Agaricus bisporus phenolic dietary fiber and preparation method and application thereof

Abstract

This invention discloses a phenolic dietary fiber from *Agaricus bisporus*, its preparation method, and its application. The preparation method includes: obtaining *Agaricus bisporus* powder; defatting the powder under light-protected and nitrogen-filled conditions; suspending the defatted powder in sterile deionized water to obtain a suspension, and inoculating it with *Lactobacillus acidophilus* activation solution for pre-fermentation; directly transferring the pre-fermented suspension to a nitrogen-purged and deoxygenated acetate buffer for enzymatic hydrolysis; rapidly inactivating the enzymes in the hydrolyzed suspension, and then separating and purifying it to obtain the phenolic dietary fiber from *Agaricus bisporus*. This invention provides the application of this phenolic dietary fiber from *Agaricus bisporus* in the preparation of foods, health products, or drugs for colonic antioxidant or anti-inflammatory purposes. This invention solves the problems of easy oxidation and degradation of polyphenols, enabling the phenolic dietary fiber to have a low polyphenol release rate in the upper digestive tract and a sustained polyphenol release during colonic fermentation, exhibiting good antioxidant and anti-inflammatory activity after colonic fermentation.

Description

(I) Technical Field

[0002] This invention belongs to the field of natural product preparation, specifically relating to a phenolic dietary fiber from Agaricus bisporus, its preparation method, and its application. (II) Background Technology

[0004] Button mushrooms (Agaricus bisporus) are one of the most widely cultivated and consumed edible fungi in the world, prized for their delicious taste and rich nutritional value. Modern nutritional and medical research shows that the substrate of button mushrooms not only contains a large amount of high-quality dietary fiber but is also rich in various polyphenolic active ingredients with antioxidant, anti-inflammatory, and intestinal-regulating properties. It is noteworthy that these polyphenols do not exist entirely in a free state; a significant portion is tightly bound to the insoluble dietary fiber backbone of the cell wall through ester bonds, glycosidic bonds, or hydrophobic interactions, forming bound polyphenol-dietary fiber (BP-DF).

[0005] Existing methods for extracting phenolic dietary fiber (such as strong acid and strong alkali hydrolysis) involve harsh conditions that easily damage the molecular structure of polyphenols. Furthermore, during the impurity removal and defatting processes, the heat exposure and oxygen contact during solvent evaporation cause polyphenols to easily oxidize and become inactive, resulting in low bound phenol content and poor activity in the final dietary fiber. To extract phenolic dietary fiber from plant or fungal matrices, researchers both domestically and internationally have conducted numerous explorations.

[0006] Patent CN202511803615.2 discloses the extraction of insoluble dietary fiber by high-pressure homogenization combined with enzymatic method. Although it improves the exposure rate of bound phenols to a certain extent, it is easily caused by severe phenolic hydroxyl self-oxidation when it is in an open air environment for a long time during the pretreatment and hydrolysis process. In addition, the mechanical heat generated during the homogenization process inevitably leads to the structural destruction of some heat-sensitive polyphenols.

[0007] Patent CN202610408580.0 discloses a method for extracting dietary fiber by coupling continuous alkali and acid methods. Such a harsh chemical treatment method is too demanding and can easily cause non-specific breakage of the covalent bonds between dietary fiber and polyphenols, resulting in the loss of a large amount of bound phenols during the washing process.

[0008] Patent CN202510230908.X focuses on using strong alkaline hydrolysis to release bound phenols. Although this method increases the yield of free phenols, it destroys the network structure of dietary fiber and cannot obtain a "dietary fiber-polyphenol" complex carrier with both high functional physicochemical properties.

[0009] Patent CN202411417117.X discloses the preparation of conjugated phenol dietary fiber using specific physical modification combined with enzymatic methods. Although this method increases the exposure of conjugated phenol, it lacks systematic antioxidant protection measures during the defatting and solvent recovery stages. Phenolic hydroxyl groups are prone to enzymatic and non-enzymatic browning when in contact with solvent residues and interfacial air.

[0010] Although patent CN202610153296.3 relates to the preparation of phenolic dietary fiber in the field of edible fungi, its core target is soluble dietary fiber, whose intermolecular interactions and physical network structure are completely different from those of insoluble dietary fiber. In addition, the conventional high-temperature enzyme inactivation technology used in this process has a long duration and slow cooling, which keeps the system in a high-temperature thermodynamic environment in the later stage of enzyme inactivation, making heat-sensitive polyphenols extremely prone to thermal decomposition.

[0011] Therefore, considering the matrix characteristics of button mushrooms, the key technical problem to be solved in the field of targeted high-value utilization of phenolic dietary fiber is to thoroughly remove impurities, maintain the loose and porous microstructure of dietary fiber, retain the natural bioactivity of bound polyphenols to the greatest extent, and actively endow the product with low release in the upper digestive tract and slow release in the colonic fermentation stage through process design. (III) Summary of the Invention

[0013] The first technical problem to be solved by the present invention is to provide a method for preparing phenolic dietary fiber from mushrooms, which solves the problems of easy oxidation and degradation of polyphenols in traditional processes, and ensures the dual stability of the physical structure of dietary fiber and the activity of polyphenols.

[0014] The second problem to be solved by the present invention is to provide a phenolic dietary fiber from Agaricus bisporus prepared according to the preparation method, which has a high total bound phenol content and high dietary fiber purity, and has the characteristics of low polyphenol release rate in the upper digestive tract environment and sustained polyphenol release during colonic fermentation.

[0015] The third technical problem to be solved by the present invention is to provide the application of the phenolic dietary fiber of Agaricus bisporus in the preparation of food, health products or drugs for colonic antioxidant or anti-inflammatory purposes, wherein the phenolic dietary fiber of Agaricus bisporus exhibits good antioxidant and anti-inflammatory activities after colonic fermentation.

[0016] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing phenolic dietary fiber from Agaricus bisporus, comprising the following steps: (1) After washing and slicing, button mushrooms were processed by vacuum freeze-drying and ultra-micro pulverization to obtain powder with a particle size of 60-80 mesh; (2) The button mushroom powder was degreased under light-proof and nitrogen protection to obtain defatted button mushroom powder; (3) The defatted mushroom powder was suspended in sterile deionized water to obtain a suspension, and then inoculated with Lactobacillus acidophilus activation solution for pre-fermentation; (4) The pre-fermented suspension was directly transferred to an acetic acid buffer solution that had been pre-purified with high-purity nitrogen for deoxygenation, and enzymatic hydrolysis was carried out under nitrogen protection to obtain the enzymatically hydrolyzed suspension. (5) Place the enzymatically hydrolyzed suspension at 80-90℃ for rapid enzyme inactivation. After enzyme inactivation, quickly place it in an ice bath to rapidly reduce the core temperature to below 50℃ in a short time. Then, perform low-temperature centrifugation to separate the cooled suspension and collect the precipitate. (6) The precipitate obtained in step (5) is suspended in deionized water and subjected to ultrasonic treatment under ice bath cooling. The suspension after ultrasonic treatment is centrifuged at low temperature and the precipitate is collected. (7) Wash and purify the precipitate obtained in step (6), and then collect the precipitate by centrifugation; (8) The precipitate after washing is freeze-dried again under vacuum. After drying, it is pulverized to obtain phenolic dietary fiber (BP-DF) of Agaricus bisporus with high bound phenol content and high antioxidant activity.

[0017] Preferably, in step (2), the degreasing solvent is petroleum ether, the material-to-liquid ratio (g / mL) is 1:8-1:12, the degreasing process is carried out under light-proof and nitrogen protection conditions, and after filtration and degreasing, the residual petroleum ether in the filter residue is removed by low-temperature nitrogen blowing technology.

[0018] Preferably, in step (3), the ratio (g / mL) of defatted button mushroom powder to sterile water is 1:10-1:20, the inoculum volume of Lactobacillus acidophilus activation solution is 3%-5% of the suspension volume, and the initial viable count of the Lactobacillus acidophilus activation solution is approximately 1×10⁻⁶. 6 -1×10 8 CFU / mL, under anaerobic conditions at 37°C, static fermentation for 4-8 hours. More preferably, the material-to-liquid ratio (g / mL) is 1:15, the inoculum size is 4%, and the initial viable count is approximately 1×10⁻⁶. 7 CFU / mL, pre-fermentation time is 4 hours.

[0019] Preferably, in step (4), the ratio (mL / mL) of the pre-fermented suspension to the acetate buffer is adjusted to 1:15-1:30. Preferably, the enzymatic hydrolysis in step (4) involves sequentially adding α-amylase, papain, and amyloglucosidase. More preferably, the amounts of α-amylase, papain, and amyloglucosidase added are 0.2-0.4% (w / w), 0.4-0.6% (w / w), and 0.1-0.3% (v / v), respectively. Those skilled in the art will understand that enzymatic hydrolysis is typically performed under the optimal pH and temperature conditions for each enzyme. For example, α-amylase derived from Bacillus subtilis has a preferred pH value between 5.0 and 6.0 and a preferred temperature between 50 and 60°C; papain derived from papaya latex has a preferred pH value between 5.0 and 7.0 and a preferred temperature between 55 and 60°C; and amylase derived from Aspergillus niger has a preferred pH value between 4.0 and 5.0 and a preferred temperature between 55 and 65°C. In a preferred embodiment of the present invention, the enzymatic hydrolysis is performed as follows: the pH of the acetate buffer is adjusted to 6.0, 0.3% (w / w) of α-amylase is added under nitrogen protection, and the enzymatic hydrolysis reaction is carried out in a water bath at 60°C for 30 minutes. Then, 0.5% (w / w) of papain is added, and the enzymatic hydrolysis reaction is carried out in a water bath at 60°C for another 30 minutes. The pH is then adjusted to 4.3-4.7, and 0.2% (v / v) of amylase is added to continue the enzymatic hydrolysis for 30 minutes.

[0020] Preferably, in step (6), the ratio of the precipitate to deionized water (g / mL) is 1:20-1:30. Preferably, the ultrasonic treatment conditions in step (6) are: ultrasonic power 400-600W, ultrasonic time 30 minutes, and working mode of ultrasonic 3 seconds interval 2 seconds.

[0021] Preferably, the washing and purification in step (6) is performed by washing thoroughly with deionized water, 80% ethanol aqueous solution and 70% acetone solution in sequence, and collecting the precipitate by low-temperature centrifugation after each washing.

[0022] The "low-temperature centrifugation" mentioned in this invention usually refers to centrifugation at 4°C.

[0023] In a second aspect, the present invention provides a phenolic dietary fiber from Agaricus bisporus prepared according to the preparation method described in the first aspect, wherein the total bound phenol content is ≥20mg / g and the dietary fiber purity is ≥85%.

[0024] Preferably, the water-holding capacity of the phenolic dietary fiber in the mushroom is ≥8.0 g / g, and the swelling capacity is ≥9.5 mL / g.

[0025] Thirdly, the present invention provides the application of the button mushroom phenolic dietary fiber described in the second aspect in the preparation of foods, health products or medicines for colonic antioxidant or anti-inflammatory purposes.

[0026] In some embodiments, the amount of polyphenols released from the upper digestive tract of the mushroom phenolic dietary fiber after simulated oral-gastric-small intestine digestion in vitro is ≤350 mgGAE / 100gDW, and the amount of polyphenols released continuously increases during in vitro colonic fermentation from 0 to 48 hours.

[0027] By employing the above-described technology, the beneficial effects of the present invention compared to the prior art are as follows: 1. This invention combines short-term pre-fermentation of Lactobacillus acidophilus with nitrogen protection throughout the process, and uses rapid enzyme inactivation followed by low-temperature cooling to reduce the oxidative loss of bound polyphenols during the preparation process.

[0028] Specifically, during the pre-fermentation stage, the short-term anaerobic metabolism of *Lactobacillus acidophilus* consumes the residual dissolved oxygen in the *Agaricus bisporus* suspension system. Simultaneously, the generated lactic acid lowers the system pH to approximately 5.0. This pH range is lower than the optimal pH for *Agaricus bisporus* endogenous polyphenol oxidase, inhibiting enzymatic oxidation. The weakly acidic, low-oxygen environment provides favorable conditions for subsequent mild enzymatic hydrolysis and retention of bound phenols. Subsequent enzymatic hydrolysis is conducted entirely under nitrogen protection. The pre-fermentation suspension is directly transferred to a pre-deoxygenated buffer system, which helps reduce the re-entry of atmospheric oxygen into the system, maintaining the low-oxygen protection state and reducing the risk of oxidative loss of bound phenols. In the enzyme inactivation stage, this invention employs rapid enzyme inactivation followed by immediate ice bath cooling, rapidly lowering the core temperature of the material. This shortens the time polyphenols are exposed to higher temperatures, reducing the risk of oxidation and thermal degradation of heat-sensitive phenolic substances. The total bound phenol content of the phenolic dietary fiber in Agaricus bisporus prepared using the above process can reach 25.4 mg / g, indicating that the combination of short-time biological pre-fermentation, multi-stage nitrogen protection, and rapid cooling treatment is beneficial to improving the stability and retention of bound polyphenols during the preparation process.

[0029] 2. This invention improves the purity of dietary fiber and its hydration properties through mild enzymatic hydrolysis and low-temperature intermittent ultrasonic treatment.

[0030] This invention employs a stepwise enzymatic hydrolysis using α-amylase, papain, and amyloglucosidase under gentle conditions to remove starch, protein, and dextrin impurities, avoiding excessive damage to the dietary fiber skeleton and the loss of large amounts of bound polyphenols caused by strong acid or alkali treatments. By controlling the reaction conditions of different enzymes stepwise, the complex state between dietary fiber and bound polyphenols can be maintained while improving the impurity removal effect. The precipitate obtained from enzymatic hydrolysis is further subjected to intermittent ultrasonic treatment under ice bath cooling conditions, which helps to disperse fiber aggregates, promotes the appropriate unfolding of fiber structure, and increases the fiber matrix's ability to bind and retain water. In the product obtained by the above method, the purity of dietary fiber can reach 88.9%, and the water-holding capacity and swelling capacity can reach 8.5 g / g and 10.8 mL / g, respectively, indicating that the combination of gentle stepwise enzymatic hydrolysis and low-temperature intermittent ultrasonic treatment can simultaneously achieve impurity removal and purification, structural regulation, and improvement of hydration properties.

[0031] 3. The phenolic dietary fiber from Agaricus bisporus obtained by this invention can reduce the premature release of polyphenols in the upper digestive tract during in vitro simulated digestion, and exhibits a continuous release characteristic in the colonic fermentation stage.

[0032] In the product obtained by this invention, polyphenols mainly exist in a bound state within the insoluble dietary fiber backbone. During in vitro simulated oral, gastric, and small intestinal digestion, digestive enzymes in the upper digestive tract are unable to fully break down the ester bonds, glycosidic bonds, and non-covalent interactions between polyphenols and dietary fiber. Therefore, premature release of polyphenols in the oral, gastric, and small intestinal stages can be reduced, allowing a significant portion of the bound polyphenols to enter the subsequent colonic fermentation system along with undigested fiber residue. During anaerobic colonic fermentation, as intestinal microorganisms gradually utilize and degrade the dietary fiber backbone, polyphenols originally embedded or bound in the fiber matrix can be gradually released, causing the phenolic content in the fermentation broth to continuously increase with prolonged fermentation time. This release behavior balances the stable retention in the upper digestive tract stage with the gradual release in the colonic fermentation stage, which is beneficial for prolonging the action time of phenolic active substances in the colonic environment and improving the colonic utilization potential of polyphenols bound to Agaricus bisporus.

[0033] 4. The phenolic dietary fiber from Agaricus bisporus obtained in this invention exhibits good antioxidant and anti-inflammatory activity after colonic fermentation.

[0034] This invention improves the retention of bound polyphenols during preparation and allows for their gradual release during colonic fermentation, resulting in a higher level of phenolic active substances and their fermentation products in the obtained colonic fermentation broth. Results show that after 48 hours of colonic fermentation, the DPPH free radical scavenging rate of the fermentation broth reaches 77.38%, indicating good free radical scavenging ability. In an LPS-induced RAW264.7 macrophage inflammation model, the obtained colonic fermentation broth inhibited excessive NO release by 59.22%, demonstrating its ability to suppress abnormal NO production caused by inflammatory stimulation to a certain extent. These results indicate that the mild preparation and activity protection process employed in this invention is beneficial for improving the retention of bound polyphenols in the phenolic dietary fiber of *Agaricus bisporus* and endowing the product with suitable digestion-fermentation release behavior, resulting in good antioxidant and anti-inflammatory activities after colonic fermentation. (iv) Description of the attached drawings

[0036] Figure 1 This refers to the amount of polyphenols released from the samples of Examples 1-3 and Comparative Examples 1-3 after digestion and fermentation. Figure 2 The DPPH free radical scavenging rate of the fermentation broth after 48 hours of colonic fermentation; Figure 3 The inhibition rate of colonic fermentation broth on LPS-induced NO release from RAW264.7 cells is [value missing]. (V) Detailed Implementation Methods

[0038] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the technical solution of the present invention will be described in detail below with reference to embodiments. The specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0039] Sources of reagents used in the examples and comparative examples: Sodium hydroxide: AR, purchased from Sinopharm Chemical Reagent Co., Ltd.; Hydrochloric acid: AR, purchased from Sinopharm Chemical Reagent Co., Ltd.; Ethanol: AR, purchased from Sinopharm Chemical Reagent Co., Ltd.; Methanol: HPLC grade, Sigma-Aldrich (Shanghai) Trading Co., Ltd.; Acetone: AR, purchased from Sinopharm Chemical Reagent Co., Ltd.; Petroleum ether: AR, purchased from Sinopharm Chemical Reagent Co., Ltd.; Lactobacillus acidophilus, purchased from China Industrial Microbial Culture Collection Center; α-Amylase: derived from Bacillus subtilis, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number A800731-100g; Papain: Derived from papaya latex, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number P799531-25g; Amylase: derived from Aspergillus niger, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number A800618-10mL; Artificial saliva: purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number R22154; Artificial gastric fluid: purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number R30386; Artificial small intestinal fluid: purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number R22156.

[0040] Example 1: Preparation of BPDF with Optimal Parameters

[0041] (1) Take 500g of fresh button mushrooms, wash them with deionized water, cut them into thin slices about 3mm thick, and freeze-dry them under vacuum at -50℃ and 0.1mbar for 48 hours. The freeze-dried sample was pulverized using an ultra-micro pulverizer under circulating cooling at -20℃ and passed through a 60-mesh sieve to obtain about 45g of freeze-dried powder. The entire process was carried out in the dark.

[0042] (2) Add the freeze-dried powder to 450 mL of petroleum ether at a ratio of 1:10 (g / mL), bubble with high-purity nitrogen to maintain an inert atmosphere, stir for 2 hours at room temperature (25°C) in the dark, filter, and repeat the degreasing process twice. Dry the filter residue at low temperature under nitrogen protection until there is no residual petroleum ether odor, to obtain approximately 38 g of degreased powder.

[0043] (3) Inoculate the frozen Lactobacillus acidophilus culture into MRS liquid medium and incubate under anaerobic conditions at 37°C for 18-24 hours until the bacterial culture OD reaches 100%. 600 The value reaches 1.0-1.5. Take the above culture medium and transfer it to fresh MRS liquid medium at an inoculation rate of 1% (v / v). Incubate under the same conditions for 12 hours for secondary activation. After activation, centrifuge the bacterial solution at 4℃ and 5000×g for 10 minutes, discard the supernatant, wash the bacterial precipitate twice with sterile physiological saline (0.85% NaCl), and finally resuspend it in sterile deionized water to the initial volume to obtain the Lactobacillus acidophilus activated solution. The initial viable count is approximately 1×10⁻⁶. 7 CFU / mL. The defatted powder was suspended in 570 mL of sterile deionized water at a material-to-liquid ratio of 1:15 (g / mL), inoculated with 4% Lactobacillus acidophilus activation solution, and fermented under anaerobic conditions at 37℃ for 4 hours. The pH of the suspension at the end of fermentation was 5.0.

[0044] (4) The fermentation suspension was directly transferred to a 0.1 mol / L acetate buffer solution that had been pre-purified with high-purity nitrogen for 30 minutes. The pH of the buffer solution was 6.0. The ratio of the fermentation suspension to the acetate buffer solution (mL / mL) was adjusted to 1:20. Under nitrogen protection, 0.3% (w / w) of α-amylase was added sequentially and enzymatically hydrolyzed at 60°C for 30 minutes, followed by 0.5% (w / w) of papain and enzymatic hydrolysis at 60°C for another 30 minutes. Subsequently, the pH of the system was adjusted to 4.5 with 1 mol / L hydrochloric acid solution, and 0.2% (v / v) of amyloglucosidase was added and enzymatically hydrolyzed at 60°C for 30 minutes.

[0045] (5) The enzyme-inactivated suspension was kept in an 85°C water bath for 10 minutes. After enzyme inactivation, it was immediately cooled in an ice bath to reduce the core temperature to below 50°C within 3 minutes. Then it was centrifuged at 4°C and 10,000×g for 15 minutes and the precipitate was collected.

[0046] (6) The precipitate was suspended in deionized water at a material-to-liquid ratio (g / mL) of 1:30 and subjected to ultrasonic treatment under ice bath cooling. The ultrasonic power was 500W, the ultrasonic time was 30 minutes, and the working mode was 3 seconds of ultrasonic treatment followed by 2 seconds of intermittent treatment. The ultrasonically treated suspension was centrifuged at 4℃ and 10000×g for 15 minutes, and the precipitate was collected.

[0047] (7) The precipitate was washed twice each with deionized water, 80% ethanol aqueous solution, and 70% acetone aqueous solution. Each time, the precipitate was collected by centrifugation at 4°C and 10000×g for 10 minutes. The final precipitate was freeze-dried under vacuum at -50°C and 0.1mbar for 48 hours. After pulverization, the phenolic dietary fiber product of Agaricus bisporus was obtained and designated as BPDF-1.

[0048] Example 2: Preparation of BPDF with lower parameters

[0049] (1) Take 500g of fresh button mushrooms, wash them with deionized water, cut them into thin slices about 3mm thick, and freeze-dry them under vacuum at -50℃ and 0.1mbar for 48 hours. The freeze-dried sample was pulverized using an ultra-micro pulverizer under circulating cooling at -20℃ and passed through a 60-mesh sieve to obtain about 45g of freeze-dried powder. The entire process was carried out in the dark.

[0050] (2) Add the freeze-dried powder to 450 mL of petroleum ether at a ratio of 1:10 (g / mL), bubble with high-purity nitrogen to maintain an inert atmosphere, stir for 2 hours at room temperature (25°C) in the dark, filter, and repeat the degreasing process twice. Dry the filter residue at low temperature under nitrogen protection until there is no residual petroleum ether odor, to obtain approximately 38 g of degreased powder.

[0051] (3) Inoculate the frozen Lactobacillus acidophilus culture into MRS liquid medium and incubate under anaerobic conditions at 37°C for 18-24 hours until the bacterial culture OD reaches 100%. 600The value reaches 1.0-1.5. Take the above culture medium and transfer it to fresh MRS liquid medium at an inoculation rate of 1% (v / v). Incubate under the same conditions for 12 hours for secondary activation. After activation, centrifuge the bacterial solution at 4℃ and 5000×g for 10 minutes, discard the supernatant, wash the bacterial precipitate twice with sterile physiological saline (0.85% NaCl), and finally resuspend it in sterile deionized water to the initial volume to obtain the Lactobacillus acidophilus activated solution. The initial viable count is approximately 1×10⁻⁶. 7 CFU / mL. The defatted powder was suspended in 570 mL of sterile deionized water at a material-to-liquid ratio of 1:15 (g / mL), inoculated with 3% Lactobacillus acidophilus activation solution, and fermented under anaerobic conditions at 37℃ for 4 hours. The pH of the suspension at the end of fermentation was 5.0.

[0052] (4) The fermentation suspension was directly transferred to a 0.1 mol / L acetate buffer solution that had been pre-purified with high-purity nitrogen for 30 minutes. The pH of the buffer solution was 6.0. The ratio of the fermentation suspension to the acetate buffer solution (mL / mL) was adjusted to 1:20. Under nitrogen protection, 0.3% (w / w) of α-amylase was added sequentially and enzymatically hydrolyzed at 60°C for 30 minutes, followed by 0.5% (w / w) of papain and enzymatic hydrolysis at 60°C for another 30 minutes. Subsequently, the pH of the system was adjusted to 4-5 with 1 mol / L hydrochloric acid solution, and 0.2% (v / v) of amyloglucosidase was added and enzymatically hydrolyzed at 60°C for 30 minutes.

[0053] (5) The enzyme-inactivated suspension was kept in an 85°C water bath for 10 minutes. After enzyme inactivation, it was immediately cooled in an ice bath to reduce the core temperature to below 50°C within 3 minutes. Then it was centrifuged at 4°C and 10,000×g for 15 minutes and the precipitate was collected.

[0054] (6) The precipitate was suspended in deionized water at a material-to-liquid ratio (g / mL) of 1:30 and subjected to ultrasonic treatment under ice bath cooling. The ultrasonic power was 400W, the ultrasonic time was 30 minutes, and the working mode was 3 seconds of ultrasonic treatment followed by 2 seconds of intermittent treatment. The ultrasonically treated suspension was centrifuged at 4℃ and 10000×g for 15 minutes, and the precipitate was collected.

[0055] (7) The precipitate was washed twice each with deionized water, 80% ethanol aqueous solution, and 70% acetone aqueous solution. Each time, the precipitate was collected by centrifugation at 4°C and 10000×g for 10 minutes. The final precipitate was freeze-dried under vacuum at -50°C and 0.1mbar for 48 hours. After pulverization, the product was obtained and designated as BPDF-2.

[0056] Example 3: Preparation of BPDF with High Limit Parameters

[0057] (1) Take 500g of fresh button mushrooms, wash them with deionized water, cut them into thin slices about 3mm thick, and freeze-dry them under vacuum at -50℃ and 0.1mbar for 48 hours. The freeze-dried sample was pulverized using an ultra-micro pulverizer under circulating cooling at -20℃ and passed through a 60-mesh sieve to obtain about 45g of freeze-dried powder. The entire process was carried out in the dark.

[0058] (2) Add the freeze-dried powder to 450 mL of petroleum ether at a ratio of 1:10 (g / mL), bubble with high-purity nitrogen to maintain an inert atmosphere, stir for 2 hours at room temperature (25°C) in the dark, filter, and repeat the degreasing process twice. Dry the filter residue at low temperature under nitrogen protection until there is no residual petroleum ether odor, to obtain approximately 38 g of degreased powder.

[0059] (3) Inoculate the frozen Lactobacillus acidophilus culture into MRS liquid medium and incubate under anaerobic conditions at 37°C for 18-24 hours until the bacterial culture OD reaches 100%. 600 The value reaches 1.0-1.5. Take the above culture medium and transfer it to fresh MRS liquid medium at an inoculation rate of 1% (v / v). Incubate under the same conditions for 12 hours for secondary activation. After activation, centrifuge the bacterial solution at 4℃ and 5000×g for 10 minutes, discard the supernatant, wash the bacterial precipitate twice with sterile physiological saline (0.85% NaCl), and finally resuspend it in sterile deionized water to the initial volume to obtain the Lactobacillus acidophilus activated solution. The initial viable count is approximately 1×10⁻⁶. 7 CFU / mL. The defatted powder was suspended in 570 mL of sterile deionized water at a material-to-liquid ratio of 1:15 (g / mL), inoculated with 5% Lactobacillus acidophilus activation solution, and fermented under anaerobic conditions at 37℃ for 8 hours. The pH of the suspension at the end of fermentation was 5.0.

[0060] (4) The fermentation suspension was directly transferred to a 0.1 mol / L acetate buffer solution that had been pre-purified with high-purity nitrogen for 30 minutes. The pH of the buffer solution was 6.0. The ratio of the fermentation suspension to the acetate buffer solution (mL / mL) was adjusted to 1:20. Under nitrogen protection, 0.3% (w / w) of α-amylase was added sequentially and enzymatically hydrolyzed at 60°C for 30 minutes, followed by 0.5% (w / w) of papain and enzymatic hydrolysis at 60°C for another 30 minutes. Subsequently, the pH of the system was adjusted to 4-5 with 1 mol / L hydrochloric acid solution, and 0.2% (v / v) of amyloglucosidase was added and enzymatically hydrolyzed at 60°C for 30 minutes.

[0061] (5) The enzyme-inactivated suspension was kept in an 85°C water bath for 10 minutes. After enzyme inactivation, it was immediately cooled in an ice bath to reduce the core temperature to below 50°C within 3 minutes. Then it was centrifuged at 4°C and 10,000×g for 15 minutes and the precipitate was collected.

[0062] (6) The precipitate was suspended in deionized water at a material-to-liquid ratio (g / mL) of 1:30 and subjected to ultrasonic treatment under ice bath cooling. The ultrasonic power was 600W, the ultrasonic time was 30 minutes, and the working mode was 3 seconds of ultrasonic treatment followed by 2 seconds of intermittent treatment. The ultrasonically treated suspension was centrifuged at 4℃ and 10000×g for 15 minutes, and the precipitate was collected.

[0063] (7) The precipitate was washed twice each with deionized water, 80% ethanol aqueous solution, and 70% acetone aqueous solution. Each time, the precipitate was collected by centrifugation at 4°C and 10000×g for 10 minutes. The final precipitate was freeze-dried under vacuum at -50°C and 0.1mbar for 48 hours. After pulverization, the product was obtained and designated as BPDF-3.

[0064] Comparative Example 1

[0065] Steps (1) and (2) are the same as in Example 1.

[0066] (3) The defatted powder obtained in step (2) was added to a 4 mol / L NaOH solution at a material-to-liquid ratio of 1:20 (g / mL). After hydrolysis in the dark at 25°C for 4 hours, the solution was centrifuged at 10000×g for 15 minutes at 4°C, and the precipitate was collected. The precipitate was washed twice each with deionized water, 80% ethanol aqueous solution, and 70% acetone aqueous solution, and then freeze-dried under vacuum at -50°C and 0.1mbar for 48 hours. After pulverization, the product was obtained. The total phenol content of the sample was less than 0.5 mg / g according to the Folin-Ciocalteu method. It can be regarded as a dietary fiber carrier that has been decontaminated with polyphenols and is denoted as IDF.

[0067] Comparative Example 2

[0068] Steps (1) and (2) are the same as in Example 1.

[0069] (3) Add the defatted powder obtained in step (2) to 80% methanol (v / v) at a material-to-liquid ratio (g / mL) of 1:20. Mix the mixture evenly by sonication at 25°C for 30 min. Centrifuge (4°C, 6000×g, 10 min) to collect the supernatant. Extract the residue twice more. Place the supernatant in a rotary evaporator and concentrate it at 35°C until dry. Redissolve the dried extract in 10 mL of 80% methanol aqueous solution and freeze-dry. This extract is denoted as FP.

[0070] Comparative Example 3

[0071] This comparative example is basically the same as Example 1, except that the short-term pre-fermentation treatment of Lactobacillus acidophilus in step (3) and the ultrasonic treatment in step (6) are omitted, and nitrogen gas is no longer used for protection during the extraction process. Specifically: during the defatting process, high-purity nitrogen gas is not introduced, and the filter residue is not dried at low temperature by nitrogen gas flow, but directly dried overnight in a fume hood; the defatted button mushroom powder obtained in step (2) is suspended in sterile deionized water at a material-to-liquid ratio (g / mL) of 1:15, and instead of inoculating with Lactobacillus acidophilus activation solution, sterile deionized water of the same volume as the inoculation amount of Lactobacillus acidophilus activation solution is added, and the mixture is left to stand for 4 hours under anaerobic conditions at 37°C; in step (4), high-purity nitrogen gas is not introduced into the acetic acid buffer to remove oxygen beforehand, and the enzymatic hydrolysis process is not protected by nitrogen gas, but is carried out in an air environment; then, the enzymatic hydrolysis without nitrogen gas protection, rapid enzyme inactivation, and ice bath quenching are carried out according to steps (4) to (5) of Example 1, and the precipitate obtained by centrifugation in step (5) is directly washed, freeze-dried and pulverized according to step (7), and the resulting product is denoted as BPDF-4.

[0072] The methods for testing the phenolic dietary fiber content of *Agaricus bisporus* prepared in the embodiments and comparative examples of this invention are as follows: 1. Combining phenol content and basic physicochemical properties The bound phenol content, dietary fiber purity, water-holding capacity, and swelling power of Examples 1-3 and Comparative Example 3 were determined to verify the feasibility of the three parameter combinations and the overall effect of the complete and mild protective process. (1) Determination of total bound phenol content The dried phenolic dietary fiber was added to 5 mol / L NaOH solution at a material-to-liquid ratio of 1:20 (g / mL) and hydrolyzed in the dark under a nitrogen atmosphere for 4 h. After alkaline hydrolysis, the pH was adjusted to 2.0 with 6 mol / L HCl solution. After centrifugation (10000×g, 4℃, 10 min), the supernatant was collected and extracted five times with an equal volume of ethyl acetate. The combined extracts were rotary evaporated to dryness at 35°C and reconstituted with pre-cooled 80% methanol aqueous solution to a final volume of 10 mL. Using gallic acid as a standard, the absorbance was measured at 760 nm using the Folin-Ciocalteu method to calculate the total bound phenol content.

[0073] (2) Dietary fiber purity

[0074] The content of insoluble dietary fiber was determined according to the method in GB5009.88-2014 "Determination of Dietary Fiber in Food".

[0075] (3) Water holding capacity

[0076] The method for determining the water adsorption capacity of the sample is as follows: Weigh 0.05g of sample (W1) and 5mL of water into a centrifuge tube, shake at 37℃ for 1h, take it out and centrifuge (8000r / min, 15min), discard the supernatant and weigh it as W2. The water holding capacity of the sample is calculated by formula (1).

[0077] (1)

[0078] (4) Expansion force

[0079] Weigh 0.5g of sample (W) into a 10mL graduated cylinder, record the dry volume V1, add 5mL of deionized water, let stand at 37℃ for 24h, record the wet volume V2, and calculate the expansion force of the sample using formula (2).

[0080] (2)

[0081] Table 1 Total phenol content and physicochemical properties of each sample

[0082] As shown in Table 1, the total bound phenol content and basic physicochemical properties of the button mushroom dietary fiber obtained in Examples 1-3 were not significantly different, but were all significantly higher than those in Comparative Example 3. This is because Comparative Example 3 lacked biological deoxygenation and weak acid protection, and also lacked nitrogen protection, which increased the oxidation loss of bound phenols due to the presence of oxygen. In addition, the structure was not loose enough due to the lack of ultrasonic treatment, and the water holding capacity and swelling capacity should be significantly lower than those in Examples 1-3.

[0083] 2. In vitro simulated digestion and colonic fermentation

[0084] Take 1g of each sample from Examples 1-3 and Comparative Examples 1-3, dissolve them in 4mL of water, and then perform the following treatments sequentially: Simulated oral digestion: Add 5 mL of artificial saliva and shake at 37°C for 2 minutes; Simulated gastric digestion: Add 10 mL of artificial gastric juice (containing pepsin), adjust the pH to 2.5 with 0.1 mol / L HCl, and shake at 37°C for 2 hours; Simulated small intestinal digestion: Adjust the pH to 6.8 with 0.1 mol / L NaOH, add 20 mL of artificial small intestinal fluid (containing pancreatic enzymes and bile salts), and shake at 37°C for 2 hours.

[0085] After digestion, immediately remove the digestive fluid and place it in an ice bath for 10 minutes to stop small intestinal digestion. Centrifuge for 10 minutes (4℃, 8000×g) and collect the precipitate. Take the supernatant to determine the polyphenol content and calculate the cumulative release rate of polyphenols in the upper digestive tract.

[0086] The precipitate was transferred to an anaerobic digester and inoculated with a 10% (v / v) suspension of fecal microbiota from healthy volunteers (prepared with 0.1 mol / L sterile PBS buffer, pH 7.0). Fermentation was carried out at 37°C for 48 hours under anaerobic conditions. Samples were taken at 0, 6, 12, 24, and 48 hours of fermentation, centrifuged for 10 minutes (4°C, 8000×g), and the supernatant was used for subsequent analysis. Fecal samples from six healthy adults who had not taken any antibiotics in the past three months and who had provided informed consent from all participants.

[0087] 3. Polyphenol release curve determination

[0088] Fermentation supernatant was collected at each time point, and the total phenol content was determined according to the Folin-Ciocalteu method. A graph showing the changes in polyphenol release was then plotted.

[0089] Depend on Figure 1 It can be seen that the polyphenol release amounts in Examples 1-3 after the completion of digestion in the upper gastrointestinal tract were all below 350 mg GAE / 100 g DW, and showed a continuous increasing trend during colonic fermentation from 0 to 48 hours. This indicates that the inoculum size, pre-fermentation time, and ultrasonic power range specified in this invention can impart good upper gastrointestinal stability and sustained colonic release performance to the obtained phenolic dietary fiber. Although there are some differences among the three examples, they all maintain the same digestion-fermentation release trend and are significantly better than Comparative Example 3. The IDF obtained in Comparative Example 1 is dephenolized dietary fiber, which contains almost no bound phenols, so the release amount remains at a low level throughout the digestion process. The FP obtained in Comparative Example 2 is a crude extract of free phenols, and its polyphenols are mainly released rapidly in the upper gastrointestinal stage, making it difficult to maintain sustained release during the colonic fermentation stage. The phenol release amount of BPDF-4 obtained in Comparative Example 3 is lower than that in Example 1 throughout the digestion process, indicating that the lack of pre-fermentation, nitrogen protection, and low-temperature intermittent ultrasonic treatment is not conducive to the preservation of bound phenols and their sustained release during colonic fermentation.

[0090] 4. Determination of antioxidant activity of colon fermentation broth

[0091] The supernatant of Example 1 and Comparative Examples 1-3 was fermented in the colon for 48 hours, and the antioxidant activity was determined using the DPPH free radical scavenging ability kit from Nanjing Jiancheng Biotechnology Institute.

[0092] Depend on Figure 2 It can be seen that after 48 hours of colonic fermentation, the BPDF-1 obtained in Example 1 exhibited a DPPH free radical scavenging rate of 77.38% in the fermentation supernatant, which was higher than that of Comparative Examples 1, 2, and 3. Comparative Example 1, being IDF, lacked bound phenols, resulting in lower antioxidant capacity in its colonic fermentation broth. Comparative Example 2, being FP, had free phenols primarily released in the upper digestive tract, making it difficult to exert significant antioxidant capacity in the colon. Although the DPPH free radical scavenging rate of Comparative Example 3 was higher than that of Comparative Examples 1 and 2, it was significantly lower than that of Example 1.

[0093] The above results indicate that the complete process consisting of short-term pre-fermentation of Lactobacillus acidophilus, nitrogen protection, and low-temperature intermittent ultrasound is beneficial to improving the retention of bound phenols and enhancing the antioxidant activity of BPDF in the colon.

[0094] 5. Determination of anti-inflammatory activity of colon fermentation broth

[0095] RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% FBS at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were harvested and cultured at a rate of 2 × 10⁻⁶ cells / cells. 5 Inoculate at a density of 100 μL / well in 96-well plates and incubate for 24 hours.

[0096] Discard the supernatant and set up the following groups: Control group: Add 100 μL of complete culture medium; Model group: Add 100 μL of complete culture medium containing LPS (final concentration 1 μg / mL); Sample groups: 100 μL of complete culture medium containing LPS (final concentration 1 μg / mL) and the supernatant of each sample after 48 h of colon fermentation (diluted to 50 μg / mL with complete culture medium) was added respectively.

[0097] Each group was divided into 3 replicates, and cultured at 37℃ for 24 hours. Cell supernatant was collected, and NO content was determined according to the instructions of the NO assay kit from Jiangsu Kaiji Biotechnology Co., Ltd. NO inhibition rate was calculated using the following formula: NO inhibition rate (%) = (NO content in the model group - NO content in the sample group) / NO content in the model group × 100% Depend on Figure 3 It can be seen that the colonic fermentation broth of Example 1 inhibited LPS-induced NO release from RAW264.7 cells by 59.22%, which was higher than that of the comparative examples. Comparative Example 1, lacking bound phenols, showed a weaker inhibitory effect on LPS-induced NO release; in Comparative Example 2, free phenols were mainly released in the upper digestive tract, with fewer phenolic substances reaching the colon to participate in fermentation. The lower NO inhibition rate of Comparative Example 3 indicates that the product obtained without short-term pre-fermentation with Lactobacillus acidophilus, nitrogen protection, and low-temperature intermittent ultrasonic treatment still possesses some anti-inflammatory activity, but its effect is significantly weaker than that of BPDF-1 obtained from the complete process.

[0098] Therefore, the NO inhibition capacity of the colonic fermentation broth samples was ranked as follows: Example 1 > Comparative Example 3 > Comparative Example 2 > Comparative Example 1. This result is basically consistent with the polyphenol release behavior and the trend of antioxidant activity of the colonic fermentation broth, indicating that a complete and mild protective process is beneficial to improving the antioxidant and anti-inflammatory effects of BPDF in the colon.

Claims

1. A method for preparing phenolic dietary fiber from Agaricus bisporus, characterized in that: The preparation method includes the following steps: (1) After washing and slicing, button mushrooms were processed by vacuum freeze-drying and ultra-micro pulverization to obtain powder with a particle size of 60-80 mesh; (2) The button mushroom powder was degreased under light-proof and nitrogen protection to obtain defatted button mushroom powder; (3) The defatted mushroom powder was suspended in sterile deionized water to obtain a suspension, and then inoculated with Lactobacillus acidophilus activation solution for pre-fermentation; (4) The pre-fermented suspension was directly transferred to an acetic acid buffer solution that had been pre-purified with high-purity nitrogen for deoxygenation, and enzymatic hydrolysis was carried out under nitrogen protection to obtain the enzymatically hydrolyzed suspension. (5) Place the enzymatically hydrolyzed suspension at 80-90℃ for rapid enzyme inactivation. After enzyme inactivation, quickly place it in an ice bath to rapidly reduce the core temperature to below 50℃ in a short time. Then, perform low-temperature centrifugation to separate the cooled suspension and collect the precipitate. (6) The precipitate obtained in step (5) is suspended in deionized water and subjected to ultrasonic treatment under ice bath cooling. The suspension after ultrasonic treatment is centrifuged at low temperature and the precipitate is collected. (7) Wash and purify the precipitate obtained in step (6), and then collect the precipitate by centrifugation; (8) The precipitate after washing is freeze-dried again under vacuum, and then pulverized to obtain the phenolic dietary fiber of button mushroom.

2. The preparation method according to claim 1, characterized in that: In step (2), the degreasing solvent is petroleum ether.

3. The preparation method according to claim 1, characterized in that: In step (3), the ratio of defatted button mushroom powder to sterile deionized water is 1:10-1:20, the inoculum volume of Lactobacillus acidophilus activation solution is 3%-5% of the suspension volume, and the initial viable count of the Lactobacillus acidophilus activation solution is 1×10⁻⁶. 6 -1×10 8 CFU / mL, and fermented under anaerobic conditions at 37℃ for 4-8 hours.

4. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis in step (4) involves sequentially adding α-amylase, papain and amyloglucosidase for enzymatic hydrolysis.

5. The preparation method according to claim 5, characterized in that: In step (4), the amounts of α-amylase, papain and amyloglucosidase added are 0.2-0.4% (w / w), 0.4-0.6% (w / w), and 0.1-0.3% (v / v), respectively.

6. A phenolic dietary fiber from *Agaricus bisporus* prepared by any one of claims 1-5, characterized in that: The total bound phenol content of the phenolic dietary fiber in the mushroom buds is ≥20mg / g, and the purity of the dietary fiber is ≥85%.

7. The button mushroom containing phenolic dietary fiber as described in claim 6, characterized in that: The button mushroom contains phenolic dietary fiber with a water-holding capacity ≥8.0 g / g and a swelling capacity ≥9.5 mL / g.

8. The use of the button mushroom phenolic dietary fiber as described in claim 6 or 7 in the preparation of foods, health products or medicines for colonic antioxidant or anti-inflammatory purposes.

9. The application as described in claim 8, characterized in that: The phenolic dietary fiber from the mushroom *Aconitum carmichaelii* showed a polyphenol release of ≤350 mgGAE / 100gDW in the upper digestive tract after simulated oral-gastric-small intestinal digestion in vitro, and the polyphenol release continuously increased during in vitro colonic fermentation from 0 to 48 hours.

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