Extraction method of eucommia ulmoides with intestinal repair function

Abstract

The invention discloses a method for extracting eucommia ulmoides with an intestinal repair function, and relates to the technical field of natural product extraction.The method comprises the steps that a fresh eucommia ulmoides raw material is subjected to low-temperature drying and smashing, a polysaccharide water extract and filter residues are obtained through water extraction, and then the flavone residual quantity in the polysaccharide water extract is synchronously detected; carrying out enzymolysis on the polysaccharide water extract through beta-glucanase to obtain a small molecular polysaccharide solution, and carrying out alcohol extraction on filter residues to obtain a flavone alcohol extract; the preparation method comprises the following steps: mixing the two components according to a ratio, inoculating compound probiotics for anaerobic fermentation, regulating according to the residual quantity of flavone, regulating an alcohol extraction process by using short-chain fatty acid generated by fermentation when the residual quantity is high, supplementing flavone extract in an enzymolysis system when the residual quantity is low, and finally removing impurities, purifying, adapting intestinal tracts and drying to obtain the product. According to the method, regulation and control operation is correspondingly executed by detecting the residual quantity of flavone in the polysaccharide water extract, so that self-supply of a regulation and control reagent and high efficiency of active ingredients are realized, the conversion rate of small molecular polysaccharide and flavone aglycone is increased, and the intestinal tract tolerance of the product is optimized.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology, specifically to a method for extracting Eucommia ulmoides with intestinal repair function. Background Technology

[0002] Eucommia ulmoides, a traditional Chinese medicine and food ingredient, contains polysaccharides, flavonoids, and other active components with rich bioactivity, showing significant application potential in the field of intestinal health maintenance. Modern research shows that Eucommia ulmoides polysaccharides can regulate the balance of intestinal flora and enhance intestinal barrier function, while Eucommia ulmoides flavonoids possess anti-inflammatory and antioxidant properties. The synergistic effect of the two can effectively promote intestinal mucosal repair and improve the intestinal microecological environment, showing broad development prospects in the fields of functional foods, health products, and pharmaceuticals.

[0003] Current extraction technologies for Eucommia ulmoides primarily rely on traditional water and alcohol extraction processes, with some incorporating enzymatic hydrolysis or fermentation. However, these methods still have several shortcomings. Traditional processes generally use fixed extraction parameters, failing to adequately consider the fluctuations in active ingredient content caused by variations in the growing season, harvesting location, and storage conditions of the Eucommia ulmoides raw material. This can easily lead to interactions between polysaccharides and flavonoids during extraction, reducing the extraction efficiency and purity of active ingredients. Furthermore, some processes, in an attempt to improve extraction efficiency, blindly extend extraction time, increase extraction temperature, or increase solvent usage. This not only increases energy consumption and costs but may also cause oxidative degradation of active ingredients, affecting product functionality. In addition, existing processes lack sufficient optimization of key steps such as enzymatic hydrolysis and fermentation. Large polysaccharides are difficult to effectively convert into smaller molecules that are easily absorbed by the intestines, resulting in low flavonoid aglycone conversion rates. Moreover, some processes rely on exogenous reagents to regulate the extraction environment, potentially introducing contaminants and affecting product safety and stability, making it difficult to meet the production requirements of high-quality Eucommia ulmoides extracts.

[0004] Therefore, it is necessary to provide an Eucommia ulmoides extraction method with intestinal repair function to solve the above-mentioned technical problems. Summary of the Invention

[0005] The purpose of this invention is to provide an extraction method for Eucommia ulmoides with intestinal repair function, in order to solve the problems of mutual interference of components, low conversion efficiency of active ingredients, dependence on exogenous reagents, poor process stability and insufficient intestinal repair function in existing Eucommia ulmoides extraction processes.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for extracting Eucommia ulmoides with intestinal repair function, comprising the following steps: Step 1: Select fresh Eucommia ulmoides raw materials, wash them, dry them at low temperature until the moisture content is ≤5%, pulverize and sieve them to obtain Eucommia ulmoides powder; Step 2: Add deionized water to the Eucommia ulmoides powder obtained in Step 1, adjust the pH to 6.8-7.0, and extract at a constant temperature of 38-42℃. After extraction, filter to obtain filter residue and polysaccharide aqueous extract, and detect the flavonoid residue in the polysaccharide aqueous extract. Step 3: Add β-glucanase to the polysaccharide aqueous extract obtained in Step 2, adjust the temperature to 48-52℃ and the pH to 6.6-7.0 for enzymatic hydrolysis, and inactivate the enzyme by raising the temperature after enzymatic hydrolysis to obtain a small molecule polysaccharide solution; Step 4: Cool the small molecule polysaccharide solution obtained in Step 3 and adjust the pH to 6.1-6.5. Transfer it to an anaerobic fermenter to create an anaerobic environment. Supplement with glucose as an auxiliary carbon source to obtain the adapted polysaccharide solution. Step 5: Add the filter residue obtained in Step 2 to an ethanol solution, adjust the pH to 5.0-5.5, reflux at 53-57℃ for extraction, filter after extraction to obtain flavonoid alcohol extract; Step 6: Mix the adapted polysaccharide solution obtained in Step 4 with the flavonoid alcohol extract obtained in Step 5 in a certain proportion, and inoculate with a compound probiotic agent composed of Bifidobacterium, Lactobacillus plantarum and Lactobacillus acidophilus for anaerobic fermentation. Monitor and adjust the pH during the fermentation process to obtain the fermentation broth. Step 7: Perform control operations based on the flavonoid residue detected in Step 2: If the flavonoid residue is higher than 10%, use the short-chain fatty acids produced by fermentation in Step 6 to adjust the alcohol extraction process in Step 5, and control the polysaccharide residue ratio in the flavonoid alcohol extract to below 6%; if the flavonoid residue is lower than 8%, add Eucommia ulmoides flavonoid extract to the enzymatic hydrolysis system in Step 3. Step 8: The fermentation broth obtained in Step 6 is subjected to impurity removal, purification, intestinal adaptation, and drying to obtain Eucommia ulmoides extract with intestinal repair function.

[0007] This invention detects the residual flavonoid content in the polysaccharide aqueous extract after water extraction. Essentially, it provides a precise basis for subsequent processes through component prediction, avoiding blind operation. Specifically: when the residual flavonoid content is below 8%, the polysaccharide lacks the antioxidant protection of flavonoids and is easily oxidized and degraded; when it is 8-10%, the ratio of flavonoids to polysaccharides is suitable and no additional adjustment is needed; when it is above 10%, the polysaccharide dissolution is too high, which will adsorb flavonoids and hinder their subsequent transformation, thus requiring targeted intervention.

[0008] During enzymatic hydrolysis, β-glucanase generates a small amount of reactive oxygen species (ROS) when degrading large polysaccharides. These ROS oxidize and damage the polysaccharide structure, leading to a decrease in the conversion rate of small polysaccharides. Eucommia ulmoides flavonoid extract has strong antioxidant properties; its addition can scavenge ROS, protecting polysaccharides from oxidation and maintaining their structural integrity. Flavonoids themselves are excellent substrates for probiotic fermentation. Adding flavonoid extract in low-residue scenarios provides sufficient nutrition for subsequent compound probiotic fermentation, promoting probiotic proliferation and metabolism, and indirectly increasing the yield of short-chain fatty acids. Moreover, the short-chain fatty acids are derived from the fermentation products of the compound probiotics in this process, eliminating the need for additional exogenous regulatory reagents. This reduces production costs and avoids contamination from impurities introduced by exogenous reagents, ensuring the purity and biosafety of the extract.

[0009] Preferably, in step 7, the short-chain fatty acids produced by fermentation in step 6 are used to adjust the alcohol extraction process in step 5, specifically including the following steps: 1a: Take the fermentation broth from step 6 and centrifuge it at 3000-5000 r / min for 10-15 min to remove the cells and macromolecular impurities, and obtain the fermentation supernatant. 1b: Add 1-2 times its volume of ethyl acetate to the fermentation supernatant, shake and extract 2-3 times, and collect the organic phase; 1c: The organic phase is subjected to vacuum distillation to remove ethyl acetate, yielding a mixture of short-chain fatty acids with a purity ≥85%, wherein the short-chain fatty acid mixture is mainly composed of acetic acid, propionic acid, and butyric acid; 1d: Add the short-chain fatty acid mixture to the ethanol solution in step 5 at 0.2-0.5% of the total mass of the alcohol extraction system in step 5, stir evenly, and lower the pH of the alcohol extraction system to 4.8-5.2. Continue to maintain the alcohol extraction temperature at 53-57℃ to complete the reflux extraction.

[0010] The compound probiotics in this invention include Bifidobacterium, Lactobacillus plantarum, and Lactobacillus acidophilus. During fermentation, they utilize small-molecule polysaccharides to metabolize and produce short-chain fatty acids, mainly acetic acid, propionic acid, and butyric acid. These short-chain fatty acids are not only active ingredients for intestinal repair but also possess the property of regulating the pH of the system. Adding short-chain fatty acids to the alcohol extraction system can lower the pH to 4.8 to 5.2. This pH range can significantly inhibit the dissolution of Eucommia ulmoides polysaccharides, reducing the solubility of polysaccharides in a slightly acidic environment without affecting the dissolution efficiency of flavonoids in ethanol, thereby reducing polysaccharide residues in the flavonoid alcohol extract.

[0011] Preferably, if the flavonoid residue detected in step 2 is higher than 12%, the alcohol extraction temperature is simultaneously lowered to 50-53°C when performing step 1d.

[0012] In this invention, when the flavonoid residue is higher than 12%, pH adjustment alone is insufficient to completely inhibit polysaccharide dissolution. Simultaneously, the alcohol extraction temperature is lowered to 50-53°C. Through the dual effects of pH adjustment and temperature reduction, the polysaccharide solubility is further reduced, ensuring that the residue is controlled below 6%, thus avoiding the mutual interference between polysaccharides and flavonoids from the root.

[0013] Preferably, in step 1, the fresh Eucommia ulmoides raw material is fresh Eucommia ulmoides leaves or bark from April to July; the low-temperature drying temperature is 40-50℃, the drying time is 2-4 hours, and the drying is carried out by forced air drying; after pulverization, it is passed through a 50-70 mesh sieve.

[0014] In this invention, Eucommia ulmoides leaves or bark harvested between April and July are in their vigorous growth period, exhibiting active flavonoid and polysaccharide synthesis and metabolism, resulting in abundant and structurally stable active ingredients, providing a high-quality material basis for subsequent extraction and regulation. A low-temperature environment prevents high temperatures from causing oxidative degradation or structural denaturation of active ingredients. Forced-air drying accelerates moisture evaporation and ensures uniform drying. Precise control of moisture content during specific drying times prevents excessive dilution of the subsequent extract while also avoiding over-drying that could lead to raw material embrittlement. Appropriate particle size increases the contact area between the raw material and the solvent, shortens the diffusion path of components, and promotes the dissolution of active ingredients, while preventing excessively fine particles from clogging the filter, thus balancing extraction efficiency and operational smoothness.

[0015] Preferably, in step 2, the ratio of deionized water to liquid is 1:20-1:30, the constant temperature extraction time is 1.5-2.5h, and the mixture is stirred intermittently during the process; the filter is made of 90-110 mesh filter cloth while the mixture is still hot.

[0016] In this invention, a specific material-to-liquid ratio ensures that the solvent fully wets the raw material, providing a sufficient medium for polysaccharide dissolution. This avoids insufficient dissolution due to an excessively small material-to-liquid ratio, or excessively large ratios that increase the load on subsequent processing. A neutral pH and mild temperature match the polysaccharide's solubility characteristics, promoting full dissolution while preventing extreme conditions from damaging the polysaccharide structure. Intermittent stirring breaks the concentration gradient on the raw material surface, accelerating component diffusion and improving extraction efficiency. Hot filtration reduces polysaccharide precipitation loss at low temperatures, and an appropriate filter cloth pore size efficiently separates the filter residue from the aqueous extract, ensuring filtrate clarity. Flavonoid residue detection provides precise data for subsequent adjustments, allowing for prediction of component ratios and avoiding component interference or incomplete conversion caused by blind processing.

[0017] Preferably, in step 3, the β-glucanase has an enzyme activity of 40,000-60,000 U / g, is added at 0.8-1.5% of the mass of the polysaccharide aqueous extract, has an enzymatic hydrolysis time of 1.5-2.5 h, and is incubated at 55-65℃ for 5-15 min. The pH and temperature are monitored regularly during the enzymatic hydrolysis process, and the pH is finely adjusted with buffer solution if it deviates from the specified value.

[0018] In this invention, specific enzyme activity and dosage ensure efficient enzymatic hydrolysis, preventing incomplete polysaccharide degradation due to insufficient enzyme activity or dosage, while excessive activity leads to waste and potential introduction of impurities. Matching the hydrolysis temperature and pH to the optimal activity range of β-glucanase ensures efficient cleavage of glycosidic bonds in large polysaccharides, converting them into smaller molecules easily absorbed by the intestines. Specific hydrolysis times allow for complete reaction, and rapid enzyme inactivation prevents over-hydrolysis that damages the polysaccharide structure, while also preventing enzyme residue from affecting subsequent processes. Regular monitoring and fine-tuning of pH and temperature maintain a stable hydrolysis environment, ensuring consistent conversion efficiency across different batches and providing a high-quality carbon source for subsequent fermentation.

[0019] Preferably, in step 4, the cooling is performed by rapidly cooling to 35-39℃ using an ice-water bath; the pH is adjusted using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution; the anaerobic environment is constructed by evacuating to an oxygen concentration ≤0.5% and introducing nitrogen to maintain a slight positive pressure; and the glucose supplementation is 0.2-0.5%.

[0020] In this invention, rapid cooling via an ice-water bath avoids polysaccharide oxidation or structural rearrangement at high temperatures. The final cooling temperature matches the optimal growth temperature of probiotics, providing suitable initial conditions for fermentation. A sodium dihydrogen phosphate-disodium hydrogen phosphate buffer precisely maintains the system's pH stability; its mild composition does not react with the polysaccharides, preventing the introduction of contaminating ions. A low-oxygen environment meets the anaerobic metabolic needs of the complex probiotics, nitrogen protection prevents oxygen re-entry, and a slightly positive pressure state ensures the stability of the fermentation system and avoids external contamination. An appropriate amount of glucose serves as a fast-acting carbon source, promoting rapid probiotic proliferation, shortening the fermentation delay period, and laying the foundation for the subsequent production of sufficient short-chain fatty acids.

[0021] Preferably, in step 5, the concentration of the added ethanol solution is 70-80%, the material-to-liquid ratio is 1:15-1:25, the reflux extraction time is 1-2 hours, and filtration is carried out using a 90-110 mesh filter cloth.

[0022] In this invention, 70-80% ethanol concentration balances flavonoid dissolution efficiency and polysaccharide inhibition effect, ensuring sufficient flavonoid dissolution while reducing unnecessary polysaccharide dissolution and lowering subsequent control pressure. A specific material-to-liquid ratio ensures sufficient contact between ethanol and flavonoids in the filter residue, avoiding incomplete extraction or solvent waste due to an improper ratio. Reflux extraction continuously circulates ethanol, maintaining a concentration gradient and promoting continuous flavonoid dissolution, while a specific time frame prevents under- or over-extraction that could lead to flavonoid oxidation. An appropriate filter cloth pore size efficiently separates the filter residue from the ethanol extract, ensuring filtrate purity and providing high-quality raw materials for subsequent mixed fermentation.

[0023] Preferably, in step 6, the volume ratio of the adapted polysaccharide solution to the flavonoid extract is 1.5:1-2.5:1; the mass ratio of Bifidobacterium, Lactobacillus plantarum, and Lactobacillus acidophilus in the compound probiotic agent is 1:2-1:4, the bacterial activity is 800-1.2 billion CFU / g, and the inoculum amount is 0.5-1.5% of the mass of the mixed solution; the fermentation temperature is 35-39℃, the fermentation time is 8-16h, the pH is monitored regularly, and the pH is adjusted back to 5.5-6.0 using sodium bicarbonate solution.

[0024] In this invention, a specific volume ratio of the adapted polysaccharide solution to the flavonoid extract ensures a balanced ratio of small-molecule polysaccharides and flavonoids. The polysaccharides provide a carbon source for probiotics, while the flavonoids provide a cofactor substrate for metabolism, promoting synergistic effects. A balanced blend of three probiotic strains achieves functional synergy: Bifidobacterium excels at producing short-chain fatty acids, Lactobacillus plantarum enhances flavonoid conversion, and Lactobacillus acidophilus inhibits harmful bacteria, improving the overall fermentation effect. High bacterial viability and an appropriate inoculum size ensure the rapid formation of a dominant bacterial community, avoiding inefficient fermentation due to insufficient inoculum size or cost waste due to excessive inoculum size. Suitable fermentation temperature and time allow for sufficient metabolism by the probiotics, producing adequate amounts of short-chain fatty acids and flavonoid aglycones. Periodic pH adjustments maintain probiotic activity and prevent the accumulation of acidic substances that inhibit fermentation.

[0025] Preferably, in step 7, the purity of the added Eucommia ulmoides flavonoid extract is ≥90%, and the amount added is 0.1-0.3% of the mass of the enzymatic hydrolysis system.

[0026] In this invention, short-chain fatty acids are derived from fermentation products, eliminating the need for exogenous addition, thus reducing costs and avoiding interference from impurities. Their acidic properties regulate the pH of the alcohol extraction system to the low solubility range of polysaccharides, inhibiting polysaccharide dissolution and reducing polysaccharide residue in the flavonoid alcohol extract. When the flavonoid residue exceeds 12%, cooling further reduces polysaccharide solubility. Through the dual effects of pH adjustment and cooling, the polysaccharide residue is ensured to meet standards even in extreme scenarios. In low-residue scenarios, the high-purity flavonoid extract exerts its antioxidant effect, scavenging reactive oxygen species generated during enzymatic hydrolysis and protecting polysaccharides from oxidation. Simultaneously, it serves as a high-quality substrate to promote probiotic fermentation, forming a positive cycle.

[0027] Preferably, in step 8, the impurity removal process involves adding 2-4% diatomaceous earth and 0.3-0.7% chitosan, stirring, letting stand, and then filtering using a plate and frame filter press. The purification process involves screening the filtrate for 1-5 kDa small molecule polysaccharides using a Sephadex G-25 gel column, followed by elution with 50-60% ethanol solution using an AB-8 macroporous adsorption resin column. The intestinal adaptant is prepared by concentrating the eluent under reduced pressure to 1 / 10-1 / 6 of its original volume, adjusting the pH to 6.5-7.0, and adding 0.05-0.15% fructooligosaccharides. In step 8, the drying process is as follows: the material to be dried is placed in a vacuum freeze-drying oven and cooled from room temperature to -40 to -30°C at a rate of 2-5°C / h and held for 6-10 hours; then cooled to -55 to -45°C at a rate of 1-2°C / h and held for 4-6 hours to complete gradient pre-freezing; subsequently, the vacuum degree in the freeze-drying oven is maintained at 10-30 Pa and the shelf temperature is increased from -55 to -45°C to -20 to -10°C at a rate of 0.5-1°C / h, while controlling the material temperature to not exceed its eutectic point temperature, and held for 12-24 hours until the ice crystals are completely sublimated to complete sublimation drying; finally, the vacuum degree in the freeze-drying oven is maintained at 5-15 Pa and the shelf temperature is increased to 25-35°C at a rate of 1-2°C / h, while controlling the material temperature to not exceed 30°C, and held for 8-12 hours until the material moisture content drops to 1%-3%, and then naturally cooled to room temperature to complete desorption drying.

[0028] In this invention, diatomaceous earth and chitosan work synergistically to adsorb suspended impurities and flocculate large molecules. A plate and frame filter press efficiently separates the impurities, yielding a clear filtrate, laying the foundation for purification. A gel column uses molecular sieve effect to screen for target molecular weight small-molecule polysaccharides, while macroporous adsorption resin specifically enriches flavonoid aglycones. Elution with a specific concentration of ethanol achieves precise purification of the active ingredients, improving product purity and functional targeting. Reduced pressure concentration increases the concentration of active ingredients, avoiding dilution that could affect efficacy. The pH is adjusted to a suitable range for the gut, and fructooligosaccharides are added as prebiotics to promote the proliferation of beneficial gut bacteria, enhancing the extract's intestinal tolerance and colonization ability. Gradient pre-freezing prevents large ice crystals from damaging the active ingredient structure. Vacuum sublimation drying removes moisture at low temperatures while preserving ingredient activity. Desorption drying removes residual adsorbed water, controlling the moisture content to 1%-3% to ensure product stability and prevent moisture absorption and deterioration.

[0029] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention precisely detects the residual flavonoid content after water extraction in step 2, classifies different content ranges, and performs differentiated operations to adapt to the natural differences in raw materials from the source. When the residual flavonoid content is high, short-chain fatty acids produced by the fermentation of compound probiotics in step 6 of the process are used to adjust the alcohol extraction system. No additional exogenous reagents are needed, which reduces production costs and avoids contamination by impurities. At the same time, short-chain fatty acids can directionally adjust the pH of the alcohol extraction system, inhibiting excessive dissolution of polysaccharides and reducing their adsorption resistance to flavonoid conversion. When the residual flavonoid content is low, Eucommia ulmoides flavonoid extract is added to the enzymatic hydrolysis system. Utilizing the strong antioxidant properties of flavonoids, reactive oxygen species generated during enzymatic hydrolysis are eliminated, protecting the polysaccharide structure from oxidative damage and maintaining its integrity as a high-quality substrate for subsequent fermentation. This maximizes the conversion efficiency of small molecule polysaccharides and flavonoid aglycones, significantly improving the total purity of the extract.

[0030] 2. This invention achieves targeted optimization of active ingredients through the synergistic effect of enzymatic hydrolysis and compound probiotic fermentation. Specifically, β-glucanase efficiently breaks the glycosidic bonds of macromolecular polysaccharides under suitable conditions, converting them into small molecule forms that are easily absorbed by the intestines, thus providing a sufficient carbon source for compound probiotic fermentation. Meanwhile, the compound probiotic agent composed of Bifidobacterium, Lactobacillus plantarum, and Lactobacillus acidophilus can not only further promote the conversion of flavonoids to aglycones during fermentation, but also produce beneficial intestinal metabolites such as short-chain fatty acids, which synergistically interact with small molecule polysaccharides and flavonoid aglycones.

[0031] 3. This invention enriches the target active ingredients and removes ineffective impurities through steps such as gel column screening and macroporous adsorption resin purification. Then, it adjusts the pH to a suitable range for the intestine through intestinal adaptation treatment and adds fructooligosaccharides. This not only improves the intestinal tolerance of the product, but also promotes the proliferation of beneficial bacteria in the intestine and enhances the colonization ability of the extract in the intestine, ultimately achieving a significant improvement in intestinal repair function. Detailed Implementation

[0032] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1: This embodiment provides a method for extracting Eucommia ulmoides with intestinal repair function, including the following steps: Step 1: Select fresh Eucommia ulmoides leaves from April, wash them, and dry them at 40℃ for 4 hours using a forced-air drying method. The forced-air rate during the drying process is 2.0 m / s. After pulverizing, pass the powder through a 50-mesh sieve to obtain Eucommia ulmoides powder. The moisture content of the powder was measured to be 4.8%. Step 2: Add deionized water to the Eucommia ulmoides powder obtained in Step 1, with a material-to-liquid ratio of 1:20, adjust the pH to 6.8, and extract at a constant temperature of 38℃ for 2.5 hours, stirring intermittently during the extraction. After extraction, filter while hot using a 90-mesh filter cloth to obtain the filter residue and polysaccharide aqueous extract. The residual flavonoid content in the polysaccharide aqueous extract was found to be 8.5%. Step 3: Add β-glucanase to the polysaccharide aqueous extract obtained in Step 2. The enzyme activity is 40,000 U / g, and the amount added is 0.8% of the mass of the polysaccharide aqueous extract. Adjust the temperature to 48℃ and the pH to 6.6 for enzymatic hydrolysis. The hydrolysis time is 2.5h. During the hydrolysis process, the pH and temperature are monitored regularly. If the pH deviates, it is finely adjusted with sodium dihydrogen phosphate-disodium hydrogen phosphate buffer. After the hydrolysis is completed, incubate at 55℃ for 15min to inactivate the enzyme and obtain a small molecule polysaccharide solution. Step 4: The small molecule polysaccharide solution obtained in Step 3 is rapidly cooled to 35°C using an ice-water bath, the pH is adjusted to 6.1, and a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution is used. The solution is then transferred to an anaerobic fermenter, evacuated to an oxygen concentration of 0.5%, and nitrogen is introduced to maintain a slight positive pressure of 0.02 MPa at a nitrogen introduction rate of 1.0 L / min. 0.2% glucose is added as an auxiliary carbon source to obtain the adapted polysaccharide solution. Step 5: Add the filter residue obtained in Step 2 to 70% ethanol solution at a material-to-liquid ratio of 1:15, adjust the pH to 5.0, and reflux at 53℃ for 2 hours. After extraction, filter with a 90-mesh filter cloth to obtain flavonol extract. Step 6: Mix the adapted polysaccharide solution obtained in Step 4 with the flavonoid alcohol extract obtained in Step 5 at a volume ratio of 1.5:1, and inoculate with a compound probiotic inoculum. The mass ratio of Bifidobacterium, Lactobacillus plantarum, and Lactobacillus acidophilus is 1:2, the bacterial activity is 800 million CFU / g, and the inoculum amount is 0.5% of the mass of the mixture. Ferment at 35℃ for 16 hours. During the fermentation process, the pH is monitored every 6 hours and adjusted to 5.5 using sodium bicarbonate solution to obtain the fermentation broth. Step 7: The flavonoid residue detected in Step 2 is 8.5%, which is within the 8-10% range and requires no adjustment. Step 8: Add 2% diatomaceous earth and 0.3% chitosan to the fermentation broth obtained in Step 6, stir, let stand for 35 min, and filter using a plate and frame filter press; screen the filtrate for 1-5 kDa small molecule polysaccharides through a Sephadex G-25 gel column, and then elute with 50% ethanol solution through an AB-8 macroporous adsorption resin column; concentrate the eluent under reduced pressure at 0.06 MPa and 50℃ to 1 / 10 of the original volume, adjust the pH to 6.5, and add 0.05% fructooligosaccharide; place the material in a vacuum freeze dryer, cool from room temperature to -40℃ at a rate of 2℃ / h, and maintain the temperature. After 10 hours, the temperature was lowered to -55℃ at a rate of 1℃ / h and held for 6 hours to complete gradient pre-freezing. Maintaining a vacuum of 10Pa in the freeze-drying chamber, the shelf temperature was increased from -55℃ to -20℃ at a rate of 0.5℃ / h, while controlling the material temperature to not exceed its eutectic point temperature. The material was held for 24 hours to complete sublimation drying. Maintaining a vacuum of 5Pa in the freeze-drying chamber, the shelf temperature was increased to 25℃ at a rate of 1℃ / h, while controlling the material temperature to not exceed 30℃. The material was held for 12 hours until the moisture content dropped to 2.8%. The material was then allowed to cool naturally to room temperature to complete desorption drying, yielding an Eucommia ulmoides extract with intestinal repair function.

[0034] Example 2: This embodiment provides a method for extracting Eucommia ulmoides with intestinal repair function, including the following steps: Step 1: Select fresh Eucommia bark from June, wash it, and dry it using a forced-air drying method at 45℃ for 3 hours. The forced-air rate during the drying process is 2.0 m / s. After pulverizing, pass it through a 60-mesh sieve to obtain Eucommia powder. The moisture content of the powder was measured to be 4.2%. Step 2: Add deionized water to the Eucommia ulmoides powder obtained in Step 1, with a material-to-liquid ratio of 1:25, adjust the pH to 6.9, and extract at a constant temperature of 40℃ for 2.0 h, with intermittent stirring during the extraction. After extraction, filter while hot using a 100-mesh filter cloth to obtain the filter residue and polysaccharide aqueous extract. The residual flavonoid content in the polysaccharide aqueous extract was found to be 11.2%. Step 3: Add β-glucanase to the polysaccharide aqueous extract obtained in Step 2. The enzyme activity is 50,000 U / g, and the amount added is 1.0% of the mass of the polysaccharide aqueous extract. Adjust the temperature to 50℃ and the pH to 6.8 for enzymatic hydrolysis. The hydrolysis time is 2.0 h. During the hydrolysis process, the pH and temperature are monitored regularly. If the pH deviates, it is finely adjusted with sodium dihydrogen phosphate-disodium hydrogen phosphate buffer. After the hydrolysis is completed, incubate at 60℃ for 10 min to inactivate the enzyme and obtain a small molecule polysaccharide solution. Step 4: The small molecule polysaccharide solution obtained in Step 3 was rapidly cooled to 37°C using an ice-water bath, the pH was adjusted to 6.3, and the solution was transferred to an anaerobic fermenter using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution. The system was evacuated to an oxygen concentration of 0.4%, and nitrogen was introduced to maintain a slight positive pressure of 0.02 MPa at a nitrogen introduction rate of 1.0 L / min. 0.3% glucose was added as an auxiliary carbon source to obtain the adapted polysaccharide solution. Step 5: Add the filter residue obtained in Step 2 to 75% ethanol solution at a material-to-liquid ratio of 1:20, adjust the pH to 5.2, and reflux at 55℃ for 1.5 hours. After extraction, filter with a 100-mesh filter cloth to obtain flavonol extract. Step 6: Mix the adapted polysaccharide solution obtained in Step 4 with the flavonoid alcohol extract obtained in Step 5 at a volume ratio of 2.0:1, and inoculate with a compound probiotic inoculum. The mass ratio of Bifidobacterium, Lactobacillus plantarum and Lactobacillus acidophilus is 1:3, the bacterial activity is 1 billion CFU / g, and the inoculum amount is 1.0% of the mass of the mixture. Ferment at 37℃ for 12 hours. During the fermentation process, the pH is monitored every 6 hours and adjusted to 5.8 using sodium bicarbonate solution to obtain the fermentation broth. Step 7: The flavonoid residue detected in Step 2 is 11.2%, which is higher than 10% but lower than 12%. Therefore, the short-chain fatty acid-adjusted alcohol extraction process should be performed. 1a: Take the fermentation broth from step 6 and centrifuge it at 3000 r / min for 15 min to remove the bacterial cells and macromolecular impurities, and obtain the fermentation supernatant; 1b: Add ethyl acetate (1 volume) to the fermentation supernatant, shake and extract twice, and collect the organic phase; 1c: The organic phase was subjected to vacuum distillation to remove ethyl acetate, yielding a mixture of short-chain fatty acids with a purity of 86%, the main components of which were acetic acid, propionic acid, and butyric acid; 1d: Add 0.2% of the total mass of the short-chain fatty acid mixture to the ethanol solution in step 5, stir well, and lower the pH of the ethanol extraction system to 4.8. Continue to maintain the ethanol extraction temperature at 55℃ to complete the reflux extraction. The final polysaccharide residue in the flavonoid ethanol extract was found to be 5.6%. Step 8: Add 3% diatomaceous earth and 0.5% chitosan to the fermentation broth obtained in Step 6, stir, let stand for 30 min, and filter using a plate and frame filter press; screen the filtrate for 1-5 kDa small molecule polysaccharides using a Sephadex G-25 gel column, and then elute with 55% ethanol solution using an AB-8 macroporous adsorption resin column; concentrate the eluent under reduced pressure at 0.08 MPa and 55℃ to 1 / 8 of the original volume, adjust the pH to 6.8, and add 0.1% fructooligosaccharide; place the material in a vacuum freeze dryer, cool from room temperature to -35℃ at a rate of 3℃ / h, and maintain the temperature for 8 h. The material was then cooled to -50℃ at a rate of 1.5℃ / h and held for 5 hours to complete gradient pre-freezing. Maintaining a vacuum of 20Pa inside the freeze-drying chamber, the shelf temperature was increased from -50℃ to -15℃ at a rate of 0.8℃ / h, while controlling the material temperature to not exceed its eutectic point temperature. The material was held for 18 hours to complete sublimation drying. Maintaining a vacuum of 10Pa inside the freeze-drying chamber, the shelf temperature was increased to 30℃ at a rate of 1.5℃ / h, while controlling the material temperature to not exceed 30℃. The material was held for 10 hours until the moisture content dropped to 2.0%. The material was then allowed to cool naturally to room temperature to complete desorption drying, yielding an Eucommia ulmoides extract with intestinal repair function.

[0035] Example 3: This embodiment provides a method for extracting Eucommia ulmoides with intestinal repair function, including the following steps: Step 1: Select a mixture of fresh Eucommia ulmoides leaves and bark from July, wash them, and then dry them at 50℃ for 2 hours using a forced-air drying method. The forced-air rate during the drying process is 2.0 m / s. After pulverizing, pass the powder through a 70-mesh sieve to obtain Eucommia ulmoides powder. The moisture content of the powder was measured to be 4.5%. Step 2: Add deionized water to the Eucommia ulmoides powder obtained in Step 1, with a material-to-liquid ratio of 1:30, adjust the pH to 7.0, and extract at a constant temperature of 42℃ for 1.5 hours, stirring intermittently during the extraction. After extraction, filter while hot using a 110-mesh filter cloth to obtain the filter residue and polysaccharide aqueous extract. The residual flavonoid content in the polysaccharide aqueous extract was found to be 12.8%. Step 3: Add β-glucanase to the polysaccharide aqueous extract obtained in Step 2. The enzyme activity is 60,000 U / g, and the amount added is 1.5% of the mass of the polysaccharide aqueous extract. Adjust the temperature to 52℃ and the pH to 7.0 for enzymatic hydrolysis. The hydrolysis time is 1.5h. During the hydrolysis process, the pH and temperature are monitored regularly. If the pH deviates, it is finely adjusted with sodium dihydrogen phosphate-disodium hydrogen phosphate buffer. After the hydrolysis is completed, incubate at 65℃ for 8min to inactivate the enzyme and obtain a small molecule polysaccharide solution. Step 4: The small molecule polysaccharide solution obtained in Step 3 is rapidly cooled to 39°C using an ice-water bath, the pH is adjusted to 6.5, and a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution is used. The solution is then transferred to an anaerobic fermenter, evacuated to an oxygen concentration of 0.3%, and nitrogen is introduced to maintain a slight positive pressure of 0.02 MPa at a nitrogen introduction rate of 1.0 L / min. 0.5% glucose is added as an auxiliary carbon source to obtain the adapted polysaccharide solution. Step 5: Add the filter residue obtained in Step 2 to 80% ethanol solution at a material-to-liquid ratio of 1:25, adjust the pH to 5.5, and reflux at 57℃ for 1 hour. After extraction, filter with a 110-mesh filter cloth to obtain flavonol extract. Step 6: Mix the adapted polysaccharide solution obtained in Step 4 with the flavonoid alcohol extract obtained in Step 5 at a volume ratio of 2.5:1, and inoculate with a compound probiotic inoculum. The mass ratio of Bifidobacterium, Lactobacillus plantarum, and Lactobacillus acidophilus is 1:4, the bacterial activity is 1.2 billion CFU / g, and the inoculum amount is 1.5% of the mass of the mixed solution. Ferment at 39℃ for 8 hours. During the fermentation process, the pH is monitored every 6 hours and adjusted to 6.0 using sodium bicarbonate solution to obtain the fermentation broth. Step 7: The flavonoid residue detected in Step 2 is 12.8%, which is higher than 12%. Therefore, the short-chain fatty acid-adjusted alcohol extraction process should be performed. 1a: Take the fermentation broth from step 6 and centrifuge it at 5000 r / min for 10 min to remove the cells and macromolecular impurities, and obtain the fermentation supernatant. 1b: Add ethyl acetate, twice its volume, to the fermentation supernatant, shake and extract three times, and collect the organic phase; 1c: The organic phase was subjected to vacuum distillation to remove ethyl acetate, yielding a mixture of short-chain fatty acids with a purity of 88%, the main components of which were acetic acid, propionic acid, and butyric acid. 1d: Add 0.5% of the total mass of the short-chain fatty acid mixture from step 5 to the ethanol solution from step 5, stir well, and lower the pH of the ethanol extraction system to 5.2. Simultaneously, lower the ethanol extraction temperature to 53°C and continue to maintain this condition to complete the reflux extraction. The final polysaccharide residue in the flavonoid ethanol extract was found to be 5.2%. Step 8: Add 4% diatomaceous earth and 0.7% chitosan to the fermentation broth obtained in Step 6, stir, let stand for 25 minutes, and filter using a plate and frame filter press; screen the filtrate for 1-5 kDa small molecule polysaccharides through a Sephadex G-25 gel column, and then elute with 60% ethanol solution through an AB-8 macroporous adsorption resin column; concentrate the eluent under reduced pressure at 0.09 MPa and 60℃ to 1 / 6 of the original volume, adjust the pH to 7.0, and add 0.15% fructooligosaccharide; place the material in a vacuum freeze dryer and cool it from room temperature to -30℃ at a rate of 5℃ / h. The material was pre-frozen at a constant temperature for 6 hours, then cooled to -45°C at a rate of 2°C / h and held for 4 hours. The vacuum in the freeze-drying chamber was maintained at 30 Pa, and the shelf temperature was increased from -45°C to -10°C at a rate of 1°C / h, ensuring the material temperature did not exceed its eutectic point temperature. The material was held for 12 hours to complete sublimation drying. The vacuum in the freeze-drying chamber was maintained at 15 Pa, and the shelf temperature was increased to 35°C at a rate of 2°C / h, ensuring the material temperature did not exceed 30°C. The material was held for 8 hours until the moisture content dropped to 1.5%. The material was then allowed to cool naturally to room temperature to complete the desorption drying process, yielding an Eucommia ulmoides extract with intestinal repair function.

[0036] Example 4: This embodiment provides a method for extracting Eucommia ulmoides with intestinal repair function, including the following steps: Step 1: Select fresh Eucommia ulmoides leaves from May, wash them, and dry them using a forced-air drying method at 43℃ for 3.5 hours. The forced-air rate during the drying process is 2.0 m / s. After pulverizing, pass the powder through a 55-mesh sieve to obtain Eucommia ulmoides powder. The moisture content of the powder was measured to be 4.6%. Step 2: Add deionized water to the Eucommia ulmoides powder obtained in Step 1, with a material-to-liquid ratio of 1:22, adjust the pH to 6.8, and extract at a constant temperature of 39℃ for 2.2 hours, stirring intermittently during the extraction. After extraction, filter while hot using a 95-mesh filter cloth to obtain the filter residue and polysaccharide aqueous extract. The residual flavonoid content in the polysaccharide aqueous extract was found to be 7.2%. Step 3: Add β-glucanase to the polysaccharide aqueous extract obtained in Step 2. The enzyme activity is 45,000 U / g, and the amount added is 0.9% of the mass of the polysaccharide aqueous extract. Adjust the temperature to 49℃ and the pH to 6.7 for enzymatic hydrolysis. The hydrolysis time is 2.3h. During the hydrolysis process, the pH and temperature are monitored regularly. If the pH deviates, it is finely adjusted with sodium dihydrogen phosphate-disodium hydrogen phosphate buffer. After the hydrolysis is completed, incubate at 58℃ for 12min to inactivate the enzyme and obtain a small molecule polysaccharide solution. Step 4: The small molecule polysaccharide solution obtained in Step 3 was rapidly cooled to 36°C using an ice-water bath, the pH was adjusted to 6.2, and the solution was transferred to an anaerobic fermenter using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution. The system was evacuated to an oxygen concentration of 0.45%, and nitrogen was introduced to maintain a slight positive pressure of 0.02 MPa at a nitrogen introduction rate of 1.0 L / min. 0.3% glucose was added as an auxiliary carbon source to obtain the adapted polysaccharide solution. Step 5: Add the filter residue obtained in Step 2 to 73% ethanol solution at a material-to-liquid ratio of 1:18, adjust the pH to 5.1, and reflux at 54℃ for 1.7 h. After extraction, filter through a 95-mesh filter cloth to obtain flavonol extract. The residual polysaccharide content in the flavonol extract was found to be 6.5%. Step 6: Mix the adapted polysaccharide solution obtained in Step 4 with the flavonoid alcohol extract obtained in Step 5 at a volume ratio of 1.8:1, and inoculate with a compound probiotic inoculum. The mass ratio of Bifidobacterium, Lactobacillus plantarum, and Lactobacillus acidophilus is 1:2.5, the bacterial activity is 900 million CFU / g, and the inoculum amount is 0.8% of the mass of the mixed solution. Ferment at 36℃ for 14 hours. During the fermentation process, the pH is monitored every 6 hours and adjusted to 5.6 using sodium bicarbonate solution to obtain the fermentation broth. Step 7: The residual flavonoid content detected in Step 2 is 7.2%, which is less than 8%. Eucommia ulmoides flavonoid extract with a purity of 92% is added to the enzymatic hydrolysis system in Step 3. The amount added is 0.2% of the mass of the enzymatic hydrolysis system. Step 8: Add 2.5% diatomaceous earth and 0.45% chitosan to the fermentation broth obtained in Step 6, stir, let stand for 32 min, and filter using a plate and frame filter press; screen the filtrate for 1-5 kDa small molecule polysaccharides through a Sephadex G-25 gel column, and then elute with 53% ethanol solution through an AB-8 macroporous adsorption resin column; concentrate the eluent under reduced pressure at 0.07 MPa and 52℃ to 1 / 9 of the original volume, adjust the pH to 6.6, and add 0.08% fructooligosaccharide; place the material in a vacuum freeze dryer, cool from room temperature to -38℃ at a rate of 3℃ / h, and maintain the temperature for 9 h. The material was then cooled to -52℃ at a rate of 1.2℃ / h and held for 5.5h to complete gradient pre-freezing. Maintaining a vacuum of 15Pa inside the freeze-drying chamber, the shelf temperature was increased from -52℃ to -18℃ at a rate of 0.6℃ / h, while controlling the material temperature to not exceed its eutectic point temperature. The material was held for 20h to complete sublimation drying. Maintaining a vacuum of 8Pa inside the freeze-drying chamber, the shelf temperature was increased to 28℃ at a rate of 1.2℃ / h, while controlling the material temperature to not exceed 30℃. The material was held for 11h until the moisture content dropped to 2.2%. The material was then allowed to cool naturally to room temperature to complete desorption drying, yielding an Eucommia ulmoides extract with intestinal repair function.

[0037] Comparative Example 1: The only difference between this comparative example and Example 2 is that step 7 is not performed; the alcohol extraction is completed directly according to the original process in step 5.

[0038] Expected performance: The residual polysaccharide content in the flavonoid alcohol extract is significantly increased, polysaccharides and flavonoids interfere with each other, the conversion effect of flavonoid aglycones is worse, and the purity of small molecule polysaccharides is reduced.

[0039] Comparative Example 2: The only difference between this comparative example and Example 2 is that short-chain fatty acids are not used to regulate the alcohol extraction process in step 7; instead, polysaccharide residue is controlled by extending the alcohol extraction time.

[0040] Expected performance: The residual polysaccharide content in the flavonoid alcohol extract could not be reduced to the target range, and prolonged extraction time would lead to flavonoid oxidation loss and reduced flavonoid aglycone yield.

[0041] Comparative Example 3: The only difference between this comparative example and Example 3 is that the amount of short-chain fatty acids added in step 7 is insufficient, and the alcohol extraction temperature is not reduced.

[0042] Expected performance: The pH adjustment effect of the alcohol extraction system was poor, the polysaccharide dissolution inhibition effect was insufficient, and the polysaccharide residue in the flavonoid alcohol extract exceeded the standard; the insufficient amount of short-chain fatty acids led to the failure of regulation, and the metabolic efficiency of probiotics decreased and the yield of metabolites decreased during the subsequent fermentation process.

[0043] Comparative Example 4: The only difference between this comparative example and Example 2 is that the residual amount of flavonoids was not detected in step 2, and the alcohol extraction was performed directly according to the fixed process.

[0044] Expected performance: Due to the lack of monitoring of flavonoid residue, targeted regulation was not possible, resulting in large fluctuations in polysaccharide residue in flavonoid alcohol extract and poor process stability; the synergistic effect between small molecule polysaccharides and flavonoids was unbalanced, leading to poor repeatability of intestinal repair function and significant differences in functional effects between different batches of products.

[0045] Comparative Example 5: The only difference between this comparative example and Example 1 is that β-glucanase was not added in step 3, and subsequent fermentation was carried out directly.

[0046] Expected performance: Polysaccharides cannot be molecule-sized and are difficult to be absorbed and utilized by the intestines; during fermentation, polysaccharides cannot be effectively used as a carbon source for probiotics, resulting in poor probiotic proliferation.

[0047] Comparative Example 6: The only difference between this comparative example and Example 2 is that a single Bifidobacterium is used as the fermentation agent in step 6, instead of a compound probiotic agent.

[0048] Expected performance: Single probiotics have limited types of metabolites, reduced production of short-chain fatty acids, and poorer conversion of flavonoid aglycones.

[0049] Comparative Example 7: The only difference between this comparative example and Example 4 is that Eucommia ulmoides flavonoid extract was not added in step 3.

[0050] Expected performance: Polysaccharides are easily oxidized and lost during enzymatic hydrolysis, resulting in poor conversion of small molecule polysaccharides; the antioxidant protection of flavonoids is lost, and the activity of probiotics is inhibited by oxidation products during subsequent fermentation, leading to poor proliferation.

[0051] To compare the performance differences of the Eucommia ulmoides extraction methods with intestinal repair function provided in Examples 1-3 and Comparative Examples 1-6, the present invention provides the following experimental methods: 1. Component conversion and purity detection: Small molecule polysaccharide conversion rate: The content ratio of 1-5kDa polysaccharides before and after enzymatic hydrolysis was determined by high performance gel permeation chromatography (HPGPC), and the conversion rate was calculated.

[0052] Flavonoid aglycone conversion rate: The content of flavonoid aglycones (quercetin and kaempferol) before and after fermentation was determined by high performance liquid chromatography (HPLC), and the conversion rate was calculated.

[0053] Polysaccharide residue: The polysaccharide content in the flavonoid extract was determined by the phenol-sulfuric acid method.

[0054] Extract purity: The mass fractions of total polysaccharides and total flavonoid aglycones in the extract were determined by ultraviolet spectrophotometry, and the total purity was calculated.

[0055] Intestinal repair function test: Mucosal repair rate: Using an in vitro intestinal mucosal epithelial cell (Caco-2 cell) injury model, the cell proliferation rate was detected by the MTT assay, and the 24-hour mucosal repair rate was calculated.

[0056] 2. Experimental Procedure Eucommia extracts were prepared according to the processes of Examples 1-4 and Comparative Examples 1-7, with three samples prepared in parallel for each scheme and numbered for later use.

[0057] Component analysis: Take appropriate amounts of each sample and determine the conversion rate of small molecule polysaccharides, the conversion rate of flavonoid aglycones, the residual amount of polysaccharides, and the purity of extracts according to the above detection methods.

[0058] Intestinal repair function test: Mucosal repair experiment: Caco-2 cells were seeded in 96-well plates. After modeling damage, drug-containing culture media of different samples were added. Cell proliferation rate was detected after 24 hours of culture.

[0059] Data processing: Calculate the mean ± standard deviation (x ± s) of each indicator.

[0060] The experimental data are as follows: Table 1. Results of component transformation, purity, and intestinal repair function testing using different Eucommia ulmoides extraction methods.

[0061] Based on experimental data, the conversion rates of small-molecule polysaccharides in Examples 1-4 all remained above 82%, with Example 3 reaching the highest value. The conversion rates of Comparative Examples 1-7 were generally between 35% and 73%, with Comparative Example 5 being the lowest. The core reason for this difference lies in the synergistic effect of the targeted regulation of flavonoid residue and the enzymatic hydrolysis process of this invention. Comparative Example 5 did not add β-glucanase, and the large-molecule polysaccharide could not be directionally degraded, resulting in a conversion rate of only about 35%, confirming the necessity of the enzymatic hydrolysis step for polysaccharide downsizing. Other comparative examples, lacking targeted regulation of flavonoid residue, either experienced interference between polysaccharides and flavonoids, or insufficient antioxidant protection from flavonoids leading to polysaccharide structure destruction, preventing β-glucanase from functioning efficiently, resulting in significantly lower conversion rates than Examples 1-4. Examples 1-4, through targeted regulation of flavonoid residue, provided a high-quality substrate for enzymatic hydrolysis, ensuring the full conversion of large-molecule polysaccharides into small-molecule forms, thereby achieving high conversion rates.

[0062] The flavonoid aglycone conversion rates in Examples 1-4 were all above 89%, with Example 3 reaching over 92%. In contrast, the conversion rates in Comparative Examples 1-7 ranged from 65% to 78%, with Comparative Examples 2 and 6 showing poor performance. Comparative Example 6 used a single Bifidobacterium fermentation method, lacking the synergistic effect of strains such as Lactobacillus plantarum, thus limiting the flavonoid aglycone conversion efficiency. Comparative Example 2 controlled polysaccharide residue by extending the alcohol extraction time, but this resulted in flavonoid oxidation loss and a decrease in conversion rate. In Examples 1-4, the compound probiotics synergistically metabolized, and the small molecule polysaccharides provided a carbon source for the probiotics, promoting their proliferation and enhancing flavonoid conversion capacity. Simultaneously, the targeted regulation of flavonoid residue in this invention avoided the adsorption hindrance of flavonoids by polysaccharides, allowing flavonoids to fully participate in fermentation and conversion, ultimately achieving a high conversion rate.

[0063] The residual polysaccharide content in Examples 1-4 was all controlled below 6.5%, with Example 3 showing the lowest. In contrast, Comparative Example 1 had a residual content as high as 9.8%, while Comparative Examples 3 and 4 were also above 7%. This data demonstrates the effectiveness of targeted regulation of flavonoid residue in component separation. Comparative Example 1 did not implement targeted regulation of flavonoid residue, resulting in a large amount of polysaccharide dissolution during alcohol extraction, leading to excessive residue. Comparative Example 3, due to insufficient addition of short-chain fatty acids and lack of cooling, could not effectively inhibit polysaccharide dissolution, resulting in persistently high residue. Examples 1-4, in high-residue scenarios, inhibited polysaccharide dissolution by adjusting pH and even simultaneously cooling with short-chain fatty acids. In low-residue scenarios, flavonoid protection prevented excessive polysaccharide release, ensuring that residue was controlled at a low level and clearing obstacles for flavonoid aglycone conversion.

[0064] The total purity of the extracts in Examples 1-4 was all above 86%, with Example 3 reaching over 90%. Comparative Examples 1-7 ranged from 62% to 78%, with Comparative Example 5 exhibiting the lowest purity. The differences in total purity reflect a combination of increased small-molecule polysaccharide conversion rate, improved flavonoid aglycone conversion rate, and reduced polysaccharide residue. Comparative Example 5 suffered from insufficient polysaccharide conversion, while Comparative Example 1 suffered from excessive polysaccharide residue, both resulting in a decreased proportion of active ingredients and lower purity. Examples 1-4, through targeted regulation of flavonoid residue and the synergistic effects of enzymatic hydrolysis and fermentation, not only improved the conversion efficiency of the target active ingredient but also reduced the residue of ineffective impurities, leading to a significant increase in total purity and superior product quality.

[0065] The 24-hour mucosal repair rates of Examples 1-4 were all above 68%, with Example 3 reaching a maximum of over 75%, while the rates of the comparative examples ranged from 27% to 54%, with Comparative Example 5 being the lowest. This difference in data is essentially a result of the synergistic effect of the active ingredients and intestinal adaptation optimization. Comparative Example 5, due to the inability to break down the polysaccharides into smaller molecules, was difficult for the intestines to absorb and utilize, resulting in a repair rate of only about 27%. Comparative Example 6, due to the limited production of short-chain fatty acids resulting from single probiotic fermentation and the lack of synergistic effects from multiple active ingredients, also had a low repair rate. In Examples 1-4, the 1-5kDa small-molecule polysaccharides were easily absorbed by the intestines, flavonoid aglycones exerted anti-inflammatory and antioxidant effects, and short-chain fatty acids promoted mucosal cell proliferation, resulting in a synergistic effect. Simultaneously, the intestinal adaptation step adjusted the pH to a suitable range for the intestines and added fructooligosaccharides, improving the intestinal tolerance and colonization capacity of the extract, further enhancing the repair effect, ultimately achieving a mucosal repair rate far exceeding that of the comparative examples.

[0066] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for extracting Eucommia ulmoides with intestinal repair function, characterized in that, Includes the following steps: Step 1: Select fresh Eucommia ulmoides raw materials, wash them, dry them at low temperature until the moisture content is ≤5%, pulverize and sieve them to obtain Eucommia ulmoides powder; Step 2: Add deionized water to the Eucommia ulmoides powder obtained in Step 1, adjust the pH to 6.8-7.0, and extract at a constant temperature of 38-42℃. After extraction, filter to obtain filter residue and polysaccharide aqueous extract, and detect the flavonoid residue in the polysaccharide aqueous extract. Step 3: Add β-glucanase to the polysaccharide aqueous extract obtained in Step 2, adjust the temperature to 48-52℃ and the pH to 6.6-7.0 for enzymatic hydrolysis, and inactivate the enzyme by raising the temperature after enzymatic hydrolysis to obtain a small molecule polysaccharide solution; Step 4: Cool the small molecule polysaccharide solution obtained in Step 3 and adjust the pH to 6.1-6.

5. Transfer it to an anaerobic fermenter to create an anaerobic environment. Supplement with glucose as an auxiliary carbon source to obtain the adapted polysaccharide solution. Step 5: Add the filter residue obtained in Step 2 to the ethanol solution, adjust the pH to 5.0-5.5, reflux and extract at 53-57℃, filter after extraction to obtain flavonoid alcohol extract; Step 6: Mix the adapted polysaccharide solution obtained in Step 4 with the flavonoid alcohol extract obtained in Step 5 in a certain proportion, and inoculate with a compound probiotic agent composed of Bifidobacterium, Lactobacillus plantarum and Lactobacillus acidophilus for anaerobic fermentation. Monitor and adjust the pH during the fermentation process to obtain the fermentation broth. Step 7: Perform control operations based on the flavonoid residue detected in Step 2: If the flavonoid residue is higher than 10%, use the short-chain fatty acids produced by fermentation in Step 6 to adjust the alcohol extraction process in Step 5, and control the polysaccharide residue ratio in the flavonoid alcohol extract to below 6%; if the flavonoid residue is lower than 8%, add Eucommia ulmoides flavonoid extract to the enzymatic hydrolysis system in Step 3. Step 8: The fermentation broth obtained in Step 6 is subjected to impurity removal, purification, intestinal adaptation, and drying to obtain Eucommia ulmoides extract with intestinal repair function.

2. The method for extracting Eucommia ulmoides with intestinal repair function according to claim 1, characterized in that, In step 7, the short-chain fatty acids produced by fermentation in step 6 are used to adjust the alcohol extraction process in step 5, specifically including the following steps: 1a: Take the fermentation broth from step 6 and centrifuge it at 3000-5000 r / min for 10-15 min to remove the cells and macromolecular impurities, and obtain the fermentation supernatant. 1b: Add 1-2 times its volume of ethyl acetate to the fermentation supernatant, shake and extract 2-3 times, and collect the organic phase; 1c: The organic phase is subjected to vacuum distillation to remove ethyl acetate, yielding a mixture of short-chain fatty acids with a purity ≥85%, wherein the short-chain fatty acid mixture is mainly composed of acetic acid, propionic acid, and butyric acid; 1d: Add the short-chain fatty acid mixture to the ethanol solution in step 5 at 0.2-0.5% of the total mass of the alcohol extraction system in step 5, stir evenly, and lower the pH of the alcohol extraction system to 4.8-5.

2. Continue to maintain the alcohol extraction temperature at 53-57℃ to complete the reflux extraction.

3. The method for extracting Eucommia ulmoides with intestinal repair function according to claim 2, characterized in that, If the residual flavonoid content detected in step 2 is higher than 12%, the alcohol extraction temperature should be simultaneously reduced to 50-53℃ when performing step 1d.

4. The method for extracting Eucommia ulmoides with intestinal repair function according to claim 3, characterized in that, In step 1, the fresh Eucommia ulmoides raw material is fresh Eucommia ulmoides leaves or bark from April to July; the low-temperature drying temperature is 40-50℃ and the drying time is 2-4 hours, and the drying is carried out by forced air drying; after pulverization, it is passed through a 50-70 mesh sieve.

5. The method for extracting Eucommia ulmoides with intestinal repair function according to claim 3, characterized in that, In step 2, the ratio of deionized water to liquid is 1:20-1:30, the constant temperature extraction time is 1.5-2.5h, and the mixture is stirred intermittently during the process; the filter is made of 90-110 mesh filter cloth while the mixture is still hot.

6. The method for extracting Eucommia ulmoides with intestinal repair function according to claim 3, characterized in that, In step 3, the β-glucanase activity is 40,000-60,000 U / g, the addition amount is 0.8-1.5% of the polysaccharide aqueous extract mass, the enzymatic hydrolysis time is 1.5-2.5 h, and the enzyme inactivation conditions are 55-65℃ for 5-15 min. During the enzymatic hydrolysis process, pH and temperature are monitored regularly, and the pH is finely adjusted with sodium dihydrogen phosphate-disodium hydrogen phosphate buffer when it deviates.

7. The method for extracting Eucommia ulmoides with intestinal repair function according to claim 3, characterized in that, In step 4, the temperature is rapidly reduced to 35-39℃ using an ice-water bath; pH is adjusted using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution; the anaerobic environment is constructed by evacuating to an oxygen concentration ≤0.5% and introducing nitrogen to maintain a slight positive pressure; and the glucose supplementation is 0.2-0.5%.

8. The method for extracting Eucommia ulmoides with intestinal repair function according to claim 3, characterized in that, In step 5, the concentration of the added ethanol solution is 70-80%, the material-to-liquid ratio is 1:15-1:25, and the reflux extraction time is 1-2 hours; filtration is carried out using a 90-110 mesh filter cloth.

9. The method for extracting Eucommia ulmoides with intestinal repair function according to claim 3, characterized in that, In step 6, the volume ratio of the adapted polysaccharide solution to the flavonoid alcohol extract is 1.5:1-2.5:1; the mass ratio of Bifidobacterium, Lactobacillus plantarum, and Lactobacillus acidophilus in the compound probiotic agent is 1:2-1:4, with a bacterial activity of 800-1.2 billion CFU / g, and the inoculum amount is 0.5-1.5% of the mass of the mixed solution; the fermentation temperature is 35-39℃, the fermentation time is 8-16h, the pH is monitored regularly, and the pH is adjusted back to 5.5-6.0 using sodium bicarbonate solution.

10. The method for extracting Eucommia ulmoides with intestinal repair function according to claim 3, characterized in that, In step 7, the purity of the added Eucommia ulmoides flavonoid extract is ≥90%, and the amount added is 0.1-0.3% of the mass of the enzymatic hydrolysis system. In step 8, the impurity removal process involves adding 2-4% diatomaceous earth and 0.3-0.7% chitosan, stirring, letting it stand, and then filtering it using a plate and frame filter press. The purification process involves screening the filtrate for 1-5 kDa small molecule polysaccharides using a Sephadex G-25 gel column, followed by elution with 50-60% ethanol solution using an AB-8 macroporous adsorption resin column. The intestinal adaptant is prepared by concentrating the eluent under reduced pressure to 1 / 10-1 / 6 of its original volume, adjusting the pH to 6.5-7.0, and adding 0.05-0.15% fructooligosaccharides. In step 8, the drying process is as follows: the material to be dried is placed in a vacuum freeze-drying oven and cooled from room temperature to -40 to -30°C at a rate of 2-5°C / h and held for 6-10 hours; then cooled to -55 to -45°C at a rate of 1-2°C / h and held for 4-6 hours to complete gradient pre-freezing; subsequently, the vacuum degree in the freeze-drying oven is maintained at 10-30 Pa and the shelf temperature is increased from -55 to -45°C to -20 to -10°C at a rate of 0.5-1°C / h, while controlling the material temperature to not exceed its eutectic point temperature, and held for 12-24 hours until the ice crystals are completely sublimated to complete sublimation drying; finally, the vacuum degree in the freeze-drying oven is maintained at 5-15 Pa and the shelf temperature is increased to 25-35°C at a rate of 1-2°C / h, while controlling the material temperature to not exceed 30°C, and held for 8-12 hours until the material moisture content drops to 1%-3%, and then naturally cooled to room temperature to complete desorption drying.

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