concentrations of 5% and below are not toxic to RAW264.7.

[0137] 2. According to the NO results, all oat fermentation product samples below 5% showed NO inhibition, and the NO inhibition rate at 5% concentration was higher than that at 0.1% concentration. Comparative experiments were conducted on samples with different added functional sugars.

[0138] The effects of different functional sugars on the growth curve of Saccharomyces cerevisiae were determined using the MGC-200 microbial growth curve analyzer.

[0139] The measurement conditions were as follows: 500 μL sample volume per well in a 48-well plate; 700 rpm pre-shaking speed; 700 rpm shaker; 2 s sampling delay; 5 xenon lamp cycles.

[0140] First fermentation: Saccharitomyces were inoculated into oat bran enzymatic hydrolysate containing 3% (w:v) functional sugars, including D-mannose, isomaltooligosaccharide, L-arabinose, D-trehalose, and galactooligosaccharide (inoculation amount: 0.01%, 50 μL).

[0141] Semi-continuous fermentation in 3 batches: Take 50 μL of the bacterial culture from the first batch that has entered the stationary phase and transfer it to the corresponding well (containing 450 μL of the corresponding culture medium). Continue fermentation in the stationary phase and repeat this operation 3 times. The measurement conditions are the same as those for the first batch of fermentation.

[0142] The results show:

[0143] In the initial fermentation stage: The growth trends of *Saccharomyces cerevisiae* were similar in media with and without added functional sugars, entering the mid-log phase around 8 hours and the stationary phase around 12 hours. The growth curves of the control group (without added functional sugars) and the isomaltooligosaccharide group were almost identical, indicating that the addition of isomaltooligosaccharide had virtually no effect on the proliferation of *Saccharomyces cerevisiae*. However, the addition of the other four functional sugars (D-mannose, L-arabinose, D-trehalose, and galactooligosaccharides) all promoted the proliferation of *Saccharomyces cerevisiae*.

[0144] Semi-continuous fermentation stage: After entering the semi-continuous fermentation stage, the time for *Saccharomyces cerevisiae* to enter the stationary phase is earlier, reaching mid-logarithmic growth around 3 hours and the stationary phase around 5 hours. In the second batch of fermentation (semi-continuous first fermentation), the addition of L-arabinose and D-trehalose promoted the proliferation of *Saccharomyces cerevisiae*. In the third batch of fermentation (semi-continuous second fermentation), the proliferation effect of *Saccharomyces cerevisiae* in the L-arabinose and D-trehalose groups was better than that in the control group. In the fourth batch of fermentation (semi-continuous third fermentation), all five added functional sugars promoted the proliferation of *Saccharomyces cerevisiae*, with D-trehalose showing the most significant promoting effect.

Claims

1. A semi-continuous fermentation method for oat fermentation combined with the addition of functional sugars, characterized in that, Includes the following steps: 1) Preparation of oat homogenate; Weigh out whole oat flour and mix it thoroughly with 8-10 times its weight of pure water to obtain oat homogenate. 2) Oat homogenate enzymatic hydrolysis; Add 0.03-0.05% (w / w) of medium-temperature α-amylase to the oat homogenate and stir at 60-75℃ for 0.6-1.5 h for enzymatic hydrolysis. Then, add 0.01-0.02% of neutral protease and maltose amylase from the oat homogenate, and stir at 55-67℃ for 0.8-1.5 hours to obtain the enzymatic hydrolysate. After centrifuging the obtained enzymatic hydrolysate to remove the precipitate, the resulting clear liquid is the oat homogenate enzymatic hydrolysate. 3) Preparation of oat culture medium; Take oat homogenate hydrolysate, add 0.5-1% fermentation nutrients and 3-8% functional sugars of oat homogenate hydrolysate by mass, put it into a fermentation tank, sterilize it and cool it to room temperature for later use. The sterilized liquid obtained is oat culture medium. 4) First fermentation stage: Add 0.005-0.02% (w / w) of brewer's yeast to the oat culture medium and ferment at 26-32℃ for 10-17 hours, maintaining an aeration ratio of 0.5-1.5 vvm and dissolved oxygen (DO) of 5-60%. Simultaneously, observe the bacterial count during fermentation to determine the fermentation status. When the bacterial count reaches 0.5-9.5 × 10⁻⁶... 7 When the concentration of CFU / mL reaches a certain level, it is recorded as the fermentation completion standard; the liquid obtained after fermentation is completed is discharged from the discharge port of the fermenter; 5) Semi-continuous fermentation stage: Prepare new oat culture medium according to steps 1)-3) above; when the first fermentation stage is completed in step 4), retain 10% of the fermentation liquid by mass in the fermentation tank and add new oat culture medium to the fermentation tank to ensure that the mass fraction ratio of the remaining fermentation liquid in the fermentation tank to the new oat culture medium is 1:

9. Maintain the fermentation parameters from the first fermentation stage, restart fermentation, and observe the number of bacteria to determine the fermentation status. When the number of bacteria reaches the same amount as in step 4), it is recorded as the fermentation completion standard. Then repeat step 5) to achieve a semi-continuous fermentation process production mode. 6) Post-processing After fermentation, the released liquid is filtered through an 80-mesh screen to remove solid impurities, and then homogenized under high pressure at 800–1,500 bar and 4°C. It is then centrifuged at 3,000–8,000 rpm and 4°C for 20 min, and the resulting supernatant is the oat fermentation product. Whole oat flour is made from whole wheat without the bran removed, with a particle size of 50-100 mesh. After centrifuging the obtained enzymatic hydrolysate at 25℃ and 8000-10,000 rpm for 10-20 min, the precipitate was removed, and the resulting clear liquid was the oat homogenate enzymatic hydrolysate. Fermented nutrients include ammonium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, glucose, and yeast extract. The functional sugar is one or a combination of several of the following: D-mannose, isomaltooligosaccharide, L-arabinose, D-trehalose, galactooligosaccharide, and fructooligosaccharide.

2. The oat fermentation method combining semi-continuous fermentation with the addition of functional sugars according to claim 1, characterized in that: Step 3) The sterilization temperature in the oat culture medium preparation is 121℃ and the time is 20 min.

3. A fermented oat product, characterized in that, It is a product prepared by the oat fermentation method of any one of claims 1-2, which combines semi-continuous fermentation with the addition of functional sugars.

4. The oat fermentation product prepared by the oat fermentation method of any one of claims 1-2, which combines semi-continuous fermentation with the addition of functional sugars, can be used in the preparation of soothing lotions, skin care essences, skin care gels, skin care lotions, skin care creams, makeup and / or facial cleansing products, and personal care products.

5. An emulsion, characterized in that: It includes 0.5%-20% fermented oats; the fermented oats are prepared by the oat fermentation method of any one of claims 1-2, which combines semi-continuous fermentation with the addition of functional sugars.

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