Specialized for oat bran insoluble dietary fiber modification of synergistic fermentation complex microbial agent and modification method and use

Abstract

The present application relates to the technical field of functional food processing and high-value utilization of grain by-products, and particularly relates to a synergistic fermentation complex microbial inoculum specially used for modification of oat bran insoluble dietary fiber and a modification method and use thereof.The synergistic fermentation complex microbial inoculum is compounded by Bacillus subtilis bacterial suspension and Lactobacillus plantarum bacterial suspension at a volume ratio of 1:2;after modification by the present application, the adsorption capacity and antioxidant activity of the obtained oat bran IDF (namely IDF-3) are significantly improved, and the inhibition effect on alpha-amylase is also enhanced.In a simulated human intestinal flora in vitro fermentation system, IDF-3 presents the fermentation characteristics of "low gas production and high acid production", the total short-chain fatty acid production, the absolute content and the relative proportion of acetic acid and butyric acid are all significantly improved under the condition of low gas production, which indicates that IDF-3 is more inclined to slow and mild acid production metabolic pathways rather than rapid gas production pathways in the fermentation and utilization process of intestinal flora.

Description

Technical Field

[0001] This invention relates to the field of functional food processing and high-value utilization of grain by-products, and particularly to a method for directional modification of oat bran insoluble dietary fiber based on dual-strain synergistic solid-state fermentation and its application in functional foods and prebiotic ingredients. It also relates to a synergistic fermentation compound microbial agent, modification method and use specifically for modifying oat bran insoluble dietary fiber. Background Technology

[0002] Oat bran is a major byproduct of oat processing, rich in various bioactive components such as β-glucan, arabinoxylan, cellulose, and bound polyphenols. Insoluble dietary fiber (IDF), the most abundant component in oat bran, accounts for over 70% of its total dietary fiber, possessing high nutritional value and potential for functional development. However, natural oat bran IDF exhibits inherent drawbacks due to its dense structure and highly cross-linked fiber network, including weak adsorption capacity for small molecules such as glucose, cholesterol, and nitrite, low antioxidant activity, and insufficient α-amylase inhibition. Furthermore, the limited efficiency of intestinal flora in its fermentation and utilization, along with low production of short-chain fatty acids (especially butyric acid), severely restricts the high-value utilization of oat bran IDF in the functional food industry.

[0003] Currently, modification technologies for oat bran IDF mainly include physical modification (such as high-pressure homogenization, ultrafine grinding, and extrusion puffing), chemical modification (such as acid treatment and alkali treatment), and enzymatic modification. However, physical modification methods are energy-intensive and require large equipment investments; chemical modification methods easily introduce residual reagents, which does not conform to the development trend of clean labeling; single enzymatic modification is costly and has a single target, making it difficult to simultaneously achieve the loosening of IDF structure and the efficient release of bound polyphenols. In contrast, bio-fermentation is green, mild, low-cost, and can simultaneously achieve structural modification and the release of active ingredients. Regarding the choice of fermentation method, liquid deep fermentation usually has problems such as high water consumption, difficulty in separating cells from the substrate, high energy consumption for subsequent dehydration, and large wastewater discharge; while solid-state fermentation has outstanding advantages such as low water content, simple equipment structure, low energy consumption, and high concentration of active substances in the fermentation end product, which is particularly in line with the industrial needs of on-site conversion of grain by-products and clean production. In terms of strain selection, Bacillus subtilis and Lactobacillus plantarum are both recognized food-grade safe strains (GRAS). The strains are widely available, the culture media are inexpensive and readily available, the growth conditions are mild, and the culture cycle is short. The enzyme production and acid production metabolism of both can be finely controlled by conventional parameters such as temperature and time. The fermentation process is easy to stably reproduce, which significantly reduces the process risks and production costs in the industrial scale-up process.

[0004] Therefore, there is an urgent need to develop a novel synergistic fermentation modification method that can simultaneously achieve targeted decomposition of IDF structure, significantly enhance adsorption performance and antioxidant activity, and effectively improve α-amylase inhibition. Furthermore, while maintaining the inherent prebiotic characteristics of IDF—slow fermentation and low gas production—this method should guide the intestinal flora metabolism towards a "low gas production, high acid production" acid-producing pathway, significantly increasing the production levels of short-chain fatty acids and butyrate in the colon. Short-chain fatty acids, as the core metabolites of this acid-producing pathway, can lower intestinal pH, inhibit the proliferation of pathogenic bacteria, and optimize the flora structure. Butyrate is the preferred energy substrate for colonic epithelial cells, strengthening the intestinal mucosal barrier and exerting anti-inflammatory effects. Acetic acid and propionic acid, absorbed via the portal vein, participate in the regulation of host glucose and lipid metabolism, helping to improve insulin sensitivity and generate a feeling of satiety.

[0005] The IDF-3 obtained by this invention can effectively avoid gastrointestinal discomfort such as bloating and abdominal pain induced by rapid fermentation, while providing a new technical path for the high-value application of oat bran in functional food fields such as assisting in lowering blood sugar, assisting in lowering blood lipids, regulating intestinal flora homeostasis, and preventing metabolic syndrome. It has important theoretical value and broad industrialization prospects.

[0006] On April 14, 2026, a search was conducted in the Chinese Patent Publication Database using "oat bran and Lactobacillus plantarum and Bacillus subtilis and solid-state fermentation" as the abstract keywords, with the option to allow synonym expansion. The search yielded patent CN120866144A. CN120866144A discloses a compound microbial agent for fermenting modified corn flour and its preparation method. This invention uses corn flour as the main ingredient and a small amount of oat bran as an auxiliary ingredient. It employs four strains of bacteria—Streptococcus thermophilus, Bacillus subtilis, Monascus purpureus, and Lactobacillus plantarum—through a two-stage liquid-solid fermentation process to obtain the modified corn flour product. Its core functional component is Monacolin K synthesized by Monascus purpureus, which exerts a cholesterol-lowering effect by inhibiting HMG-CoA reductase activity. The product is primarily targeted at the development of cholesterol-lowering health foods. In contrast, this patent uses pure oat bran as the sole fermentation substrate and selects only two strains of bacteria—Bacillus subtilis and Lactobacillus plantarum—for single-stage solid-state fermentation, offering significant advantages such as simplified strains, a streamlined process, and lower energy consumption. The core technology of this patent lies in the synergistic effect of enzyme and acid production by two bacteria to directionally loosen and enhance the structure of IDF fibers. This is a directional modification at the fiber structure level, rather than the enrichment and synthesis of new functional components. Therefore, it differs fundamentally from CN120866144A in terms of technical approach and mechanism of action. Furthermore, this patent systematically characterizes the differentiated fermentation characteristics of IDF-3, namely "low gas production, high acid production, and high butyrate," clarifying that it effectively avoids gastrointestinal discomfort while possessing unique prebiotic properties. This technical dimension is also completely absent from CN120866144A.

[0007] On April 14, 2026, using "oat bran and Lactobacillus plantarum and Bacillus subtilis and solid-state fermentation" as the abstract keywords and with the option to allow synonym expansion, a search was conducted on CNKI (China National Knowledge Infrastructure), but no relevant patents were found.

[0008] On April 14, 2026, a search was conducted on the website of the United States Patent and Trademark Office for the term "Oat bran WITH mixed bacteria WITH solid statefermentation" at https: / / ppubs.uspto.gov / pubwebapp / , but no relevant patent was found.

[0009] On April 14, 2026, a search was conducted on WIPO's website https: / / patentscope2.wipo.int / for the term "Oat bran and mixed bacteria and solid-statefermentation.", but no relevant patents were found. Summary of the Invention

[0010] The purpose of this invention is to provide a method for modifying insoluble dietary fiber (IDF) in oat bran through synergistic solid-state fermentation using Bacillus subtilis and Lactobacillus plantarum. This significantly improves the adsorption performance, antioxidant activity, and α-amylase inhibition capacity of oat bran IDF. Simultaneously, while maintaining the inherent prebiotic characteristics of slow fermentation and low gas production of oat bran IDF, it further guides the intestinal flora metabolism towards a "low gas production, high acid production" acid-producing pathway, significantly promoting the production of short-chain fatty acids in the colon and effectively avoiding gastrointestinal discomfort such as bloating and abdominal pain induced by rapid fermentation. The method described in this invention uses GRAS-grade food-safe bacterial strains for solid-state fermentation, offering significant advantages such as wide availability of strains, low cost, simple process, low energy consumption, easy parameter control, and suitability for industrial-scale production. To achieve the above objectives, this invention adopts the following technical solution: (I) A co-fermentation compound microbial agent specifically for modifying insoluble dietary fiber in oat bran: It is composed of Bacillus subtilis suspension and Lactobacillus plantarum suspension in a volume ratio of 1:2; wherein the viable cell concentration of Bacillus subtilis suspension is not less than 1×10⁻⁶. 8 The cfu / mL concentration of the *Lactobacillus plantarum* suspension should not be less than 1×10⁻⁶. 8 cfu / mL.

[0011] (II) A method for targeted modification of insoluble dietary fiber in oat bran based on synergistic solid-state fermentation by two microorganisms, characterized by comprising the following steps: After the dry oat bran is crushed and sieved, it is autoclaved at 121 ℃ for 15 minutes and then cooled to room temperature for later use. (2) Inoculate the above compound microbial agent into the oat bran matrix obtained in step (1) at a total inoculation amount of 10% (v / w) and a liquid-to-material ratio of 0.8:1 (mL / g), and mix evenly under aseptic conditions; (3) Place the material obtained in step (2) in a 37 ℃ constant temperature incubator and let it undergo solid-state fermentation for 3 days; (4) After fermentation, insoluble dietary fiber was separated by enzyme-alkali method. The filter residue was freeze-dried for 48 h to obtain modified oat bran insoluble dietary fiber (IDF-3).

[0012] A further technical solution of the present invention is that the inoculation amount of the compound microbial agent in step (2) is 10% (v / w) of the dry basis mass of oat bran, and the liquid-to-material ratio is 0.8:1 (mL / g); the fermentation temperature in step (3) is 37 ℃, and the fermentation time is 3 days; the modified product obtained under these conditions is labeled as IDF-3, and its comprehensive performance is significantly better than that of fermented for 1 day (IDF-1), fermented for 5 days (IDF-5) and unfermented control (IDF-0).

[0013] A further technical solution of the present invention is that the process parameters of the method are optimized by combining the AHP-CRITIC weighted method with Box-Behnken response surface design, and the optimal combination is determined by using IDF degradation rate, SDF growth rate and free polyphenol content as comprehensive evaluation indicators.

[0014] A further technical solution of the present invention is that the synergistic mechanism of the fermentation process is as follows: Bacillus subtilis secretes hydrolytic enzymes such as cellulase, hemicellulase, xylanase, and β-glucosidase during solid-state fermentation, which can directionally degrade the dense fibrous network structure of IDF, gradually transforming the original dense blocky morphology into a highly loose and porous honeycomb-like three-dimensional spatial structure; at the same time, Lactobacillus plantarum creates a weakly acidic microenvironment through acid production metabolism, inhibiting contamination by other bacteria and maintaining the stability of the fermentation system; the two strains complement each other nutritionally in terms of carbon source utilization and metabolites, thereby achieving synergistic regulation of IDF structure loosening and active ingredient release.

[0015] A further technical solution of the present invention is that the modified oat bran insoluble dietary fiber has the following physicochemical characteristics: (a) It has excellent small molecule adsorption performance, wherein the adsorption capacity for glucose is not less than 16 mg / g, the adsorption capacity for cholesterol is not less than 18 mg / g, and the adsorption capacity for nitrite under pH=2 conditions is not less than 880 μg / g. (b) It possesses significant in vitro antioxidant activity, with a scavenging rate of not less than 55% against ABTS free radicals; (c) It has an inhibitory effect on α-amylase, with an inhibition rate of not less than 24% when the IDF concentration is 20 mg / ml; (d) During in vitro fecal microbial fermentation, it exhibits extremely low gas production and gradual pH changes in the fermentation broth, and can simultaneously promote the production of total short-chain fatty acids, as well as acetic acid and butyric acid. Attached Figure Description

[0016] To further illustrate the present invention, the following description is provided in conjunction with the accompanying drawings: Figure 1 This is a comparison chart showing the adsorption capacity of oat bran insoluble dietary fiber obtained in the embodiments and comparative examples of the present invention for glucose, nitrite and cholesterol. Figure 2 This is a comparison chart of the ABTS free radical scavenging capabilities of oat bran insoluble dietary fiber obtained from the embodiments and comparative examples of the present invention; Figure 3 This is a comparison chart of the α-amylase inhibition rate of insoluble dietary fiber from oat bran obtained in the embodiments and comparative examples of the present invention; Figure 4 These are the gas production change curves of the embodiments and comparative examples of the present invention during the in vitro fecal microbial fermentation process; Figure 5 These are the pH change curves of the fermentation broth during the in vitro fecal microbial fermentation process in the embodiments and comparative examples of the present invention; Figure 6 This is a comparison chart of the total short-chain fatty acids and the amounts of acetic acid, propionic acid, and butyric acid produced during the in vitro fecal fermentation process of the embodiments and comparative examples of the present invention. Detailed Implementation

[0017] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0018] Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. All raw materials and instruments used are commercially available conventional products, including but not limited to those used in the embodiments of this application.

[0019] Examples 2, 5, and 6 are essentially comparative examples; Example 1 (IDF-3)

[0020] This embodiment provides a method for preparing a synergistic fermentation compound microbial agent for the targeted modification of insoluble dietary fiber in oat bran, as detailed below: Bacillus subtilis was inoculated into nutrient broth liquid medium and cultured at 37 ℃ and 180 rpm for 8 hours with shaking. A bacterial suspension was prepared by adding freeze-dried Lactobacillus plantarum powder to sterile water at a material-to-liquid ratio of 1:10, with a viable Lactobacillus plantarum concentration of not less than 1 × 10⁻⁶. 8 cfu / mL.

[0021] (2) Preparation of bacterial suspension: Centrifuge the activated Bacillus subtilis suspension at 6000 rpm for 10 minutes, discard the supernatant, wash twice with sterile physiological saline, and resuspend. Adjust the concentration of live Bacillus subtilis to not less than 1×10⁻⁶. 8 cfu / mL.

[0022] (3) Compound preparation: Under aseptic conditions, the two bacterial suspensions above are mixed evenly at a volume ratio of 1:2 to obtain the synergistic fermentation compound bacterial agent of the present invention.

[0023] (4) Take 10 g of oat bran, autoclave at 121 °C for 15 minutes, and cool to room temperature for later use.

[0024] (5) Inoculate the compound microbial agent obtained in Example 1 (IDF-3) into the oat bran matrix obtained in step (4) at a liquid-to-material ratio of 0.8:1 (mL / g). Mix thoroughly under aseptic conditions, and then seal the fermentation vessel with a breathable microporous sealing film to maintain gas exchange during solid-state fermentation and effectively block external contamination.

[0025] (6) Place the material obtained in step (5) in a 37 ℃ constant temperature incubator and let it stand for solid fermentation for 3 days.

[0026] (7) After fermentation, add distilled water at 55°C at a ratio of 1:10 and stir well.

[0027] (8) The pH of the material obtained in step (7) is adjusted to about 6.0 using 0.5M HCl solution, and 10% of Bacillus subtilis α-amylase is added. The Bacillus subtilis α-amylase activity is 4000U / g. The enzyme is hydrolyzed at 70℃ for 120min.

[0028] (9) Adjust the pH of the material obtained in step (8) to about 9.0 using 0.5M NaOH solution, add 1% by mass of alkaline protease with a protease activity of 200U / mg, and enzymatically hydrolyze at 40℃ for 120min.

[0029] (10) After centrifuging the material obtained in step (9), remove the supernatant. Wash the residue with 80% ethanol at 60-70℃ 3-4 times, then wash with distilled water at 60-70℃ 3-4 times, and freeze dry to obtain modified oat bran insoluble dietary fiber (labeled as IDF-3).

[0030] Example 2: This example is a comparative example, referred to as Comparative Example 1: Unfermented oat bran insoluble dietary fiber (labeled as IDF-0), its preparation method refers to the inoculation and fermentation treatment in steps (5)-(6) of Example 1 (IDF-3).

[0031] Example 3: (IDF-1); A method for directional modification of oat bran insoluble dietary fiber based on dual-strain synergistic solid-state fermentation is provided (labeled as IDF-1). The preparation method is the same as in Example 1 (IDF-3), except that the fermentation time in step (6) is 1 day, and the other processing steps are the same. The modified oat bran insoluble dietary fiber obtained is labeled as IDF-1.

[0032] Example 4: (IDF-5); A method for directional modification of oat bran insoluble dietary fiber based on dual-strain synergistic solid-state fermentation is provided (labeled as IDF-5). The preparation method is the same as in Example 1 (IDF-3), except that the fermentation time in step (6) is 5 days, and the other processing steps are the same. The modified oat bran insoluble dietary fiber is labeled as IDF-5.

[0033] Example 5: (FOS); This example is a comparative example, specifically Comparative Example 2, serving as a positive control during the fermentation process of intestinal flora. The sample is commercially available fructooligosaccharide (FOS).

[0034] Example 6: This example is a comparative example, specifically Comparative Example 3, serving as a blank control during the fermentation process of intestinal flora, i.e., without the addition of any dietary fiber or fermentation products.

[0035] Determination of glucose adsorption capacity: Accurately weigh 0.1 g of oat bran insoluble dietary fiber (IDF) sample and place it in a stoppered centrifuge tube. Add 10 mL of phosphate buffer to fully disperse the sample, then add 10 mL of glucose solution of a certain concentration. Stir magnetically in a 37 ℃ constant temperature water bath for 6 h to allow the sample to fully react with the glucose solution. Subsequently, centrifuge at 4500 r / min for 20 min, and take the supernatant to determine the glucose concentration after the reaction using the 3,5-dinitrosalicylic acid (DNS) method. The glucose adsorption capacity of the sample is obtained by multiplying the difference in glucose concentration before and after the reaction by the solution volume and then dividing by the sample mass. The result is expressed in mg / g.

[0036] Determination of cholesterol adsorption capacity: Accurately weigh 50 mg of oat bran IDF sample and place it in a stoppered centrifuge tube. Add 20 mL of a cholesterol solution of a certain concentration and mix thoroughly to ensure uniform dispersion of the sample. Shake at 120 r / min for 2 h in a constant temperature shaker at 37 ℃ to ensure sufficient contact and reaction between the sample and the cholesterol solution. Then centrifuge at 5000 r / min for 15 min, and use the supernatant to determine the cholesterol concentration after the reaction at a wavelength of 550 nm using the phthalaldehyde method. The cholesterol adsorption capacity of the sample is obtained by multiplying the difference in cholesterol concentration before and after the reaction by the solution volume and then dividing by the sample mass. The result is expressed in mg / g.

[0037] Determination of nitrite adsorption capacity: Accurately weigh 50 mg of oat bran IDF sample and place it in a stoppered centrifuge tube. Adjust the pH to approximately 2, add 10 mL of sodium nitrite solution of a certain concentration, and magnetically stir in a 37 ℃ constant temperature water bath for 2 h to ensure sufficient contact and reaction between the sample and the sodium nitrite solution. Subsequently, centrifuge at 4500 r / min for 20 min, and use the supernatant to determine the nitrite concentration after the reaction using the naphthylethylenediamine hydrochloride method. The adsorption capacity of the sample for nitrite is obtained by multiplying the difference in nitrite concentration before and after the reaction by the solution volume and then dividing by the sample mass, expressed in μg / g.

[0038] Solid-state fermentation significantly enhanced the adsorption capacity of oat bran insoluble dietary fiber (IDF) for glucose, cholesterol, and nitrite, reaching its peak in Example 1 (IDF-3). Specifically, the glucose adsorption capacity of Comparative Example 1 (IDF-0) was only 5.2 mg / g, while that of IDF-1 increased to 8.8 mg / g, and the glucose adsorption capacity of Example 1 (IDF-3) reached a peak of 16.0 mg / g. Meanwhile, the cholesterol adsorption capacity of Comparative Example 1 (IDF-0) was 10.46 mg / g, that of IDF-1 increased to 12.98 mg / g, and that of IDF-3 in Example 1 (IDF-3) further increased to 18.72 mg / g, while that of IDF-5 decreased to 16.35 mg / g. This indicates that moderate dual-strain synergistic fermentation can significantly enhance the adsorption performance of IDF; however, excessively long fermentation times can lead to excessive degradation of some active structures, resulting in a decrease in adsorption capacity. Furthermore, under simulated gastric fluid conditions at pH = 2, Example 1 (IDF-3) achieved a maximum adsorption capacity of 888.43 μg / g for nitrite, significantly higher than the comparative group. This significant improvement in adsorption performance can be attributed to the dual effects of structural modification and chemical modification during the synergistic fermentation by two microorganisms. The cellulase and hemicellulase systems secreted by Bacillus subtilis directionally degraded the dense fibrous network structure of IDF, gradually transforming the original compact blocky morphology into a highly porous, honeycomb-like three-dimensional structure. This significantly increased the specific surface area, exposing more hydrophilic functional groups such as hydroxyl and carboxyl groups, thereby enhancing the adsorption of glucose molecules through physical retention and hydrogen bonding. On the other hand, the development of the pore structure during fermentation improved the accessibility of lipophilic regions, significantly enhancing the hydrophobic interaction and physical encapsulation between IDF and cholesterol molecules. Simultaneously, the free polyphenols released during fermentation could further promote adsorption and fixation by forming hydrogen-bonded complexes with cholesterol. Furthermore, the weakly acidic microenvironment created by *Lactobacillus plantarum* through acid-producing metabolism further promotes the loosening of the cell wall matrix, increasing the protonation degree of polar groups such as carboxyl and hydroxyl groups on the IDF surface under acidic conditions. This allows for the effective binding and fixation of nitrite ions through electrostatic interactions and ion exchange mechanisms. From a physiological perspective, dietary fiber's adsorption of glucose can effectively slow down the diffusion and transmembrane transport rate of glucose in the gastrointestinal tract, reducing postprandial blood glucose peaks and helping to improve insulin sensitivity. Adsorption of cholesterol can reduce cholesterol absorption in the small intestine and promote its excretion in feces. Simultaneously, by interfering with the enterohepatic circulation of bile acids and accelerating the liver's metabolic conversion of endogenous cholesterol, it plays an auxiliary role in lowering blood lipids. The efficient adsorption of nitrite can effectively reduce the concentration of free nitrite in the acidic environment of the stomach, inhibiting its reaction with secondary amines to form highly carcinogenic N-nitrosamines, which is of positive significance in preventing the occurrence of gastrointestinal tumors.Therefore, Example 1 (IDF-3) showed significantly better adsorption performance than other control examples for three types of small molecules: glucose, cholesterol, and nitrite. This indicates that IDF-3 has multiple potential physiological functions in assisting in lowering blood sugar, assisting in lowering blood lipids, and preventing the formation of nitrosamine carcinogens, laying a solid experimental foundation for its high-value application as a functional food ingredient.

[0039] In Example 2 (Comparative Example 1), the ABTS radical scavenging rate of IDF-0 was 21.60%. The ABTS radical scavenging rate of IDF-1 significantly increased to 30.60%, while the ABTS radical scavenging rate of Example 1 (IDF-3) reached a peak of 56.17%, and the ABTS radical scavenging rate of IDF-5 was 32.98%. The trend of IDF antioxidant activity showed a peak on day 3 of fermentation followed by a certain degree of decline, which may be related to the further degradation of the IDF structure in the later stages of fermentation. In the early stages of fermentation, microbial enzymatic hydrolysis gradually exposed the bound polyphenols on and inside the IDF surface, increasing the content of phenolic substances carried by the IDF and thus increasing antioxidant activity. In the later stages of fermentation, with the destruction of the fiber structure, some exposed polyphenols may detach from the IDF and transfer to a free state, leading to a reduction in the phenolic substances bound to the IDF itself and a decrease in antioxidant activity.

[0040] The method for determining the inhibition rate of α-amylase activity was as follows: Fermented oat bran samples of different masses (10, 20, 30, 40, and 50 mg) were accurately weighed into 50 mL centrifuge tubes. 2 mL of distilled water and 0.5 mL of enzyme solution were added, and the mixture was preheated at 37 ℃ for 5 min. Then, 2 mL of cooked starch solution (20 mg / mL) was added to begin digestion, and the reaction was carried out at 37 ℃ for 15 min. 0.3 mL of the digest was added to 1.2 mL of anhydrous ethanol to inactivate the enzyme, and the mixture was centrifuged at 10000 r / min for 10 min. 0.1 mL of the supernatant was added to 3 mL of PAHBAH reagent, and the mixture was heated in a boiling water bath for 10 min for color development. After cooling, the absorbance at 410 nm was measured.

[0041] α-Amylase is a key enzyme in starch digestion, and its activity directly affects postprandial blood glucose levels. Inhibiting α-amylase activity through natural food components is considered an effective strategy for regulating postprandial blood glucose elevation. Therefore, evaluating the α-amylase inhibitory ability of fermented oat bran is of great significance for revealing its in vitro hypoglycemic function. Oat bran samples at different fermentation stages all showed some inhibitory effect on α-amylase, and the inhibition rate increased with increasing addition amount.

[0042] The inhibition rate of IDF-0 in Comparative Example 1 was the lowest among all addition levels, reaching only 1.76% at 5 mg / mL and only 17.25% even at 20 mg / mL. Example 1 (IDF-3) exhibited the highest inhibitory activity, reaching 12% at 5 mg / mL, steadily increasing with increasing addition level, peaking at 24.24% at 20 mg / mL, and then stabilizing. IDF-1 and IDF-5 showed similar inhibition rates against α-amylase. At 5 mg / mL, the inhibition rates of IDF-1 and IDF-5 were 8.84% and 8.15%, respectively, while at 20 mg / mL, the inhibition rates were 20.38% and 22.73%, respectively. IDF typically inhibits α-amylase activity through physical trapping or binding to enzymes via intermolecular forces. Further evidence confirms that moderate fermentation can effectively improve the structural characteristics of IDF and enhance its interaction with α-amylase. The inhibition rates of IDF-5 and fermented IDF-0 were comparable, indicating that excessive fermentation negatively impacts the inhibitory effect of α-amylase. This may be because the degradation of dietary fiber or polyphenols weakens the inhibitory effect. The IDF-3 group showed the best inhibition rate against α-amylase, demonstrating that the modified oat bran IDF method described in this paper can effectively improve its inhibitory activity against α-amylase, suggesting its potential hypoglycemic activity.

[0043] In vitro fermentation experiment: A carbonate-phosphate buffer solution was prepared, and cysteine ​​hydrochloride was added to it to a final concentration of 0.25 g / L to obtain a reducing buffer solution. All necessary equipment, including but not limited to beakers, gauze, pipette tips, anaerobic culture flasks, and the prepared buffer solution, were placed in an autoclave and sterilized at 121°C for 20 min. After sterilization, the equipment was transferred to an oven for drying and prepared for later use. Fecal samples were collected from three healthy volunteers aged 20–30 years. These volunteers had a reasonable diet, no history of gastrointestinal diseases, and had not taken antibiotics or consumed probiotic-containing yogurt products in the month prior to sample collection. The collected fecal samples were placed in sterile collection tubes and quickly transferred to an anaerobic chamber for overnight incubation to eliminate oxygen interference. Under anaerobic conditions, pretreated carbonate-phosphate buffer was mixed with fecal samples from the three volunteers at a ratio of 5:1 (v:w, mL:g). The resulting mixture was filtered through sterile gauze, and the filtrate was collected as the fecal suspension used in subsequent fermentation experiments. 50 mg of the example sample and the comparative sample were weighed and placed in anaerobic culture flasks. 1 mL of the prepared fecal suspension and 4 mL of carbonate-phosphate buffer were added to each anaerobic culture flask sequentially, mixed thoroughly, and immediately sealed. The airtightness of the seal was confirmed to maintain the anaerobic environment. The sealed anaerobic culture flasks were placed in a 37°C constant temperature water bath for fermentation culture, with four time gradients set at 4 h, 8 h, 12 h, and 24 h. At each preset time point, the corresponding anaerobic culture flask was removed, and the gas produced by the fermentation reaction was collected using a graduated syringe. The gas production data at each time point was recorded based on the syringe piston displacement reading.

[0044] Gas production is one of the indicators reflecting the fermentation rate of dietary fiber, because gut microbiota produce CO2 while breaking down and utilizing dietary fiber. 2 H 2 Gases. From Figure 4As can be seen, the gas production rate of Comparative Example 2 (FOS) group was the fastest, with a rapid increase in gas production within 0-8 hours, reaching 14.33 mL at 8 hours, and gradually stabilizing from 8 to 24 hours. This indicates that FOS has rapid fermentation characteristics, and the nutrient substrate is almost completely utilized after 8 hours. This leads to rapid fermentation in the proximal colon, preventing it from reaching the distal colon, which can easily cause adverse symptoms related to intestinal gas production such as bloating and abdominal pain. During the entire 24-hour in vitro fermentation, the gas production values ​​of oat bran insoluble dietary fiber obtained in Example 1 (IDF-3) and the three examples (IDF-0, IDF-1, and IDF-5) were all between 3-4 ml, far lower than that of Comparative Example 2 (FOS). This proves that dietary fiber can be metabolized by intestinal flora during fermentation, indicating that oat bran insoluble dietary fiber has slow fermentation characteristics, produces less gas, and is less likely to cause gastrointestinal discomfort such as bloating and abdominal pain. At the same time, it can also transport nutrients to the distal colon, maintaining the homeostasis of the distal colonic intestinal microenvironment.

[0045] The sealed anaerobic culture flasks were placed in a 37°C constant temperature water bath for fermentation culture, with four time gradients set at 4 h, 8 h, 12 h, and 24 h. At each preset time point, the corresponding anaerobic culture flask was removed, and the gas generated by the fermentation reaction was collected using a graduated syringe. The pH value of the fermentation solution was then measured using a pH meter, and the data was recorded.

[0046] The pH value of the fermentation broth is an important indicator that directly reflects the microbial metabolic activity and the accumulation of acidic metabolites during in vitro intestinal fermentation. For example... Figure 4 As shown, during the initial fermentation stage, the pH values ​​of all groups were within the slightly alkaline range of 7.7 to 7.8, indicating a consistent initial fermentation environment. The control group (blank) exhibited minimal pH fluctuations throughout the fermentation cycle, consistently remaining between 7.55 and 7.78, demonstrating that in the absence of an external carbon source, the basal metabolic activity of the gut microbiota was insufficient to significantly alter the acid-base environment of the fermentation system. The pH value of the control group (FOS) showed a typical trend of a sharp initial decrease followed by a slow recovery, which is highly consistent with the characteristic of FOS as an easily fermentable short-chain carbohydrate being rapidly degraded and utilized by the gut microbiota.

[0047] The pH of the fermentation broth in Comparative Example 1 (IDF-0) decreased to 7.41 after 24 hours of in vitro fermentation, while the pH changes in Examples (IDF-1), (IDF-5), and (IDF-3) were smaller. This indicates that the fermentation characteristics of IDF changed after solid-state fermentation. Example 1 (IDF-3) had a higher pH, with the fermentation broth pH ranging from 7.56 to 7.77 after 24 hours of fermentation. This demonstrates that the oat bran insoluble dietary fiber prepared by solid-state fermentation for 3 days can effectively reduce the fermentation rate of the microbial community, thereby slowing down the production of short-chain fatty acids.

[0048] The content of short-chain fatty acids in fermentation broth was determined by the following method:

[0049] First, the supernatant of the fermentation broth was centrifuged at 13,000 rpm for 10 minutes. 800 µL of the supernatant was then mixed with 200 µL of internal standard solution. Subsequently, the sample was drawn using a syringe and filtered through a 0.22 µm aqueous filter membrane into a sample vial. The types and contents of short-chain fatty acids in the fermentation broth were analyzed using a gas chromatograph equipped with a polar ZB-FFAP capillary column and a combustion ionization detector (FID). The initial temperature of the column oven was set to 80 °C, and the temperatures of the sample inlet and pre-detector were set to 230 °C. Nitrogen gas was supplied at a fixed flow rate of 1 mL / min. The types of short-chain fatty acids were determined based on fatty acid standards. The correction factor was determined by the ratio of the peak areas of tetramethylvaleric acid (internal standard) to the target fatty acid in the fatty acid standards, thus quantifying the short-chain fatty acids in the fermentation broth.

[0050] Short-chain fatty acids (SCFAs) are the main end products of dietary fiber fermentation by anaerobic microorganisms in the gut. These primarily include acetic acid, propionic acid, and butyric acid, which play an irreplaceable role in maintaining intestinal homeostasis and regulating systemic metabolism. In Example 6 (blank group), the SCFA content after 24 hours of in vitro fermentation was 6.82 mM. In Comparative Example 1 (IDF-0), the total SCFA content after 24 hours of in vitro fermentation was 40.66 mM, while in Example 1 (IDF-3), the total SCFA content reached 44.31 mM. The butyric acid content after 24 hours of in vitro fermentation in IDF-0 was 4.07 mM, while in Example 1 (IDF-3), the butyric acid content was significantly higher at 5.41 mM. This indicates that although solid-state fermentation significantly improved the ability of IDF-3 to produce SCFAs in vitro, it did not cause a synchronous increase in the total gas production of oat bran. This suggests that after solid-state fermentation, oat bran IDF tends to utilize a low-gas-producing acid-producing metabolic pathway during the fermentation process by intestinal flora. Simultaneously, solid-state fermentation significantly and positively regulates the butyrate-producing capacity of IDF-3. This may be related to the fact that solid-state fermented oat bran (IDF) promotes the accumulation of butyrate-producing bacteria. Butyrate is the preferred energy source for colonic epithelial cells; its oxidation can meet the energy needs of colonic cells and maintain the anaerobic microenvironment of the intestinal lumen through oxygen consumption. Butyrate can also promote the expression of tight junction proteins to strengthen intestinal barrier integrity, and has anti-inflammatory and immunomodulatory functions, providing protection against inflammatory bowel disease and colorectal cancer. This demonstrates that Example 1 (IDF-3) has potential application value in maintaining intestinal barrier function, regulating intestinal immune homeostasis, and preventing related intestinal diseases.

[0051] Example 7

[0052] This embodiment describes a co-fermentation compound microbial agent specifically for modifying insoluble dietary fiber in oat bran. The compound microbial agent is characterized by being a mixture of Bacillus subtilis suspension and Lactobacillus plantarum suspension at a volume ratio of 1:2, wherein the viable bacterial concentration of the Bacillus subtilis suspension is not less than 1×10⁻⁶. 8 The cfu / mL concentration of the *Lactobacillus plantarum* suspension should not be less than 1×10⁻⁶. 8 CFU / mL. Compared to existing technologies where the modification effect of a single bacterial species is limited and the synergistic effect is lost due to unreasonable ratios of multiple bacterial species, this patent innovatively and non-obviously combines Bacillus subtilis and Lactobacillus plantarum in a 1:2 volume ratio, strictly controlling the concentration of viable bacteria in both suspensions to be no less than 1×10⁻⁶. 8 CFU / mL, Bacillus subtilis can secrete hydrolytic enzymes to loosen the dense structure of insoluble dietary fiber in oat bran, while Lactobacillus plantarum can produce acid to maintain the stability of the fermentation system. The two complement each other metabolically, solving the fundamental problem of dense structure and poor functional activity of insoluble dietary fiber in natural oat bran from the perspective of strain compatibility.

[0053] Example 8

[0054] The embodiment described here, as in Example 7, is characterized in that the volume ratio of Bacillus subtilis to Lactobacillus plantarum is 1:2. Compared to the shortcomings of existing technologies where the ratio of compound microbial agents is arbitrary and cannot achieve optimal synergistic effects, this patent innovatively and non-obviously fixes the volume ratio of Bacillus subtilis to Lactobacillus plantarum at 1:2. This ratio allows for precise matching of the enzyme and acid production metabolic rates of the two strains, ensuring that Bacillus subtilis fully degrades the fiber network while avoiding excessive acid production by Lactobacillus plantarum that inhibits enzyme activity, thus maximizing the synergistic modification effect of the two bacteria and solving the problem of low modification efficiency caused by improper ratios.

[0055] Example 9

[0056] Example of this embodiment: A method for directional modification of oat bran insoluble dietary fiber based on dual-strain synergistic solid-state fermentation, characterized by the following steps: (1) Matrix pretreatment: Oat bran is crushed, sieved, sterilized at 121 ℃ for 15 minutes, cooled to room temperature, and set aside; (2) Based on the dry weight of oat bran, the compound microbial agent described in Example 7 is inoculated into the oat bran matrix obtained in step (1) at an inoculation amount of 10% (v / w) and a liquid-to-material ratio of 0.8:1 (mL / g). The mixture is thoroughly stirred and mixed evenly under aseptic conditions. Then, the fermentation vessel is sealed with a breathable microporous sealing film to maintain gas exchange during solid-state fermentation and effectively block external contamination. (3) The material obtained in step (2) is placed in a 37 ℃ constant temperature incubator for static solid-state fermentation for 3 days; (4) After fermentation, insoluble dietary fiber is obtained by enzymatic separation. The obtained filter residue is freeze-dried for 48~60 h to obtain oat bran insoluble dietary fiber modified by solid-state fermentation with compound microbial agent. Compared to the shortcomings of existing technologies such as high energy consumption in physical modification, reagent residues in chemical modification, and high cost and single target of enzymatic modification, this patent innovatively applies dual-strain synergistic solid-state fermentation to the modification of insoluble dietary fiber from oat bran. By eliminating interference from miscellaneous bacteria through pretreatment sterilization, precisely controlling the inoculum amount, liquid-to-material ratio, fermentation temperature and time, and ensuring a stable fermentation environment with a breathable sealing film, the product is then purified by enzymatic separation and freeze-drying. The entire process is green, mild, and low-cost, simultaneously achieving fiber structure loosening and activity enhancement, thus solving various drawbacks of traditional modification methods.

[0057] Example 10

[0058] The embodiment described here is as follows: The modification method described in Example 9 is characterized in that the inoculation amount of the compound microbial agent in step (2) is 10% (v / w) of the dry basis of oat bran. Compared with the defects of the prior art, where the inoculation amount is too high, increasing costs, or too low, resulting in incomplete modification, this patent innovatively sets the inoculation amount of the compound microbial agent to 10% (v / w) of the dry basis of oat bran. This inoculation amount allows the strain to be fully distributed in the oat bran matrix, ensuring uniform fermentation. It avoids both incomplete fiber modification caused by insufficient inoculation amount and waste of raw materials and accumulation of metabolites caused by excessive inoculation amount, thus achieving the optimal balance between modification effect and cost.

[0059] Example 11

[0060] The embodiment described here, as in Example 9, is characterized in that the process parameters of the method are optimized using the AHP-CRITIC weighted average method combined with Box-Behnken response surface methodology. The optimal process conditions are: a volume ratio of Bacillus subtilis to Lactobacillus plantarum in the compound microbial agent of 1:2, a liquid-to-solid ratio of 0.8:1, and a fermentation time of 3 days. Compared to the shortcomings of existing technologies where modification process parameters are set based on experience and lack scientific optimization, leading to unstable product performance, this patent innovatively combines the AHP-CRITIC weighted average method with Box-Behnken response surface methodology for process optimization. This accurately determines the optimal parameter combination, solving the problems of strong subjectivity and inability to consider multiple indicators in traditional process optimization methods, making the modification process controllable and ensuring stable and compliant product performance.

[0061] Example 12

[0062] The embodiment described here is as follows: The modification method described in Example 9 is characterized in that the specific steps of the enzyme-alkali separation in step (4) are as follows: Modified oat bran is added to α-amylase for enzymatic hydrolysis at a material-to-liquid ratio of 1:10, and then treated with protease to remove starch and protein. The residue is then washed 3-4 times with 80% ethanol at 60-70℃, and then washed 3-4 times with distilled water at 60-70℃. The residue is then freeze-dried to obtain modified oat bran insoluble dietary fiber. Compared with the defects of low purity and easy destruction of functional structure in the dietary fiber separation method of the prior art, this patent innovatively applies the enzyme-alkali method to the separation of modified oat bran insoluble dietary fiber. Starch is removed by α-amylase and protein is removed by protease. Then, the product is purified by washing with gradient ethanol and distilled water. The whole process is mild and without damage from strong chemical reagents, which solves the problem of residual impurities and destruction of functional structure during the separation process, resulting in a decline in product performance.

[0063] Example 13

[0064] Example of this embodiment: A modified oat bran insoluble dietary fiber prepared by the method described in Examples 9-12, characterized in that the insoluble dietary fiber in the oat bran fermented by the compound microbial agent for 3 days has the following physicochemical characteristics: (a) adsorption capacity for glucose is not less than 16 mg / g, adsorption capacity for cholesterol is not less than 18 mg / g, and adsorption capacity for nitrite is not less than 880 μg / g under pH=2 conditions; (b) scavenging capacity for ABTS free radicals is not less than 55%; (c) it has inhibitory activity against α-amylase, with an inhibition rate of up to 24%; (d) it has extremely low gas production during in vitro fecal fermentation, and the pH change of the fermentation broth is gradual, while promoting the production of total short-chain fatty acids, acetic acid and butyric acid. Compared to the shortcomings of existing natural oat bran insoluble dietary fiber, such as weak adsorption capacity, low antioxidant activity, poor α-amylase inhibition, and high gas production during intestinal fermentation, this patent innovatively and non-obviously endows the product with multiple superior physicochemical properties through dual-strain synergistic solid-state fermentation. The loose and porous structure enhances the adsorption capacity of small molecules, releases bound polyphenols to enhance antioxidant properties, and structural changes strengthen the α-amylase inhibition effect. At the same time, it achieves fermentation characteristics of low gas production and high acid production, comprehensively solving the functional defects of natural fiber.

[0065] Example 14

[0066] The embodiments described herein: One use, characterized in that the use is any one of the following: ① Use of the modified oat bran insoluble dietary fiber of Example 13 in the preparation of foods, health products, or drugs with α-amylase inhibitory activity and auxiliary hypoglycemic function; ② Use of the modified oat bran insoluble dietary fiber of Example 13 in the preparation of foods, health products, or drugs with glucose adsorption, cholesterol adsorption, and nitrite adsorption functions; ③ Use of the modified oat bran insoluble dietary fiber of Example 13 in the preparation of intestinal flora regulators or prebiotic ingredients; ④ Use of the modified oat bran insoluble dietary fiber of Example 13 as a substance with slow fermentation, low gas production, and avoidance of gastrointestinal discomfort symptoms such as bloating and abdominal pain; ⑤ Use of the modified oat bran insoluble dietary fiber of Example 13 in the preparation of a substance that promotes the production of total short-chain fatty acids, acetic acid, and butyric acid in the colon; ⑥ Use of the modified oat bran insoluble dietary fiber of Example 13 in the preparation of foods and health products for the prevention or improvement of metabolic syndromes such as obesity, diabetes, and hyperlipidemia. Compared to the shortcomings of existing dietary fiber, such as its single function, limited application scenarios, and tendency to cause gastrointestinal discomfort, this patent innovatively expands the application of modified oat bran insoluble dietary fiber to multiple fields in a non-obvious way. Relying on its functions of lowering blood sugar, regulating blood lipids, adsorbing harmful substances, regulating intestinal flora, reducing gas production, and promoting the production of short-chain fatty acids, it solves the problems of narrow application range and poor tolerance of traditional fiber, and realizes the high-value and multi-functional application of grain by-products.

[0067] Overall concept: The essential difference between this invention and the technical path of CN120866144A

[0068] This invention (a method for modifying oat bran IDF based on dual-strain fermentation) uses pure oat bran as the sole substrate and employs a single-stage solid-state fermentation using only Bacillus subtilis and Lactobacillus plantarum. The core of this invention is to perform targeted structural decomposition and functional enhancement of oat bran insoluble dietary fiber (IDF), focusing on improving adsorption, antioxidant, and α-amylase inhibitory activities, while simultaneously achieving the prebiotic characteristics of "low gas production, high acid production, and high butyrate" in intestinal fermentation. In contrast, CN120866144A uses corn flour as the main ingredient and oat bran as the auxiliary ingredient, employing a two-stage fermentation with four strains of bacteria. The core of this invention is the synthesis of Monacolin K to achieve cholesterol reduction. The two inventions are completely different in terms of fermentation substrate, strain system, process mode, modification target, and mechanism of action, and their technical approaches have no overlap or continuity.

[0069] CN120866144A: Main technical content, defects, and targeted solutions of the present invention; (I) Main technical contents of CN120866144A This patent discloses a compound microbial agent, method, and application for fermentation-modified corn flour. It adopts a two-stage fermentation process of liquid and solid: the liquid fermentation uses Streptococcus thermophilus and Bacillus subtilis, and the solid fermentation uses Monascus purpureus and Lactobacillus plantarum. After the corn flour is microwave-expanded, it is mixed with oat bran. The supernatant of the liquid fermentation is centrifuged and then sprayed back onto the residue for solid fermentation. Finally, modified corn flour rich in Monacolin K and soluble dietary fiber is obtained and used in cholesterol-lowering health foods.

[0070] (ii) Deficiencies compared to existing technologies

[0071] Substrate and process redundancy: Corn flour is used as the main material and oat bran is only an auxiliary material. No targeted modification of pure oat bran IDF is carried out, which makes it impossible to achieve the specific high-value utilization of oat bran by-products. The liquid-solid two-stage fermentation requires centrifugation and supernatant respraying, which is complicated, consumes a lot of water and energy.

[0072] The microbial system is complex: it uses four strains of bacteria, namely Streptococcus thermophilus, Bacillus subtilis, Monascus purpureus and Lactobacillus plantarum, and adapts to different metabolic conditions of different strains in stages. The fermentation parameters are difficult to control and the industrial stability is low.

[0073] The modification targets are singular: focusing only on Monacolin K synthesis to achieve cholesterol reduction, without improving the adsorption, antioxidant, and α-amylase inhibition properties of oat bran IDF, and without paying attention to prebiotic properties such as intestinal fermentation gas production and short-chain fatty acid generation.

[0074] The fermentation mode has poor adaptability: liquid fermentation requires high temperature and high humidity, while solid fermentation requires room temperature and low humidity. The switching between the two stages of the environment can easily lead to enzyme activity loss and increased risk of contamination by miscellaneous bacteria, and the fermentation cycle is long (total time 42-72 hours).

[0075] (iii) This patent solves the above-mentioned technical problems in a non-obvious and inventive manner.

[0076] This patent innovatively and non-obviously uses pure oat bran as the sole fermentation substrate, eliminating corn flour as an auxiliary material, and focuses on modifying oat bran IDF to achieve precise and high-value utilization of grain by-products. At the same time, it simplifies the process to a single-stage solid-state fermentation, eliminating centrifugation and supernatant respraying processes, significantly reducing energy and water consumption, and making the process simpler and easier to industrialize.

[0077] This patent innovatively simplifies the bacterial strain to a combination of Bacillus subtilis and Lactobacillus plantarum, with an optimized 1:2 volume ratio. It eliminates the need for staged adaptation of metabolic conditions and achieves synergistic effects through static fermentation at a constant temperature of 37°C. Parameters are easily controlled and fermentation stability is significantly improved.

[0078] This patent innovatively breaks through the single cholesterol-lowering target in a non-obvious way. Through the synergistic enzymatic hydrolysis and acid production of two bacteria, it simultaneously enhances the glucose / cholesterol / nitrite adsorption capacity, antioxidant activity and α-amylase inhibition rate of IDF. At the same time, it endows the product with the intestinal prebiotic characteristics of "low gas production, high acid production and high butyrate", solving the pain points of bloating and discomfort caused by traditional high-fiber diets.

[0079] This patent innovatively adopts a single-stage constant-temperature solid-state fermentation mode, which avoids the risk of enzyme activity loss and contamination by miscellaneous bacteria during the two-stage environmental switching. The fermentation cycle is shortened to 3 days, and the fermentation conditions are mild, which completely solves the problems of long cycle and poor adaptability of existing technologies.

[0080] Figure 1 visually illustrates the effect of solid-state fermentation time on the adsorption capacity of oat bran IDF for glucose, cholesterol, and nitrite. All three curves peaked at 3 days of fermentation (IDF-3), with limited improvement at 1 day and a slight decline at 5 days. This result strongly demonstrates that synergistic solid-state fermentation with dual microorganisms can significantly improve IDF adsorption performance, with 3 days being the optimal fermentation time. This precisely validates the core points of the patent: "modified IDF adsorption of glucose ≥16mg / g, cholesterol adsorption ≥18mg / g, and nitrite adsorption ≥880μg / g at pH=2." It also verifies the modification mechanism of "Bacillus subtilis loosening the fiber structure and Lactobacillus plantarum optimizing the microenvironment, synergistically enhancing adsorption."

[0081] Figure 2 shows that the ABTS free radical scavenging rate first increases and then decreases with fermentation time. The antioxidant activity of IDF-3 is significantly higher than that of IDF-0, IDF-1, and IDF-5, reaching the patent requirement of ≥55%. This result strongly demonstrates that moderate dual-strain fermentation can release bound polyphenols, significantly enhancing the antioxidant capacity of IDF. Excessive fermentation leads to polyphenol loss and decreased activity, directly validating the technical effect of "modified IDF possessing significant in vitro antioxidant activity" in the patent, and providing data support for its antioxidant function application.

[0082] Figure 3 shows that IDF-3 exhibited the highest α-amylase inhibition rate across all addition levels, reaching over 24% at 20 mg / mL, significantly superior to unfermented samples and samples with other fermentation durations. This result strongly demonstrates that 3 days of dual-strain fermentation maximizes the inhibitory effect of IDF on key starch digestion enzymes, precisely validating the core argument in the patent that "modified IDF has strong inhibitory activity against α-amylase and possesses the potential to assist in lowering blood sugar," thus clarifying its target and efficacy advantages in blood sugar reduction.

[0083] Figure 4 shows that the gas production of IDF-0 / 1 / 3 / 5 is significantly lower than that of the positive control FOS, with only 3-4 mL of gas produced in 24 hours, and the gas production rate is slow. This result strongly proves that modified oat bran IDF is a slowly fermentable dietary fiber with very little gas production, directly demonstrating the core advantages of the patent: "low gas production and avoidance of gastrointestinal discomfort such as bloating and borborygmus." Compared with the rapid gas production characteristics of FOS, it highlights its high gastrointestinal tolerance.

[0084] Figure 5 shows that the pH of the IDF-3 fermentation broth decreased the least, remaining near neutral for 24 hours, which is much gentler than the rapid acid-base fluctuations of FOS. This result strongly demonstrates that the modified IDF fermentation produces acid at a moderate rate, without causing sudden changes in intestinal pH, thus supporting the patent's claim of "slow acid production and maintenance of intestinal acid-base homeostasis," and providing direct evidence for its adaptation to the intestinal microenvironment.

[0085] Figure 6 shows that the total short-chain fatty acid, acetic acid, and butyric acid production of IDF-3 were all higher than those of other IDF samples, with a particularly significant increase in butyric acid content. This result strongly demonstrates that modified IDF achieves targeted fermentation with "low gas production, high acid production, and high butyric acid," accurately demonstrating the core functions of the patent in "promoting the production of beneficial short-chain fatty acids, maintaining the intestinal mucosal barrier, and regulating metabolism," and clarifying its prebiotic value.

[0086] Figures 1-6 collectively reveal a non-linear performance pattern of "increase followed by decrease" in the performance of dual-strain fermentation at 1, 3, and 5 days, indicating that longer fermentation times do not necessarily lead to better results. Furthermore, IDF-3 achieves optimal performance simultaneously across five dimensions: adsorption, anti-oxidation, enzyme inhibition, low gas production, and high acid production, rather than a single performance improvement. Compared to commercially available FOS, IDF-3 possesses the dual advantages of "low gas production" and "high butyrate," overcoming the inherent defects of traditional prebiotics such as "rapid gas production and gastrointestinal discomfort." This precise, targeted modification, synergistic optimization of multiple performance aspects, and differentiated superiority over commercially available products are effects that cannot be reasonably expected by those skilled in the art based on existing solid-state fermentation and single-strain fermentation technologies, fully demonstrating the non-obviousness and outstanding technical contribution of this invention.

[0087] This invention relates to the field of functional food processing and high-value utilization of grain by-products, and discloses a method for modifying insoluble dietary fiber from oat bran based on synergistic solid-state fermentation by two bacteria. The modification method of this invention uses Bacillus subtilis (… Bacillus subtilis ) and Lactobacillus plantarum ( Lactobacillus plantarum A compound microbial agent prepared at a volume ratio of 1:2 was used as a fermentation agent to modify oat bran using solid-state fermentation. The optimal conditions were determined through process optimization as follows: inoculum amount of 10% (v / w), liquid-to-material ratio of 0.8:1 (mL / g), fermentation temperature of 37℃, and fermentation time of 3 days.

[0088] The oat bran IDF (i.e., IDF-3) obtained after modification by the present invention has significantly improved adsorption capacity and antioxidant activity, and its inhibitory effect on α-amylase is also enhanced.

[0089] In an in vitro fermentation system simulating human gut microbiota, IDF-3 exhibited fermentation characteristics of "low gas production and high acid production." While gas production was low, the total production of short-chain fatty acids and the absolute and relative content of butyric acid were significantly increased. This indicates that IDF-3 tends to utilize a slow and mild acid-producing metabolic pathway rather than a rapid gas-producing pathway during the fermentation and utilization of gut microbiota.

[0090] This fermentation method can effectively avoid common gastrointestinal discomfort symptoms such as bloating, flatulence, and increased gas that are common in high-fiber diets, and improve the product's gastrointestinal tolerance and dietary compliance. On the other hand, the continuous production of abundant short-chain fatty acids, especially butyric acid, helps to lower the pH of the intestinal lumen, inhibit the colonization of pathogenic bacteria, promote the proliferation of beneficial bacteria such as Bifidobacteria and Lactobacillus, and provide high-quality energy substrates for colonic epithelial cells. Thus, it plays a positive role in regulating the structure of the intestinal flora, maintaining the function of the intestinal mucosal barrier, and assisting in the regulation of glucose and lipid metabolism.

[0091] The product obtained by this invention can be widely used in the development of functional foods and prebiotic ingredients that help lower blood sugar, lower blood lipids, and regulate intestinal health.

[0092] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claims.

Claims

1. A synergistic fermentation compound microbial agent specifically for modifying insoluble dietary fiber in oat bran, characterized in that, The compound microbial agent is composed of Bacillus subtilis suspension and Lactobacillus plantarum suspension in a volume ratio of 1:2, wherein the viable bacteria concentration of the Bacillus subtilis suspension is not less than 1×10⁻⁶. 8 The cfu / mL concentration of the *Lactobacillus plantarum* suspension should not be less than 1×10⁻⁶. 8 cfu / mL.

2. The co-fermentation compound microbial agent specifically for modifying insoluble dietary fiber in oat bran as described in claim 1, characterized in that, The volume ratio of Bacillus subtilis to Lactobacillus plantarum is 1:

2.

3. A method for targeted modification of insoluble dietary fiber in oat bran based on synergistic solid-state fermentation by two microorganisms, characterized in that, Includes the following steps: (1) Substrate pretreatment: After crushing and sieving, oat bran is autoclaved at 121 °C for 15 minutes and cooled to room temperature for later use; (2) Based on the dry weight of oat bran, the compound microbial agent described in claim 1 is inoculated into the oat bran matrix obtained in step (1) at an inoculation amount of 10% (v / w) and a liquid-to-material ratio of 0.8:1 (mL / g). Under aseptic conditions, the mixture is thoroughly stirred and mixed evenly. Then, the fermentation vessel is sealed with a breathable microporous sealing film to maintain gas exchange during solid-state fermentation and effectively block external contamination. (3) Place the material obtained in step (2) in a 37 ℃ constant temperature incubator and let it undergo solid-state fermentation for 1-3 days; (4) After fermentation, insoluble dietary fiber was obtained by enzymatic separation. The filter residue was freeze-dried for 48-60 h to obtain insoluble dietary fiber of oat bran modified by solid fermentation of compound microbial agent.

4. The method for targeted modification of insoluble dietary fiber in oat bran based on synergistic solid-state fermentation by two microorganisms as described in claim 3, characterized in that, In step (2), the inoculation amount of the compound microbial agent is 10% (v / w) of the dry basis of oat bran.

5. The method for targeted modification of insoluble dietary fiber in oat bran based on dual-strain synergistic solid-state fermentation as described in claim 3, characterized in that, The process parameters of the method were optimized by combining the AHP-CRITIC weighted average method with Box-Behnken response surface design. The optimal process conditions were: a volume ratio of Bacillus subtilis to Lactobacillus plantarum in the compound bacterial agent of 1:2, a liquid-to-solid ratio of 0.8:1, and a fermentation time of 3 days.

6. The method for targeted modification of insoluble dietary fiber in oat bran based on dual-strain synergistic solid-state fermentation as described in claim 3, characterized in that, The specific steps of the enzyme-alkali separation method described in step (4) are as follows: Modified oat bran is added to α-amylase for enzymatic hydrolysis at a material-to-liquid ratio of 1:10, and then treated with protease to remove starch and protein. The residue is washed 3-4 times with 80% ethanol at 60-70℃, and then washed 3-4 times with distilled water at 60-70℃. The modified oat bran insoluble dietary fiber is obtained by freeze drying.

7. A modified oat bran insoluble dietary fiber prepared by the method according to claims 3 to 6.

8. The modified oat bran insoluble dietary fiber prepared by the method described in claims 3-6 as in claim 7, characterized in that, The insoluble dietary fiber in oat bran fermented with the compound microbial agent for 3 days has the following physicochemical characteristics: (a) The adsorption capacity for glucose is not less than 16 mg / g, the adsorption capacity for cholesterol is not less than 18 mg / g, and the adsorption capacity for nitrite under pH=2 conditions is not less than 880 μg / g. (b) The scavenging ability against ABTS free radicals is not less than 55%; (c) It exhibits inhibitory activity against α-amylase, with an inhibition rate reaching up to 24%; (d) During in vitro fecal microbial fermentation, the gas production is extremely low, the pH of the fermentation broth changes slowly, and the production of total short-chain fatty acids, acetic acid and butyric acid is promoted.

9. An application characterized in that, This application can be any of the following: ①The use of the modified oat bran insoluble dietary fiber according to claim 7 in the preparation of food, health products or drugs with α-amylase inhibitory activity and auxiliary hypoglycemic function; ②The use of the modified oat bran insoluble dietary fiber according to claim 7 in the preparation of food, health products or drugs with glucose adsorption, cholesterol adsorption and nitrite adsorption functions; ③The use of the modified oat bran insoluble dietary fiber according to claim 7 in the preparation of intestinal flora regulators or prebiotic ingredients; ④ The use of the modified oat bran insoluble dietary fiber as described in claim 7 as a substance that slowly ferments, produces little gas, and avoids gastrointestinal discomfort symptoms such as bloating and abdominal pain; ⑤ Use of the modified oat bran insoluble dietary fiber according to claim 7 in the preparation of substances that promote the production of total short-chain fatty acids, acetic acid and butyric acid in the colon; ⑥ The use of the modified oat bran insoluble dietary fiber according to claim 7 in the preparation of foods and health products for the prevention or improvement of metabolic syndromes such as obesity, diabetes, and hyperlipidemia.

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