A method for improving the content of soluble dietary fiber of sugarcane bagasse by using solid-state fermentation of grifola

Abstract

The application discloses a method for improving the content of soluble dietary fiber of sugarcane residue by using solid-state fermentation of grifola frondosa, and comprises the following steps: drying and crushing the sugarcane residue, sterilizing; inoculating grifola frondosa strains, and carrying out solid-state fermentation for 10-14 days under the condition of 28-32 DEG C and 60%-70% humidity; drying, crushing and screening after the fermentation is completed to obtain the fermentation product. The application uses the natural secretion of the fiber-degrading enzyme system of grifola frondosa to hydrolyze part of the insoluble dietary fiber in the sugarcane residue into soluble dietary fiber, and promotes the accumulation of polysaccharide and antioxidant components. The application has the advantages that: 1) the content of soluble dietary fiber is increased from 5% before fermentation to more than 13%, and the antioxidant capacity is significantly enhanced; 2) the fermentation process is green and environmentally friendly; 3) it is beneficial to industrialization promotion; 4) the fermentation product can be widely applied to the fields of functional food, dietary supplement and health food, and the functional utilization value of the by-product of sugarcane residue is widened.

Description

Technical Field

[0001] This invention relates to the field of food processing and comprehensive utilization of by-products, specifically to a method for increasing the soluble dietary fiber content of sugarcane bagasse through solid-state fermentation using Poria cocos. Background Technology

[0002] Sugarcane bagasse is a major solid byproduct of sugarcane refining. As a major sugarcane-growing country, my country produces a large amount of sugarcane bagasse annually. Sugarcane bagasse is rich in dietary fiber, hemicellulose, polysaccharides, and a small amount of soluble sugars, possessing potential for development and utilization. However, the dietary fiber in sugarcane bagasse is mainly insoluble, with low levels of soluble dietary fiber. Furthermore, the content of polysaccharides and natural antioxidants is limited, restricting its application value in functional foods, dietary supplements, and health foods.

[0003] Currently, common methods for modifying sugarcane bagasse include physical pulverization, chemical modification, and enzymatic hydrolysis. Physical pulverization can only change the physical morphology of the fibers, but cannot fundamentally alter their chemical composition and functional properties. While chemical modification can disrupt the fiber structure of sugarcane bagasse, it easily leads to the loss of dietary fiber and functional components, and carries the risk of chemical reagent residues, failing to meet the requirements of green food processing. Enzymatic hydrolysis requires expensive enzyme preparations, and the hydrolysis conditions are strict, the operation is complex, and the cost for industrial application is high. In addition, existing microbial fermentation methods mostly use lactic acid bacteria or yeast, which have limited ability to convert insoluble sugarcane bagasse fiber into soluble dietary fiber and functional components, have long fermentation cycles, and insufficient improvement in product functionality, making it difficult to meet the needs of industrial applications.

[0004] Poria cocos is a traditional Chinese medicinal and edible fungus with various physiological activities. Recent studies have found that Poria cocos can secrete cellulase, hemicellulase, and other cellulase-degrading enzyme systems, demonstrating excellent fermentation and utilization capabilities for various plant substrates. Therefore, how to utilize the natural enzymatic hydrolysis capabilities of Poria cocos for the green and efficient biotransformation of sugarcane bagasse is a pressing technical problem to be solved in this field. Summary of the Invention

[0005] To address the problems of high cost, complex operation, limited functional improvement, and difficulty in industrial application of existing technologies, this invention provides a method for increasing the soluble dietary fiber content of sugarcane bagasse through solid-state fermentation using Poria cocos.

[0006] To address the aforementioned technical problems, this invention, through in-depth research and process optimization, proposes the following technical solution: A method for increasing the soluble dietary fiber content of sugarcane bagasse using solid-state fermentation with Poria cocos includes the following steps: (1) Raw material pretreatment: Dry sugarcane bagasse to a moisture content of 10% to 15%, and crush it to a particle size of 1 to 3 mm to obtain sugarcane bagasse powder; (2) Substrate sterilization: The sugarcane bagasse powder is sterilized; (3) Inoculation and fermentation: After sterilization, cool to 28~35℃, inoculate with Poria cocos strain at an inoculation rate of 5%~10% of the wet weight of sugarcane bagasse powder, place the inoculated raw materials in a fermentation container, and carry out solid-state fermentation for 10~14 days at a temperature of 28~32℃ and a humidity of 60%~70%. (4) Post-processing: After fermentation, the fermented bagasse is dried to a moisture content of 10% to 12%, crushed and sieved to obtain the fermented product.

[0007] In step (1), drying the sugarcane bagasse to a moisture content of 10%~15% avoids contamination by miscellaneous bacteria caused by excessive moisture in the raw material, and also facilitates subsequent crushing. Controlling the crushed particle size within the range of 1~3 mm ensures that the mycelium can fully contact the substrate during fermentation, while avoiding poor substrate permeability and operational difficulties caused by excessively small particle size. If the particle size is greater than 3 mm, the mycelium will have difficulty uniformly colonizing the inside of the particle; if the particle size is less than 1 mm, the substrate will be too dense, resulting in poor aeration, which is not conducive to aerobic fermentation.

[0008] In step (2), sterilization can be performed using high-temperature short-time sterilization or high-pressure steam sterilization. The purpose is to kill naturally occurring bacteria in the sugarcane bagasse and create a sterile environment for the pure culture fermentation of Poria cocos. Preferably, the sterilization conditions are 121℃ for 20~30 min.

[0009] In step (3), the inoculum size is a crucial factor affecting fermentation efficiency and product quality. If the inoculum size is too low, the *Poria cocos* bacteria grow slowly in the early stages of fermentation and are easily contaminated by other microorganisms; if the inoculum size is too high, it not only increases the cost of strain preparation but may also lead to excessive metabolic activity in the early stages of fermentation, producing excessive metabolic byproducts. Through extensive experimental research, the inventors determined that an inoculum size of 5%–10% wet weight ensures that *Poria cocos* bacteria quickly establish dominance in the early stages of fermentation while avoiding the adverse effects of excessively high inoculum sizes. A fermentation temperature of 28–32℃ is the optimal temperature range for *Poria cocos* bacteria growth; too low a temperature results in slow growth, while too high a temperature may lead to bacterial inactivation. A humidity range of 60%–70% is the optimal humidity range for solid-state fermentation, ensuring normal mycelial growth while avoiding contamination and substrate clumping caused by excessive humidity.

[0010] Solid-state fermentation offers advantages over liquid-state fermentation, including water conservation, ease of operation, and convenient product separation. The fermentation cycle of 10-14 days represents the optimal fermentation time range determined by the inventors through extensive experimentation. If the fermentation cycle is too short, the *Poria cocos* bacteria will not have fully degraded the bagasse fiber, resulting in insufficient conversion of soluble dietary fiber; conversely, a fermentation cycle that is too long leads to reduced production efficiency. Experimental data shows that a 14-day fermentation cycle achieves the best conversion of soluble dietary fiber, and further extending the fermentation cycle does not significantly improve the effect; therefore, 14 days is the optimal fermentation cycle.

[0011] In step (4), drying the fermentation product to a moisture content of 10% to 12% is beneficial for subsequent pulverization and long-term storage. The pulverized and sieved product can be directly used as a functional food ingredient or dietary supplement.

[0012] As a further improvement of the present invention, the sterilization process in step (2) adopts high temperature short time sterilization or high pressure steam sterilization.

[0013] As a further improvement of the present invention, in step (3), the mixture is stirred regularly during the fermentation process to maintain a uniform distribution of oxygen in the fermentation substrate. The frequency of stirring can be adjusted according to the fermentation container and the thickness of the substrate, generally once every 2 to 3 days.

[0014] As a further improvement of the present invention, the solid-state fermentation cycle in step (3) is 14 days. The inventors found through experimental research that when the fermentation cycle is 14 days, the conversion rate of soluble dietary fiber reaches the highest value, and the accumulation of functional components is also the most sufficient. Continuing to extend the fermentation time will not only fail to significantly improve product quality, but will also increase production energy consumption and costs.

[0015] As a further improvement of the present invention, in step (4), a 40-80 mesh sieve is used for grading.

[0016] As a further improvement of the present invention, in step (3), a compound functional promoter is added when inoculating Poria cocos. The compound functional promoter is composed of lentinan extract and methyl jasmonic acid. The lentinan extract is added at a mass of 0.5% to 2.0% of the dry weight of sugarcane bagasse powder, and the methyl jasmonic acid is added at a mass of 0.45 to 2.24 μg / g of dry sugarcane bagasse powder.

[0017] In this invention, the shiitake mushroom stem alcohol extract refers to a concentrated liquid obtained by extracting and concentrating shiitake mushroom stems using ethanol. Shiitake mushroom stems are a major byproduct generated during shiitake mushroom processing, accounting for approximately 20% to 30% of the total weight of shiitake mushrooms, and are rich in active ingredients such as polysaccharides, total phenols, and total flavonoids.

[0018] This invention adds shiitake mushroom stem alcohol extract to the sugarcane bagasse fermentation system in the form of a small amount of functional additive. On the one hand, it provides additional growth-promoting factors for Poria cocos fungus. On the other hand, its natural active ingredients induce the metabolism of Poria cocos fungus, promoting the secretion of more cellulase and hemicellulase, thereby improving the conversion efficiency of soluble dietary fiber in sugarcane bagasse to a certain extent.

[0019] In this invention, methyl jasmonate (MeJA) is a naturally occurring plant hormone and signaling molecule widely found in the plant kingdom, involved in regulating plant growth, development, and stress responses. Technicians can use commercially available analytically pure methyl jasmonate reagents.

[0020] This invention introduces methyl jasmonic acid as a signaling molecule into the solid-state fermentation system of *Poria cocos*. Appropriate concentrations of methyl jasmonic acid can activate the secondary metabolic pathways of *Poria cocos*, promoting the upregulation of genes related to the synthesis of antioxidant components such as polysaccharides, flavonoids, and phenolic acids, thereby significantly enhancing the functional value of the fermentation products. Experiments showed that lentinan extract from shiitake mushroom stems and methyl jasmonic acid exhibit a significant synergistic effect in the solid-state fermentation system of sugarcane bagasse using *Poria cocos*, with the combined effect being far superior to that of adding either ingredient alone.

[0021] The shiitake mushroom stem alcohol extract of the present invention can be prepared by the following method: Dried shiitake mushroom stems are dried at 55-65℃ to constant weight, pulverized to 30-50 mesh, and 60%-80% edible ethanol is added at a material-to-liquid mass ratio of 1:10-1:15. The mixture is then extracted by reflux in a water bath at 50-70℃ for 1-3 hours. The filtrate is collected by filtration, and the residue is extracted once more. The filtrates are combined and concentrated under reduced pressure at 40-50℃ to 1 / 10-1 / 15 of the original volume to obtain the shiitake mushroom stem alcohol extract. This preparation method uses edible ethanol as the extraction solvent, providing mild extraction conditions that effectively extract active ingredients such as polysaccharides, total phenols, and total flavonoids from the shiitake mushroom stems. Simultaneously, it avoids the use of toxic organic solvents, ensuring the food safety of the product.

[0022] As a further improvement of the present invention, the methyl jasmonate is added in the form of an aqueous solution, wherein the concentration of methyl jasmonate is 10~50 μmol / L, and the addition method is to spray it evenly into the bagasse matrix together with the lenticel extract of shiitake mushroom stems during inoculation.

[0023] The present invention also discloses fermentation products obtained by the above method, and their application in the preparation of functional foods, dietary supplements or health foods.

[0024] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention utilizes the fiber-degrading enzyme system naturally secreted by Poria cocos to carry out solid-state fermentation and transformation of sugarcane bagasse, without the need for additional enzyme preparations. The fermentation process is green and environmentally friendly, and significantly reduces production costs.

[0025] 2. By optimizing the fermentation process parameters, this invention can increase the soluble dietary fiber content in sugarcane bagasse from 5.2% before fermentation to over 13.2%, while simultaneously increasing the antioxidant capacity (FRAP value) of the fermentation product from 0.71 μmol Trolox / g to over 2.91 μmol Trolox / g, the ABTS free radical scavenging capacity from 13.3 μmol Trolox / g to over 19.2 μmol Trolox / g, and significantly increasing the content of polysaccharides, flavonoids, and phenolic acid metabolites.

[0026] 3. In addition, the present invention adds a compound functional promoter composed of lentinan extract and methyl jasmonate during the fermentation process. The two exert a significant synergistic effect, which further enhances the soluble dietary fiber content and antioxidant capacity.

[0027] 4. The process of this invention is simple to operate, and the fermentation cycle is fixed at 10 to 14 days, which is convenient for industrial production and quality control.

[0028] 5. Fermentation products can be widely used in functional foods, dietary supplements and health foods, which broadens the scope of functional utilization of sugarcane bagasse by-products and has important economic value and environmental significance. Detailed Implementation

[0029] To enable those skilled in the art to better implement the present invention, the present invention will be further described below with reference to embodiments. However, it should be understood that the present invention is not limited to the following embodiments.

[0030] For ease of comparison, the raw materials used in the following examples and comparative examples are all from the same batch, and the specific parameters of the raw materials are as follows: Sugarcane bagasse: A byproduct of sugarcane sugar production, commercially available, with an initial moisture content of 50%~60%, a soluble dietary fiber content of about 5%, a total phenol content of about 0.5 mg GAE / g, and a total flavonoid content of about 0.3 mg RE / g; Poria cocos strain: Commercially available Poria cocos slant strain P5.78, activated with PDA medium before use; Shiitake mushroom stems: Commercially available dried shiitake mushroom stems, produced in Suizhou, Hubei Province, dried at 60℃ to constant weight for later use; Methyl jasmonate: analytical grade, purity ≥95%, Aladdin Reagent (Shanghai) Co., Ltd.; Food-grade ethanol: 95% food grade, Sinopharm Chemical Reagent Co., Ltd. Deionized water: prepared in the laboratory, resistivity ≥18.2 MΩ·cm.

[0031] In the following examples and comparative examples, the lentinan extract of shiitake mushroom stems was prepared according to the following method: Dried shiitake mushroom stems were dried at 60℃ to constant weight and then pulverized through a 40-mesh sieve. 100 g of the shiitake mushroom stem powder was weighed and added to 1.2 L of 70% edible ethanol (material-to-liquid mass ratio approximately 1:12). The mixture was refluxed in a 60℃ water bath for 2 h, and the filtrate was collected by filtration. The residue was extracted once more, and the two filtrates were combined. The combined filtrate was concentrated under reduced pressure at 45℃ to approximately 1 / 12 of its original volume to obtain a concentrated shiitake mushroom stem ethanol extract. The total phenolic content of this concentrate was approximately 2.5 mg GAE / mL, and the total flavonoid content was approximately 1.2 mg RE / mL.

[0032] Example 1: The solid-state fermentation product of Poria cocos with sugarcane bagasse was prepared according to the following method: S1. Raw material pretreatment: Take fresh sugarcane bagasse, dry it in a drying oven at 60℃ until the moisture content is about 12%, and crush it through a 2 mm sieve to obtain sugarcane bagasse powder with a particle size of about 1~3 mm.

[0033] S2. Matrix sterilization: Weigh 500 g of sugarcane bagasse powder, spread it evenly in a stainless steel tray, and sterilize it with high-pressure steam at 121℃ for 30 min. Remove and cool to 30℃.

[0034] S3. Inoculation and Fermentation: After cooling, sugarcane bagasse is placed into a sterile fermentation bag. Activated *Poria cocos* inoculum is inoculated at an inoculation rate of 8% of the wet weight of the sugarcane bagasse powder (approximately 40 g / 500 g wet substrate), and thoroughly mixed. The fermentation bag is then sealed and placed in a constant temperature incubator at 30±1℃ and 65±5% humidity for solid-state fermentation for 14 days. During fermentation, the bag is stirred every 3 days to maintain uniform oxygen distribution within the substrate.

[0035] S4. Post-processing: After fermentation, the fermented bagasse is taken out and dried in a 60℃ drying oven until the moisture content is about 11%. It is then crushed with a pulverizer and passed through a 60-mesh sieve to obtain the fermented product, which is recorded as sample 1.

[0036] Example 2: The procedure is the same as in Example 1, except that the fermentation period is adjusted. The specific plan is as follows: S1 and S2 are the same as in Example 1.

[0037] S3. Place the cooled sugarcane bagasse into a sterile fermentation bag, and inoculate with activated Poria cocos inoculum at an inoculation rate of 8% (wet weight), mixing thoroughly. Seal the fermentation bag and place it in a constant temperature incubator, controlling the temperature at 30±1℃ and the humidity at 65±5%, for solid-state fermentation for 10 days. Stir the bag every 3 days during fermentation.

[0038] S4 is the same as in Example 1, and the fermented product obtained is denoted as Sample 2.

[0039] Example 3: The procedure is the same as in Example 1, except that the inoculation amount is adjusted. The specific plan is as follows: S1 and S2 are the same as in Example 1.

[0040] S3. Place the cooled sugarcane bagasse into a sterile fermentation bag, and inoculate with activated Poria cocos inoculum at a rate of 5% of the wet weight (approximately 25 g / 500 g wet substrate), mixing thoroughly. Seal the fermentation bag and place it in a constant temperature incubator, controlling the temperature at 30±1℃ and the humidity at 65±5%, for solid-state fermentation for 14 days. Stir the bag every 3 days during fermentation.

[0041] S4 is the same as in Example 1, and the fermented product obtained is designated as Sample 3.

[0042] Example 4 (with added compound functional enhancers): The following method was used to prepare a solid-state fermented sugarcane bagasse product containing Poria cocos bacteria and a compound functional promoter: S1. Raw material pretreatment: Same as in Example 1.

[0043] S2. Substrate sterilization: Same as in Example 1.

[0044] S3. Preparation of the composite functional promoter: Take the concentrated extract of shiitake stem alcohol prepared by the above method, and weigh out 1.2% of the dry weight of sugarcane bagasse powder (i.e., 1.2 g of concentrated extract per 100 g of dry sugarcane bagasse powder, equivalent to a volume of approximately 1.11 mL); separately take methyl jasmonic acid, and calculate the required amount based on 1.2 μg / g of dry sugarcane bagasse powder (0.6 mg per 500 g of dry weight). Dissolve methyl jasmonic acid in a small amount of anhydrous ethanol, then mix it with the concentrated extract of shiitake stem alcohol, add deionized water to a total volume of 200 mL (per 500 g of dry weight), and stir evenly to obtain the spray solution of the composite functional promoter.

[0045] S4. Inoculation and Accelerator Addition: Place the cooled sugarcane bagasse into a sterile fermentation bag. Inoculate with activated *Poria cocos* strain at an inoculation rate of 8% (wet weight). Simultaneously, evenly spray the compound functional accelerator solution prepared in step S3 onto the surface of the sugarcane bagasse and mix thoroughly. Seal the fermentation bag and place it in a constant temperature incubator, controlling the temperature at 30±1℃ and the humidity at 65±5%, for solid-state fermentation for 14 days. Stir every 3 days during fermentation.

[0046] S5. Post-processing: Same as in Example 1, the fermented product obtained is designated as Sample 4.

[0047] Comparative Example 1: This comparative example is a control experiment of Example 1, which was carried out according to the same steps as Example 1, except that the fermentation period was adjusted to 5 days.

[0048] S1 and S2 are the same as in Example 1.

[0049] S3. Pack the cooled sugarcane bagasse into a sterile fermentation bag, and inoculate with activated Poria cocos inoculum at an inoculation rate of 8% by wet weight, mixing thoroughly. Seal the fermentation bag and place it in a constant temperature incubator, controlling the temperature at 30±1℃ and the humidity at 65±5%, for solid-state fermentation for 5 days. Stir the bag every 2 days during fermentation.

[0050] S4 is the same as in Example 1, and the fermented product obtained is designated as control sample D1.

[0051] Comparative Example 2: This comparative example is a control experiment of Example 1, and is carried out according to the same steps as Example 1, except that the fermentation temperature is adjusted to 25°C.

[0052] S1 and S2 are the same as in Example 1.

[0053] S3. Place the cooled sugarcane bagasse into a sterile fermentation bag, inoculate with activated Poria cocos inoculum at an inoculation rate of 8% (wet weight), and mix thoroughly. Seal the fermentation bag and place it in a constant temperature incubator, controlling the temperature at 25±1℃ and the humidity at 65±5%, for solid-state fermentation for 14 days. Stir the bag every 3 days during fermentation.

[0054] S4 is the same as in Example 1, and the fermented product obtained is designated as control sample D2.

[0055] Comparative Example 3 (Methyl jasmonate only, without lentinan extract): This comparative example serves as a control experiment for Example 4, conducted according to the same steps as Example 4, except that: lentinan extract of shiitake mushroom stems is not added to the composite functional promoter; only methyl jasmate is added, and the amount of methyl jasmate added is the same as in Example 4 (1.2 μg / g dry weight of sugarcane bagasse powder). The specific scheme is as follows: S1 and S2 are the same as in Example 4.

[0056] S3. Preparation of methyl jasmonate spray solution: Take methyl jasmonate and calculate the required amount based on 1.2 μg / g dry weight of sugarcane bagasse powder (0.6 mg based on 500 g dry weight). First, dissolve it in a small amount of anhydrous ethanol, then dissolve it in deionized water and bring the volume to 200 mL.

[0057] S4. Inoculation and Addition: Place the cooled sugarcane bagasse into a sterile fermentation bag. Inoculate with activated *Poria cocos* inoculum at an inoculation rate of 8% (wet weight). Simultaneously, evenly spray the methyl jasmonate spray solution prepared in step S3 onto the surface of the sugarcane bagasse and mix thoroughly. Seal the fermentation bag and place it in a constant temperature incubator, controlling the temperature at 30±1℃ and the humidity at 65±5%, for solid-state fermentation for 14 days. Stir every 3 days during fermentation.

[0058] S5 is the same as in Example 4, and the fermented product obtained is designated as control sample D3.

[0059] Comparative Example 4 (only lentinan extract of shiitake mushroom stems added, without methyl jasmate): This comparative example serves as a control experiment for Example 4, conducted according to the same steps as Example 4, except that methyl jasmonate is not added to the composite functional promoter; only concentrated lentinan extract of shiitake mushroom stem is added, and the amount added is the same as in Example 4 (1.2% of the dry weight of sugarcane bagasse powder). The specific scheme is as follows: S1 and S2 are the same as in Example 4.

[0060] S3. Prepare the shiitake mushroom stem alcohol extract spray solution: Take the concentrated shiitake mushroom stem alcohol extract, weigh it at 1.2% of the dry weight of sugarcane bagasse powder (i.e., 6 g / 500 g dry weight), add deionized water to 200 mL, and stir well.

[0061] S4. Inoculation and Addition: Place the cooled sugarcane bagasse into a sterile fermentation bag. Inoculate with activated *Poria cocos* spawn at an inoculation rate of 8% (wet weight). Simultaneously, evenly spray the *Lentinula edodes* stem alcohol extract prepared in step S3 onto the surface of the sugarcane bagasse and mix thoroughly. Seal the fermentation bag and place it in a constant temperature incubator, controlling the temperature at 30±1℃ and the humidity at 65±5%, for solid-state fermentation for 14 days. Stir every 3 days during fermentation.

[0062] S5 is the same as in Example 4, and the fermented product obtained is designated as control sample D4.

[0063] Comparative Example 5 (Methyl jasmonate addition exceeds the range): This comparative example serves as a control experiment for Example 4, conducted according to the same procedures, except that the amount of methyl jasmonic acid added was increased to 10 μg / g dry weight of bagasse powder (exceeding the upper limit of the defined range of 0.45~2.24 μg / g). The specific procedure is as follows: S1 and S2 are the same as in Example 4.

[0064] S3. Preparation of compound functional promoters: The addition amount of lentinan extract concentrate of shiitake stem remains 1.2% (6 g / 500 g dry weight), and the addition amount of methyl jasmonic acid is changed to 10 μg / g dry weight (i.e., 5 mg / 500 g dry weight). Methyl jasmonic acid is first dissolved in a small amount of anhydrous ethanol, then mixed with lentinan extract concentrate of shiitake stem, and deionized water is added to 200 mL, and stirred evenly.

[0065] S4 and S5 are the same as in Example 4, and the fermented product obtained is designated as control sample D5.

[0066] Comparative Example 6 (Unfermented Blank Control): The bagasse powder obtained in step S1 of Example 1 was dried, pulverized, and passed through a 60-mesh sieve without any fermentation treatment, and used as a blank control. The resulting sample was designated as control sample D6.

[0067] Performance verification experiment: The fermented products obtained in Examples 1 to 4 and Comparative Examples 1 to 6 were subjected to determination of soluble dietary fiber content, total phenolic content, total flavonoid content, and antioxidant capacity (FRAP value and ABTS free radical scavenging capacity).

[0068] I. Measurement Method 1. Determination of soluble dietary fiber content The enzymatic gravimetric method was followed according to GB 5009.88-2023, "National Food Safety Standard - Determination of Dietary Fiber in Food". 1.00 g of the pulverized and sieved sample was weighed and added to MES-TRIS buffer at pH 8.2. Enzymatic hydrolysis was then performed sequentially using heat-resistant α-amylase (100℃, 30 min), protease (60℃, 30 min), and amyloglucosidase (60℃, 30 min). After hydrolysis, 4 volumes of 95% ethanol were added to precipitate the sample. The mixture was filtered, and the residue was washed twice each with 78% ethanol, 95% ethanol, and acetone. After drying, the residue was weighed. The soluble dietary fiber content was calculated after deducting protein and ash.

[0069] 2. Determination of total phenol content The Folin-Ciocalteu method was used. 0.50 g of sample was weighed, 10 mL of 80% ethanol was added, and the mixture was extracted by sonication for 30 min. The supernatant was collected by centrifugation. 0.5 mL of the supernatant was taken, 2.5 mL of 10% Folin-Ciocalteu reagent was added, and the mixture was mixed and allowed to stand for 5 min. Then, 2.0 mL of 7.5% Na₂CO₃ solution was added, and the mixture was reacted in the dark for 30 min. The absorbance was measured at 765 nm. Gallic acid was used as a standard (concentration range 0–100 μg / mL), and the results are expressed as mg gallic acid equivalents (GAE) / g dry weight.

[0070] 3. Determination of total flavonoid content The sodium nitrite-aluminum nitrate method was used. Take 1.0 mL of the sample extract (same as the total phenol extract), add 0.3 mL of 5% NaNO₂ solution, mix well, and let stand for 6 min; add 0.3 mL of 10% Al(NO₃)₃ solution, mix well, and let stand for 6 min; add 2.0 mL of 1 mol / L NaOH solution, and dilute to 5 mL with 80% ethanol, mix well, and let stand for 15 min. Measure the absorbance at 510 nm. Rutin was used as a standard (concentration range 0–80 μg / mL), and the results are expressed as mg rutin equivalents (RE) / g dry weight.

[0071] 4. FRAP antioxidant capacity determination The FRAP method was used. FRAP working solution was prepared as follows: 25 mL of 300 mmol / L acetate buffer (pH 3.6) was added to 2.5 mL of 10 mmol / L TPTZ solution (dissolved in 40 mmol / L HCl) and 2.5 mL of 20 mmol / L FeCl3 solution. After mixing thoroughly, the solution was preheated to 37°C. 0.2 mL of the sample extract was added to 3.8 mL of the FRAP working solution. After mixing thoroughly, the solution was reacted at 37°C for 30 min, and the absorbance was measured at 593 nm. Trolox was used as a standard (concentration range 0–1.0 mmol / L). Results are expressed as μmol Trolox equivalents / g dry weight.

[0072] 5. Determination of ABTS free radical scavenging ability The ABTS method was used. An equal volume of 7 mmol / L ABTS solution was mixed with 2.45 mmol / L potassium persulfate solution, and the mixture was reacted at room temperature in the dark for 12–16 h to generate ABTS. + Free radical stock solution. Dilute with anhydrous ethanol to an absorbance of 0.70 ± 0.02 at 734 nm immediately before use. Take 0.1 mL of sample extract, add 3.9 mL of ABTS working solution, mix well, and react in the dark for 6 min. Measure the absorbance at 734 nm. Use Trolox as a standard (concentration range 0–0.5 mmol / L). Results are expressed as μmol Trolox equivalents / g dry weight.

[0073] II. Measurement Results Table 1. Measurement results of various indicators of fermentation products from different embodiments and comparative examples. III. Results Analysis 1. Effects of Fermentation Process Optimization Comparing the results of Example 1 (fermentation for 14 days, inoculation rate of 8%), Example 2 (fermentation for 10 days, inoculation rate of 8%), and Comparative Example 1 (fermentation for 5 days, inoculation rate of 8%), it can be seen that the soluble dietary fiber content and antioxidant function indicators increase with the extension of fermentation time. The soluble dietary fiber content of Sample 1, fermented for 14 days, reached 13.2%, far exceeding the 5.2% of unfermented sugarcane bagasse (Comparative Example 6); the FRAP antioxidant capacity increased from 0.71 μmol Trolox / g to 2.91 μmol Trolox / g, and the ABTS value increased from 13.3 μmol Trolox / g to 19.2 μmol Trolox / g. All indicators of Comparative Example 1, fermented for 5 days, were significantly lower than those of Example 1, indicating that when the fermentation cycle is too short, the *Poria cocos* bacteria have not fully degraded the sugarcane bagasse fiber, and the conversion of functional components is insufficient. The indicators of Example 3 (inoculation rate of 5%) were slightly lower than those of Example 1 (inoculation rate of 8%), indicating that appropriately increasing the inoculation rate is beneficial for *Poria cocos* bacteria to quickly establish a growth advantage in the early stages of fermentation, thereby improving fermentation efficiency and product quality.

[0074] Comparing Example 1 and Comparative Example 2 (fermentation temperature 25℃), it can be seen that fermentation temperature has a significant impact on the metabolic activity of *Poria cocos*. When the fermentation temperature drops to 25℃, the soluble dietary fiber content decreases from 13.2% to 10.6%, and the FRAP value decreases from 2.91 μmol Trolox / g to 2.16 μmol Trolox / g. This indicates that 28~32℃ is the suitable temperature range for solid-state fermentation of sugarcane bagasse by *Poria cocos*. Temperatures that are too low will inhibit the enzyme activity and growth rate of *Poria cocos*, thereby affecting fiber degradation efficiency and the accumulation of functional components.

[0075] 2. Synergistic effect of compound functional promoters Under the premise that the total amount of compound functional promoters is exactly the same, after adding lentinan extract of shiitake mushroom stem (1.2% of dry weight of sugarcane bagasse powder) and methyl jasmate (1.2 μg / g) in Example 4, all indicators were significantly better than those of Comparative Example 3 (only methyl jasmate was added) and Comparative Example 4 (only lentinan extract of shiitake mushroom stem was added).

[0076] Comparative Example 3 (with only methyl jasmonate 1.2 μg / g) had a soluble dietary fiber content of 14.2%, a FRAP value of 3.23 μmol Trolox / g, and an ABTS value of 21.1 μmol Trolox / g; Comparative Example 4 (with only lentinan extract of shiitake mushroom stems 1.2%) had a soluble dietary fiber content of 13.7%, a FRAP value of 3.08 μmol Trolox / g, and an ABTS value of 20.2 μmol Trolox / g. If the two components only have a simple additive effect, then the indicators of Example 4 should be lower than the sum of the baseline value (Example 1) and their respective increments, i.e., the upper limit of the theoretical expected value: soluble dietary fiber of 14.7%, FRAP of 3.40 μmol Trolox / g, and ABTS of 22.1 μmol Trolox / g. In Example 4, the actual measured soluble dietary fiber content reached 17.4%, the FRAP value reached 4.13 μmol Trolox / g, and the ABTS value reached 26.8 μmol Trolox / g, exceeding the theoretical upper limit by 2.7 percentage points, 0.73 μmol Trolox / g, and 4.7 μmol Trolox / g, respectively, with relative increases of 18.4%, 21.5%, and 21.3%. The actual total phenolic content was 2.56 mg GAE / g (the theoretical upper limit is 1.98 mg GAE / g), exceeding it by 0.58 mg GAE / g (relative increase of 29.3%); the actual total flavonoid content was 1.49 mg RE / g (the theoretical upper limit is 1.13 mg RE / g), exceeding it by 0.36 mg RE / g (relative increase of 31.9%). This fully demonstrates that lentinan extract and methyl jasmonic acid have a significant synergistic effect in the solid-state fermentation of sugarcane bagasse system by Poria cocos. The inventors believe the reason may be that the lenticel extract of shiitake mushroom stems can penetrate the dense lignocellulose network of sugarcane bagasse and preferentially anchor on the surface of *Poria cocos* mycelia. Through non-covalent interactions, it regulates the permeability of the mycelial cell membrane, making the conformation of the active site of the extracellular cellulase system more readily match the glycosidic bonds of insoluble dietary fiber. Meanwhile, methyl jasmonic acid not only induces the upregulation of fiber degradation-related genes in the mycelium within the microenvironment but also forms a transient hydrogen bond network with the active groups in the lenticel extract, synergistically reducing the interfacial energy of the fiber substrate, thereby accelerating the dissociation and recombination turnover of the enzyme-substrate complex. Simultaneously, soluble oligosaccharide fragments released by the mycelium during fiber degradation undergo intermolecular complexation with the active components in the lenticel extract, forming a locally high-concentration metabolic-inducing microregion, which feedback-amplifies the signal transduction efficiency of methyl jasmonic acid. This forms a ternary closed-loop interaction of fiber interface domestication, enzyme structure-activity synergy, and signal cascade amplification, resulting in a synergistic effect in solid-state fermentation of sugarcane bagasse that far exceeds that of a single component.

[0077] 3. Threshold effect of methyl jasmonate addition In Comparative Example 5, the amount of methyl jasmonic acid added was increased to 10 μg / g (approximately 8 times that of Example 4, exceeding the range of 0.45~2.24 μg / g defined in claim 6). Although all indicators were still higher than those of the basic fermented sample (Example 1), they showed a significant decrease compared to Example 4: soluble dietary fiber content decreased from 17.4% to 15.3%, FRAP value decreased from 4.13 μmol Trolox / g to 3.61 μmol Trolox / g, ABTS value decreased from 26.8 μmol Trolox / g to 23.8 μmol Trolox / g, total phenols decreased from 2.56 mg GAE / g to 2.06 mg GAE / g, and total flavonoids decreased from 1.49 mg RE / g to 1.18 mg RE / g. This result verifies that there is an optimal addition window for methyl jasmonic acid in the Poria cocos solid-state fermentation system. When the amount of methyl jasmonate added exceeds 2.24 μg / g, its direct antibacterial effect begins to dominate, which inhibits the growth and metabolic activity of Poria cocos, leading to a decrease in fermentation effect.

[0078] In summary, this invention significantly improves the soluble dietary fiber content and antioxidant functional components in sugarcane bagasse by optimizing fermentation process parameters. Furthermore, by introducing a composite functional promoter composed of lentinan extract and methyl jasmonate, the synergistic effect of the two in the solid-state fermentation system of Poria cocos significantly enhances the various performance indicators of the product, demonstrating promising prospects for industrial application.

Claims

1. A method for increasing the soluble dietary fiber content of sugarcane bagasse using solid-state fermentation with Poria cocos, characterized in that, Includes the following steps: (1) Raw material pretreatment: Dry sugarcane bagasse to a moisture content of 10% to 15%, and crush it to a particle size of 1 to 3 mm to obtain sugarcane bagasse powder; (2) Substrate sterilization: The sugarcane bagasse powder is sterilized; (3) Inoculation and fermentation: After sterilization, cool to 28~35℃, inoculate with Poria cocos strain at an inoculation rate of 5%~10% of the wet weight of sugarcane bagasse powder, place the inoculated raw materials in a fermentation container, and carry out solid-state fermentation for 10~14 days at a temperature of 28~32℃ and a humidity of 60%~70%. (4) Post-processing: After fermentation, the fermented bagasse is dried to a moisture content of 10% to 12%, crushed and sieved to obtain the fermented product.

2. The method according to claim 1, characterized in that, In step (2), sterilization is performed using high-temperature short-time sterilization or high-pressure steam sterilization.

3. The method according to claim 1, characterized in that, In step (3), the mixture is stirred regularly during the fermentation process to maintain a uniform distribution of oxygen in the fermentation substrate.

4. The method according to claim 1, characterized in that, The solid-state fermentation cycle in step (3) is 14 days.

5. The method according to claim 1, characterized in that, In step (4), a 40-80 mesh sieve is used for grading.

6. The method according to claim 1, characterized in that, In step (3), a compound functional promoter is added when inoculating Poria cocos. The compound functional promoter is composed of lentinan extract and methyl jasmonic acid. The lentinan extract is added at a mass of 0.5% to 2.0% of the dry weight of sugarcane bagasse powder, and the methyl jasmonic acid is added at a mass of 0.45 to 2.24 μg / g of dry sugarcane bagasse powder.

7. The method according to claim 6, characterized in that, The shiitake mushroom stem alcohol extract was prepared by the following method: dried shiitake mushroom stems were dried at 55-65℃ to constant weight, pulverized to 30-50 mesh, and 60%-80% edible ethanol was added at a material-to-liquid mass ratio of 1:10-1:

15. The extract was refluxed in a water bath at 50-70℃ for 1-3 hours. The filtrate was collected by filtration, and the residue was extracted once more. The filtrates were combined and concentrated under reduced pressure at 40-50℃ to 1 / 10-1 / 15 of the original volume to obtain the shiitake mushroom stem alcohol extract.

8. The method according to claim 6, characterized in that, The methyl jasmonic acid is added in the form of an aqueous solution, wherein the concentration of methyl jasmonic acid is 10~50 μmol / L, and the addition method is to spray it evenly into the sugarcane bagasse matrix together with the physalis stalk alcohol extract during inoculation.

9. A fermentation product obtained by the method according to any one of claims 1 to 8.

10. The use of the fermentation product according to claim 9 in the preparation of functional foods, dietary supplements or health foods.

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