Application of Hericium erinaceus He-02-06 in increasing the soluble dietary fiber content in corn

Abstract

The present invention discloses the application of Hericium erinaceus He-02-06 in increasing the soluble dietary fiber content in corn. The Hericium erinaceus He-02-06 has a strain collection number of CCTCC NO: 20221043; Application of the described Hericium erinaceus He‑02‑06 in increasing the soluble dietary fiber content in corn; A method for preparing corn bran soluble dietary fiber, which comprises: preparing a corn bran solid-state fermentation medium, inoculating Hericium erinaceus He‑02‑06, and culturing at 22-30°C for 5-10 days; weighing the fermented sample, sieving, adding water, extracting in a high-temperature water bath for 1.5-2.5 hours, cooling, centrifuging, and retaining the supernatant; adding ethanol, standing at low temperature for 6-12 hours, and drying; Beneficial effects: After fermentation, the water holding capacity, swelling capacity, and oil holding capacity of the corn bran SDF are increased to 1.57 times, 1.95 times, and 1.80 times, respectively, the adsorption properties of the corn bran SDF are enhanced, and the adsorption capacity for nitrite, cholesterol, and bile salts are all improved to a certain extent.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to the application of Hericium erinaceus He-02-06 in increasing the content of soluble dietary fiber in corn. Background Art

[0002] Corn is one of my country's important grain crops. Corn husks are a byproduct of wet starch production, accounting for 14%-20% of corn mass. They contain 10%-13% protein, 15%-20% starch, 1.0%-3.7% fat, 15%-35% cellulose, 38%-57% hemicellulose, and 0.6%-2.0% ash. They also contain vitamins, minerals, sterols, and other nutrients. Corn husks contain a variety of bioactive substances, such as dietary fiber, corn fiber oil, arabinoxylan, and ferulic acid, which have numerous physiological functions. Dietary fiber refers to a polymer of ten or more carbohydrate units that is not digested and absorbed by the human body. It has physiological functions such as lowering blood lipids and blood sugar, promoting weight loss, removing toxic and harmful substances, improving intestinal flora, and preventing cancer. Hu et al. used xylanase to modify corn husk dietary fiber. The study found that the serum total cholesterol, triglyceride and low-density lipoprotein cholesterol levels of rats that consumed modified and unmodified dietary fiber were significantly reduced, and the reduction effect of the modified dietary fiber was more significant after the 6th week, indicating that corn husk dietary fiber has a good effect on lowering cholesterol and losing weight.

[0003] Dietary fiber modification involves using certain technical means to process raw materials to convert some IDF into SDF, thereby improving the structure of dietary fiber and enhancing its physical and chemical properties, making it more beneficial to human health and enabling better applications. Currently, methods for modifying corn husk dietary fiber include physical, chemical, biological, and combined methods, with physical and biological methods being the most commonly used in research. Microbial modification of dietary fiber utilizes microbial growth and fermentation to consume carbon and nitrogen sources in the raw materials, reducing protein, starch, fat, and other substances. The fermentation process then hydrolyzes the raw materials through enzyme or acid production, achieving the desired modification. This method utilizes safe and non-toxic microorganisms and offers advantages such as safety, efficiency, pollution-free operation, and low cost. It is amenable to industrial production and produces dietary fiber of high purity. Existing microbial methods for dietary fiber modification primarily use lactic acid bacteria as the primary bacteria, while commonly used fungi are molds that secrete extracellular cellulases, such as Rhizopus oryzae, Trichoderma viride, Monascus purpureus, and Aspergillus niger. Other fungi, such as Pleurotus ostreatus and Ganoderma lucidum, produce enzymes such as proteases, amylases, and cellulases during growth.

[0004] As a new nutrient, dietary fiber's physiological functions and impact on human health are gradually attracting attention. Jilin Province is a major corn-growing province. Corn processing and production produces many byproducts, such as corn husks, corn slurry, and corn gluten meal, and Jilin Province has abundant dietary fiber resources. Research on corn husk dietary fiber has an important role in promoting the deep processing of corn in my country. Modification of corn husk dietary fiber can improve the extraction rate of corn husk dietary fiber, enhance the physiological activity of corn husk dietary fiber, increase the comprehensive utilization rate of corn, and improve social and economic benefits. It is of great significance to promote human health and reduce environmental pollution. The method of modifying dietary fiber using fungal solid-state fermentation can also increase the utilization rate of microorganisms. Microbial fermentation has the advantages of short production cycle, low cost, no geographical or seasonal restrictions, large-scale production, and environmental pollution-free. It is a good way to modify dietary fiber. Summary of the Invention

[0005] The present invention aims to provide Hericium erinaceus He-02-06 capable of modifying dietary fiber and its application in increasing the content of soluble dietary fiber in corn.

[0006] Hericium erinaceus Hericium erinaceus He-02-06, strain collection number CCTCC NO: M 20221043.

[0007] The application of Hericium erinaceus He-02-06 in increasing the soluble dietary fiber content in corn;

[0008] The corn is corn husk.

[0009] The preparation method of corn husk soluble dietary fiber comprises:

[0010] 1) Corn husk processing: wash the corn husk until the pH is neutral, dry it, and crush it;

[0011] 2) Solid-state fermentation: a solid-state fermentation medium was prepared with corn husks, inoculated with the Hericium erinaceus He-02-06 at an inoculum concentration of 5-10%, and cultured at 22-30°C for 5-10 days;

[0012] 3) Preparation of soluble dietary fiber: Weigh the fermented sample from step 2), sieve it, add water, and extract it in a water bath at 75-85°C for 1.5-2.5 hours. Cool it, centrifuge it, and retain the supernatant. Add ethanol, let it stand at 0-4°C for 6-12 hours, and dry it to obtain corn husk soluble dietary fiber.

[0013] The solid-state fermentation medium of step 2) is prepared according to the proportions of 100 g corn husk, 150-250 g water, 2-4 g anhydrous glucose, 0.05-0.15 g potassium dihydrogen phosphate, and 0.02-0.05 g magnesium sulfate heptahydrate;

[0014] The solid-state fermentation medium in step 2) comprises 100 g corn husk, 200 g water, 3 g anhydrous glucose, 0.1 g potassium dihydrogen phosphate, and 0.04 g magnesium sulfate heptahydrate;

[0015] The inoculum size in step 2) was 8%, and the cells were cultured at 25°C for 7 days;

[0016] Step 3) The water bath temperature is 80°C and the time is 2 hours;

[0017] The centrifugation in step 3) is carried out at 4000 r / min for 20 min; the ethanol concentration is 95%, and the standing temperature is 4°C.

[0018] The present invention provides Hericium erinaceus He-02-06, whose strain deposit number is CCTCC NO: M 20221043; use of the Hericium erinaceus He-02-06 in increasing the soluble dietary fiber content in corn; and a method for preparing corn husk soluble dietary fiber, which comprises: 1) washing corn husks until the pH value is neutral, drying, and crushing; 2) preparing a solid-state fermentation medium, inoculating Hericium erinaceus He-02-06, and culturing at 22-30°C for 5-10 days; 3) preparing the soluble dietary fiber by weighing the fermented sample obtained in step 2), sieving, adding water, extracting in a water bath at 75-85°C for 1.5-2.5 hours, cooling, centrifuging, and retaining the supernatant; adding ethanol, standing at 0-4°C for 6-12 hours, and drying to obtain the corn husk soluble dietary fiber;

[0019] The present invention takes corn husk as the main research object, adopts fungal solid-state fermentation to modify corn husk dietary fiber, carries out strain screening, optimization of solid-state fermentation medium and culture conditions, and fermentation kinetics research, explores the relationship between extracellular enzymes and changes in related nutrients during solid-state fermentation, and compares and analyzes the changes in structural properties and physicochemical properties of corn husk soluble dietary fiber before and after fermentation, provides a new research idea for the comprehensive utilization of corn husk, and is of great significance for the study of biotransformation of corn processing by-products and product development; beneficial effects: after fermentation, the water holding capacity, swelling capacity and oil holding capacity of corn husk SDF are increased from 2.33±0.11 g / g, 2.21±0.07 mL / g and 2.18±0.06 g / g to 3.65±0.12 g / g, 4.31±0.04 mL / g and 3.93±0.09 g / g, respectively, which are increased by 1.57 times, 1.95 times and 1.80 times, respectively. Through solid-state fermentation modification, the adsorption properties of corn bran SDF have been enhanced. Studies have found that the adsorption capacity of fermented corn bran SDF for nitrite, cholesterol and bile salt has been improved to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Soluble dietary fiber content in corn husk;

[0021] Figure 2 Phylogenetic tree of the strains;

[0022] Figure 3 Results of culture medium response surface analysis;

[0023] Figure 4 Results of response surface analysis of culture conditions;

[0024] Figure 5 Changes in enzyme activity during fermentation;

[0025] Figure 6 Changes in nutrient content during fermentation;

[0026] Figure 7 Electron microscope scanning images of SDF before and after fermentation;

[0027] Figure 8 Infrared spectra of SDF before and after fermentation;

[0028] Figure 9 SDF adsorption capacity before and after fermentation. DETAILED DESCRIPTION

[0029] Experimental materials and reagents: 1) Corn husks were provided by the National Engineering Research Center for Deep Processing of Wheat and Corn, and the corn variety was (Jidan 66); 2) Based on the results of the research team's previous experiments, strains with better (cellulose biodegradation) indicators were selected, and Morchella Me-01 ( Morchella esculenta )、Hericium erinaceus He-02-06( Hericium erinaceus ) and Armillaria mellea Am-07, provided by the National Engineering Center for Deep Processing of Wheat and Corn; all three fungi were isolated from seed bodies collected in Changbai Mountain, Morchella Me-01 was isolated from Morchella fruiting bodies, Hericium erinaceus He-02-06 was isolated and induced from Hericium erinaceus fruiting bodies, and Armillaria mellea Am-07 was isolated from Armillaria fruiting bodies; the Morchella Me-01 was disclosed in the "Research on Solid-State Fermentation of Corn Gluten Powder by Morels" reported by Tong Weina et al.; Hericium erinaceus He-02-06 was deposited in the China Center for Type Culture Collection on July 6, 2022, with the strain collection number CCTCC NO: M20221043; Armillaria Am-07 was disclosed in the "Research on Isolation, Purification and Functional Activity of Active Proteins in Corn Gluten Powder Products Fermented by Armillaria" reported by He Yinhua.

[0030] Example 1 Screening of mutant strains

[0031] 1. Mutation method of bacterial strains

[0032] The laboratory preserved Morchella Me-01 ( Morchella esculenta )、Hericium erinaceus He-02( Hericium erinaceus ) and Armillaria mellea Am-07 were used as starting strains. Protoplast suspensions of the strains to be mutated were placed on plates, agitated with a rotor, and then placed 30 cm below a 15W UV lamp for UV mutagenesis using an irradiation dose of 20-90 seconds. The induced bacterial suspension was spread on regeneration medium and incubated in a 23°C incubator in the dark for 10 days. The colonies that grew were counted, and a lethality curve was plotted. Strains with good growth were selected as mutants and cultured under appropriate conditions. Mutant strains with soluble dietary fiber content exceeding 15% of the starting strain were selected as positive mutants. These strains were serially passaged 10 times, with fermentation tests conducted every other generation. Mutants with stable production performance after mutagenesis were selected for sequencing and strain identification, and stored in a refrigerator at 4°C.

[0033] 2. Comparison of mutagenic effects

[0034] After the He-02 strain was subjected to UV mutagenesis treatment, solid-state fermentation was performed to investigate its effect on the soluble dietary fiber content in corn husk dietary fiber. Figure 1 After solid-state fermentation of corn husks using its mutant strain, the soluble dietary fiber content in the corn husks was increased.

[0035] The isolated and purified bacterial solution was sent to Jilin Kumei Biotechnology Co., Ltd. for testing. The company extracted the DNA genome of the bacterial solution and then amplified the ribosomal DNA and ITS sequences, as shown in the table below, to obtain the PCR products, which were then sequenced.

[0036]

[0037] The bacterial liquid DNA sent for testing was amplified using ITS4 and ITS5 universal fungal primers and successfully spliced ​​into a 623 bp linker fragment. The splicing results are shown below. The sequence was measured and the tree diagram of the strain was drawn using MEGA7.1 software, as shown below. Figure 2 The figure shows that the mutant strain of He-02 has a sequence similarity of up to 99% with AY 534590.1. The strain was identified as Hericium erinaceus; Hericium erinaceus He-02-06 was deposited in the China Center for Type Culture Collection on July 6, 2022, with the strain accession number CCTCC NO: M 20221043.

[0038]

[0039] Example 2 Bacteria culture

[0040] 1. Solid slant culture of bacteria

[0041] Me-01, He-02-06 and Am-07 strains were inoculated onto malt agar medium, respectively, and cultured in a constant temperature incubator at 27°C for 7 days.

[0042] 2. Liquid Activation Culture of Bacteria

[0043] 1. Preparation of primary bacterial cultures: Aliquot 30 mL of liquid activated culture medium into each 100 mL conical flask and sterilize for later use. Inoculate the activated solid slant bacterial cultures Me-01, He-02-06, and Am-07 into their corresponding liquid activated culture medium, respectively. Incubate at 27°C and 160 rpm for 6 days to obtain primary fermentation cultures. Store in a refrigerator at 4°C until use.

[0044] 2. Preparation of Secondary Culture: Aliquot 200 mL of liquid activated culture medium into each 500 mL conical flask and sterilize for later use. Inoculate the primary fermentation culture into the liquid activated culture medium and culture at 27°C and 160 rpm for 6 days to obtain the secondary fermentation culture. Store in a refrigerator at 4°C until use.

[0045] Me-01 liquid activation medium: anhydrous glucose 2.0%, yeast extract powder 1.0%, potassium dihydrogen phosphate 0.12%, magnesium sulfate heptahydrate 0.075%, ferrous sulfate 0.001%;

[0046] He-02-06 liquid activation medium: 3.0% anhydrous glucose, 1.0% yeast extract powder, 2.0% soluble starch, 0.3% potassium dihydrogen phosphate, 0.06% magnesium sulfate heptahydrate;

[0047] Am-07 liquid activation medium: silkworm pupa powder 0.5%, potato 20.0%, glucose 1.0%, yeast extract powder 2.0%, sucrose 1.0%, magnesium sulfate heptahydrate 0.075%, potassium dihydrogen phosphate 0.15%, VB10.001%.

[0048] Example 3 Screening of solid-state fermentation-modified corn husk dietary fiber strains

[0049] 1. Corn husk processing and composition testing

[0050] Corn husk pretreatment: Wash the corn husk with deionized water until neutral, dry and crush at 50°C, and store in a drying dish for later use; basic component detection method is as follows:

[0051] 1) Moisture: GB5009.3-2016 direct drying method;

[0052] 2) Total protein: GB5009.5-2016 Kjeldahl method;

[0053] 3) Total starch: GB5009.9-2016 enzymatic hydrolysis method;

[0054] 4) Crude fat: GB5009.6-2016 Soxhlet extraction method;

[0055] 5) Dietary fiber: GB5009.88-2014 enzymatic gravimetric method;

[0056] 6) Ash content: GB5009.4-2016 ignition method;

[0057] The basic component test results of corn husk are shown in Table 3. It can be seen from Table 3 that the main component of corn husk is dietary fiber, which contains carbon source, nitrogen source, water, fat, a small amount of ash and other components required for microbial fermentation growth, and can be used as a culture medium for microbial solid-state fermentation.

[0058]

[0059] 2. Solid-state fermentation culture method

[0060] 1. Preparation of solid-state fermentation medium

[0061] The culture medium components are:

[0062] 1) Me-01: 15 g corn husk, 2.0% anhydrous glucose, 0.15% potassium dihydrogen phosphate, 0.15% magnesium sulfate heptahydrate;

[0063] 2) He-02-06: corn husk 15 g, anhydrous glucose 3.0%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.06%;

[0064] 3) Am-07: corn husk 15 g, anhydrous glucose 2.4%, potassium dihydrogen phosphate 0.25%, magnesium sulfate heptahydrate 0.15%;

[0065] 2. Strain screening

[0066] The solid-state fermentation medium was prepared according to the composition of the three bacterial strains. After sterilization, the secondary fermentation strains of each of the three bacterial strains were inoculated into the solid-state fermentation medium. After fermentation, the medium was dried and crushed for later use. Changes in nutritional components such as total starch, reducing sugars, total protein, free amino acids, soluble protein, and dietary fiber content before and after fermentation were compared to identify the optimal strain for solid-state fermentation of modified corn husk dietary fiber.

[0067] The specific corn husk solid-state fermentation culture method is as follows:

[0068] Mix corn husks and water in a 1:2 ratio. Add 3.0% anhydrous glucose, 0.1% potassium dihydrogen phosphate, and 0.06% magnesium sulfate heptahydrate (based on the weight of the corn husks). Mix thoroughly and sterilize at 121°C for 20 minutes. After cooling, inoculate the mixture at 10% of the inoculum size and incubate at 27°C for 7 days.

[0069] 3. Nutritional testing

[0070] 1) Total starch: GB5009.9-2016 enzymatic hydrolysis method;

[0071] 2) Reducing sugar content determination method

[0072] The reducing sugar content was determined by referring to Zeng Zhiheng's method and modifying it.

[0073] Preparation of the standard curve: Pipette 0.00, 0.20, 0.40, 0.80, 1.00, and 1.20 mL of 1 mg / mL glucose standard solution into a 10-mL centrifuge tube, add distilled water to 2.00 mL, add DNS reagent to 4.00 mL, heat in a boiling water bath, and cool to room temperature. Dose to volume, shake well, and measure the OD value at 540 nm to draw the glucose standard curve.

[0074] Sample preparation and reducing sugar detection: Accurately weigh 1.00 g corn husk powder (accurate to 0.0001 g), add a small amount of water, and extract in an 80°C water bath for 10 min. Wash with distilled water and transfer to a 25 mL volumetric flask, make up to volume, and filter. Take 5.00 mL of the filtrate and transfer to a 25 mL volumetric flask, make up to volume, and then take 1.00 mL of the sample solution in a 10 mL centrifuge tube and add distilled water to 2.00 mL. The determination method is the same as the standard curve. After measuring the OD value, calculate the reducing sugar concentration according to the standard curve.

[0075] 3) Total protein: GB5009.5-2016 Kjeldahl method;

[0076] 4) Free amino acid content detection

[0077] The free amino acid content was detected by ninhydrin colorimetry;

[0078] To prepare the standard curve, pipette 1.60, 3.20, 6.40, 8.00, and 12.80 mL of a 1 mg / mL L-leucine standard solution into a 25 mL volumetric flask and dilute to volume with distilled water. To a 10 mL centrifuge tube, add 200 μL of acetic acid-sodium acetate buffer, 200 μL of the L-leucine standard solution, and 400 μL of ninhydrin solution, sequentially. Shake well and heat in a boiling water bath. Cool to room temperature. Add 4.80 mL of distilled water and measure the OD at 570 nm to prepare the L-leucine standard curve.

[0079] Sample preparation and free amino acid detection: Accurately weigh 0.10 g corn husk powder (accurate to 0.0001 g), add 5.00 mL of distilled water and mix well. Incubate at 90°C for 10 min. The determination method is the same as the standard curve. Measure the OD value at 570 nm and calculate the free amino acid content based on the standard curve.

[0080] 5) Soluble protein content detection

[0081] Soluble protein content was determined using the Coomassie Brilliant Blue G-250 colorimetric method. Accurately weigh 1.00 g of corn husk powder (accurate to 0.0001 g) and add normal saline at a ratio of 1:9 (w / v). Mix thoroughly and centrifuge at 2500 rpm for 10 minutes. Dilute the supernatant fivefold with normal saline for later use. Prepare three test tubes and add 0.05 mL of distilled water, a protein standard, and a sample to serve as the blank, standard, and assay groups, respectively. Add 3.00 mL of Coomassie Brilliant Blue colorimetric solution to each test tube, mix thoroughly, and let stand for 10 minutes. Measure the OD at 595 nm to calculate the soluble protein content.

[0082] 6) Dietary fiber: GB5009.88-2014 enzymatic gravimetric method;

[0083] 4. Results

[0084] The effects of solid-state fermentation of three strains on the nutritional components and dietary fiber content of corn husks are shown in Table 4. As can be seen from the table, the total starch content of corn husks decreased after solid-state fermentation by the three fungi, among which He-02-06 had the largest decrease, from 9.88±0.33g / 100g to 5.01±0.13g / 100g, indicating that He-02-06 had the highest starch utilization rate. The reducing sugar content of the three strains increased significantly after fermentation, among which He-02-06 had the highest reducing sugar content after fermentation, which was 2.10 times that before fermentation. The total protein content of the matrix after fermentation of Me-01 was 1.4477mg / L, which was 1.3377mg / L. The content of corn bran dietary fiber did not change significantly after fermentation with the other two fungi, while the total protein content increased after fermentation with the other two fungi. The free amino acid content increased significantly after fermentation, with He-02-06 showing the greatest increase. The soluble protein content decreased to varying degrees after fermentation with the three fungi. The IDF content decreased after fermentation with the three fungi, likely due to the partial conversion of IDF to SDF during fermentation, which reduced the IDF content. The SDF content of corn bran increased to a certain extent after fermentation with the three fungi, with He-02-06 showing the highest SDF content of 2.15±0.03 g / 100g, followed by Am-07 and Me-01. Based on the experimental results, He-02-06 was determined to be the optimal strain for solid-state fermentation of corn bran dietary fiber, and further experimental research was conducted.

[0085]

[0086] Note: Different letters in the same row represent different significant differences ( P <0.05).

[0087] Example 4 Optimization of culture conditions for solid-state fermentation of modified corn husk dietary fiber

[0088] 1. Culture medium optimization

[0089] 1. Single-factor experiment

[0090] 1) Effect of corn husk addition on SDF content

[0091] Corn husk powder (5.0, 10.0, 15.0, 20.0, and 25.0 g) was added to conical flasks, respectively. The addition amounts of other culture medium components were fixed. The SDF content was determined after constant culture at 27°C for 7 days at a solid-liquid ratio of 1:2 and an inoculum size of 10%.

[0092] 2) Effect of material-liquid ratio on SDF content

[0093] After adding the culture medium components, deionized water was added to the conical flask at a mass-to-volume ratio of 1:1, 1:1.5, 1:2, 1:2.5, and 1:3, respectively. The SDF content was determined after constant culture at 27°C for 7 days with an inoculum size of 10%.

[0094] 3) Effect of anhydrous glucose on SDF content

[0095] After adding other culture medium components, the addition amounts of anhydrous glucose were determined to be 1.5%, 2.0%, 2.5%, 3.0%, and 3.5%, respectively. The SDF content was determined after constant temperature culture at 27°C for 7 days at a solid-liquid ratio of 1:2 and an inoculum size of 10%.

[0096] 4) Effect of potassium dihydrogen phosphate on SDF content

[0097] After adding other culture medium components, the addition amounts of potassium dihydrogen phosphate were determined to be 0.05%, 0.10%, 0.15%, 0.20%, and 0.25%, respectively. The SDF content was determined after constant temperature cultivation at 27°C for 7 days at a solid-liquid ratio of 1:2 and an inoculum size of 10%.

[0098] 5) Effect of magnesium sulfate heptahydrate on SDF content

[0099] After adding other culture medium components, the addition amounts of magnesium sulfate heptahydrate were determined to be 0.02%, 0.04%, 0.06%, 0.08%, and 0.10%, respectively. The SDF content was determined after constant temperature cultivation at 27°C for 7 days with a solid-liquid ratio of 1:2 and an inoculum size of 10%.

[0100] Results: The effect of corn bran addition on SDF content showed an initial increase followed by a decrease. The highest SDF content, reaching 6.81 g / 100 g, was achieved when the corn bran addition was 15 g. When the corn bran addition was less than 15 g, the SDF content was low, likely because the nutrients in the corn bran were insufficient for bacterial growth and fermentation. As the corn bran addition increased, the space within the conical flask decreased, reducing oxygen content and hindering bacterial growth and metabolism, which also affected SDF content. Therefore, 15 g of corn bran was selected as the optimal addition amount.

[0101] The SDF content reached its maximum at material-to-liquid ratios of 1:1.5 and 1:2, with no significant difference, reaching 6.57 g / 100 g and 6.81 g / 100 g, respectively. Fungal solid-state fermentation requires a certain amount of moisture in the substrate. Low moisture content impairs normal bacterial growth and enzyme production; high moisture content leads to clumping of the culture medium, impairing aeration and negatively impacting bacterial growth. Therefore, a material-to-liquid ratio of 1:2 was used as the primary focus for subsequent experiments.

[0102] The effect of anhydrous glucose on SDF content showed a trend of first decreasing, then increasing, and then decreasing again. The SDF content reached its highest level at 7.16 g / 100 g when added at a 3.0% concentration. When anhydrous glucose addition exceeded 3.0%, the excessively high carbon-nitrogen ratio inhibited bacterial growth and metabolism. An appropriate anhydrous glucose concentration facilitated SDF conversion. Therefore, a 3.0% anhydrous glucose addition was selected for subsequent experiments.

[0103] The effect of potassium dihydrogen phosphate on SDF content showed a trend of first increasing and then decreasing. When the addition amount was 0.10%, the SDF content was the highest, at 6.52 g / 100g. If the addition amount of inorganic salts was too low, it would not be enough to support the growth of fungi. If the addition amount was too high, the excessive heavy metal ion content would be toxic to fungal cells, reducing the physiological activity of fungi and leading to a decrease in SDF content.

[68] Therefore, 0.10% potassium dihydrogen phosphate was selected as the core for subsequent experiments.

[0104] The effect of magnesium sulfate heptahydrate on SDF content showed a trend of increasing first and then decreasing. When the addition amount was 0.04%, the SDF content was the highest, which was 12.53 g / 100g. 2+ It stimulates fungal cell division and enzyme production, and can promote bacterial growth. 2+Excessively high concentrations can damage bacterial cells, reduce bacterial activity, and lead to a decrease in SDF content. Therefore, a concentration of 0.04% magnesium sulfate heptahydrate was selected as the core for subsequent experiments.

[0105] 2. Response surface experiment

[0106] Based on single-factor experiments, 15 g of corn husk and 3.0% glucose were added to the fixed medium. Using the SDF content of corn husk as an indicator, a response surface design (RSD) experiment was conducted using three factors: A: solid-liquid ratio; B: potassium dihydrogen phosphate; and C: magnesium sulfate heptahydrate. The design table of experimental factor levels is shown in Table 5. A response surface optimization of the solid-state fermentation medium components was performed using Design-Expert 12. The optimal process was determined and experimentally verified.

[0107]

[0108] result

[0109] Based on the results of the single-factor test of culture medium components and the response surface design, a Box-Behnken experimental design was performed to obtain the optimal conditions for fermentation-modified SDF. The experimental scheme and results are shown in Table 6, and the variance analysis results are shown in Table 7. Design-Expert 12 software was used to perform a multiple regression fitting analysis on the data in Table 6, and the regression equation between the three factors of solid-liquid ratio (A), potassium dihydrogen phosphate (B), and magnesium sulfate heptahydrate (C) and SDF content was obtained:

[0110] SDF content (g / 100g) = 7.01 + 0.5175 A +0.3125 B -0.4400 C -0.8900 AB -0.4350 A +0.0950 BC - 2.19 A ²-1.15 B ²-2.01 C ²

[0111] From the results of the model regression coefficient and variance analysis in Table 7, we can see that the model is extremely significant ( P <0.0001), determination coefficient R 2=0.9841, the test situation has a good fit with the actual situation, the predicted value has a strong correlation with the experimental value, and the lack-of-fit error is 0.1880, which is not significant, indicating that the model can fit the test situation well and can well explain the relationship between the three influencing factors and SDF content. In this experiment, the order of the influence of the three factors of solid-liquid ratio, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate on SDF content is: A (solid-liquid ratio) > C (magnesium sulfate heptahydrate) > B (potassium dihydrogen phosphate).

[0112]

[0113]

[0114] Note: “*” indicates significant impact on the results ( P <0.05); “**” indicates that the effect on the results is extremely significant ( P <0.01).

[0115] (2) Response surface analysis

[0116] The response surface analysis showed that the interaction between the three factors affected the SDF content. Figure 3 As shown. Figure 3 It can be seen that the SDF content shows a trend of first increasing and then decreasing with the increase of the level of each factor, and the interaction between the factors is significant, indicating that this factor has the greatest impact on the SDF content, indicating that the interaction between the material-liquid ratio and potassium dihydrogen phosphate during the fermentation process is the most significant.

[0117] Analysis using Design-Expert 12 software revealed that the optimal medium composition predicted by the response surface optimization model was: 15 g corn bran, a solid-liquid ratio of 1:2.056, 3.0% anhydrous glucose, 0.104% potassium dihydrogen phosphate, and 0.038% magnesium sulfate heptahydrate. The theoretically calculated maximum SDF content was 7.077 g / 100 g. To facilitate experimental analysis, the fermentation medium composition was adjusted to 15 g corn bran, a solid-liquid ratio of 1:2, 3.0% anhydrous glucose, 0.10% potassium dihydrogen phosphate, and 0.04% magnesium sulfate heptahydrate. Three parallel experiments were conducted under these conditions to verify the measured SDF content, resulting in a small error between the predicted values ​​and 7.14 ± 0.131 g / 100 g. This demonstrates that the model effectively optimizes SDF content.

[0118] 2. Optimization of culture conditions

[0119] Based on the optimization experiment of solid-state fermentation modified corn husk dietary fiber culture medium, the culture conditions of solid-state fermentation were studied by single factor and response surface optimization method to determine the optimal fermentation process parameters.

[0120] 1. Single-factor experiment

[0121] 1) Effect of inoculum size on SDF content

[0122] The culture medium was prepared according to the optimal culture medium composition. After sterilization, the secondary liquid fermentation strain was inoculated into the culture medium at an inoculum rate of 6%, 8%, 10%, 12%, and 14%, respectively. The SDF content was determined after constant temperature culture at 27°C for 7 days.

[0123] 2) Effect of culture temperature on SDF content

[0124] The culture medium was prepared according to the optimal culture medium composition. After sterilization, the secondary liquid fermentation strain was inoculated into the culture medium at a 10% inoculation rate. The culture medium was cultured at 23°C, 25°C, 27°C, 29°C, and 31°C for 7 days, and the SDF content was determined.

[0125] 3) Effect of culture time on SDF content

[0126] The culture medium was prepared according to the optimal culture medium composition. After sterilization, the secondary liquid fermentation strain was inoculated into the culture medium at a 10% inoculum rate. The culture was cultured at 27°C for 3, 5, 7, 9, and 11 days, and the SDF content was determined after that.

[0127] 4) Results

[0128] The highest SDF content, 8.76 g / 100 g, was achieved when the inoculum size was 8%. Low SDF content below 8% may be due to a small bacterial strain, low enzyme production, and low bacterial activity, resulting in a weaker ability to modify corn husk dietary fiber. The decrease in SDF content when the inoculum size exceeded 8% may be due to an excessively high inoculum size, insufficient culture medium nutrients for bacterial growth, and a consequent impact on the fungal fermentation capacity. Therefore, an inoculum size of 8% was selected as the primary inoculum for subsequent experiments.

[0129] The effect of culture temperature on SDF content showed an initial increase followed by a decrease, with the highest SDF content at 25°C, reaching 7.24 g / 100g. At temperatures below 25°C, bacterial growth was slow, and extracellular enzyme activity was low, resulting in lower SDF content. At temperatures exceeding 25°C, the culture environment became increasingly unfavorable for bacterial growth, and the significant water loss hindered mycelial accumulation and the synthesis of related enzymes, leading to a decrease in SDF content. Therefore, a culture temperature of 25°C was selected as the central temperature for subsequent experiments.

[0130] The effect of culture time on SDF content showed an initial increase followed by a decrease, with SDF content reaching its highest values ​​on the 7th and 9th days of culture, at 7.40 g / 100 g and 7.38 g / 100 g, respectively. SDF content gradually increased with culture time, but decreased after 9 days. This may be because the nutrients in the culture medium became insufficient for bacterial growth, leading to the consumption of SDF by bacterial growth. Furthermore, with prolonged culture time, the water content in the culture medium decreased, the soluble components decreased, bacterial viability and extracellular enzyme activity decreased, and metabolic capacity weakened, resulting in a decrease in SDF content. Therefore, a 7-day culture time was selected as the central focus for subsequent experiments.

[0131] 2. Response surface experiment

[0132] Based on the single-factor experiments, a response surface design (RSD) experiment was conducted using corn bran SDF content as an indicator for the three factors. The design table of experimental factor levels is shown in Table 8. A response surface optimization method was used to optimize the culture conditions for solid-state fermentation using Design-Expert 12. The optimal process parameters for the fermentation conditions were determined and experimentally verified.

[0133]

[0134] IBM SPSS Statistics 23 software was used for data analysis, Graph Pad Prism 8 software was used for plotting, and Design-Expert 12 software was used for response surface design and plotting. All experiments were repeated three times, and the results are expressed as mean ± standard deviation.

[0135] (1) Response surface optimization experimental design and results

[0136] Based on the single-factor experimental results of solid-state fermentation and the response surface design, a Box-Behnken experimental design was conducted to obtain the optimal conditions for solid-state fermentation-modified SDF. The experimental scheme and results are shown in Table 9, and the variance analysis is shown in Table 10. Design-Expert 12 software was used to perform a multiple regression fitting analysis on the data in Table 2-9, and the regression equation between the three factors of inoculation size (A), culture temperature (B), and culture time (C) and the SDF content was obtained:

[0137] SDF content (g / 100g) = 8.13 + 0.7150 A +0.6737 B +0.2363 C -0.8750 AB -0.5200 AC +0.2725 BC -

[0138] 2.48A ²-1.53 B ²-1.80 C ²

[0139] From the results of the model regression coefficient and variance analysis, we can see that the model is extremely significant ( P <0.05), determination coefficient R 2 =0.9557, indicating that the predicted value fits the actual situation well. In this experiment, the order of the influence of the three factors of inoculum size, culture temperature, and culture time on SDF content is: A (inoculum size) > B (culture temperature) > C (culture time).

[0140]

[0141]

[0142] Note: “*” indicates significant impact on the results ( P <0.05)

[0143] (2) Response surface analysis

[0144] The response surface analysis showed that the interaction between the three factors affected the SDF content. Figure 4 As shown in the figure, the SDF content showed a trend of first increasing and then decreasing with the increase of the level of each factor. The interaction between the factors was significant, indicating that the inoculation amount had the greatest impact on the SDF content, and that the interaction between the inoculation amount and the culture temperature during the fermentation process was the most significant.

[0145] Analysis by Design-Expert 12 software showed that the optimal culture and fermentation conditions predicted by the response surface optimization model were: inoculation size 8.205%, culture temperature 25.395 ℃, and culture time 7.132 d. The theoretically calculated maximum SDF content was 8.245 g / 100 g. To facilitate the experiment, the fermentation conditions were adjusted to inoculation size 8%, culture temperature 25 ℃, and culture time 7 d. Under these conditions, three parallel experiments were carried out for verification. The measured SDF content was 8.39±0.14 g / 100 g, which had a small error with the predicted value. This indicated that the model had a good optimization fitting effect on the SDF content, and the SDF content was 24.42 times higher than that before modification.

[0146] Example 5 Fermentation Kinetics of Corn Husk Solid-State Fermentation by He-02-06

[0147] The preparation of the culture medium and the solid-state fermentation culture method were carried out using the results optimized in Example 3.

[0148] 1. Detection of extracellular enzyme activity during fermentation

[0149] 1. Extraction and preservation of crude enzyme solution

[0150] Add 20 mL of citric acid buffer solution (0.05 mol / L pH 4.6) to 2 g of fermented solid culture medium, extract in a constant temperature water bath at 30 °C for 2 h, centrifuge the extract at 4 °C and 8000 r / min for 15 min, and the obtained supernatant is the crude enzyme solution. Dispense into EP tubes and store at -80 °C for later use.

[69] .

[0151] 2. Amylase activity determination

[0152] Add 0.5 mL of a five-fold diluted crude enzyme solution (preheated at 40°C for 15 min) to 1.0 mL of preheated 1% soluble starch solution (prepared with 0.1 mol / L, pH 5.6 citrate buffer) and mix thoroughly. Incubate in a 40°C water bath for 30 min. Add 2.00 mL of DNS reagent and boil in a boiling water bath for 5 min. Cool to terminate the reaction. Dilute to 25 mL and measure the OD at 520 nm. Calculate the glucose concentration based on the standard curve. Use the inactivated crude enzyme solution as a control. Enzyme activity is expressed in U / mL using the following formula:

[0153]

[0154] Where: C: glucose concentration in the reaction system, mg / mL; V: total volume of the reaction system, mL; W: dilution factor of crude enzyme solution; 1000: conversion factor of mg glucose to μg; t: reaction time, min; V1: amount of enzyme involved in the reaction, mL.

[0155] 3. Acid protease activity determination

[0156] The test was performed with reference to Yuan Shengdong's experimental method. 1.0 mL of crude enzyme solution diluted five times was mixed with 1.0 mL of 1% casein solution preheated at 40°C for 5 minutes. The mixture was placed in a 40°C constant temperature water bath and kept warm for 10 minutes. After removal, 2.00 mL of 0.4 mol / L trichloroacetic acid solution was added. After incubation for 15 minutes, the mixture was centrifuged at 4000 r / min for 10 minutes. 0.5 mL of the supernatant was taken, 2.5 mL of Na2CO3 solution and 0.5 mL of Folin-phenol reagent were added and shaken. The mixture was incubated in a 40°C water bath for 20 minutes, cooled to room temperature, and the OD value at 680 nm was measured. The L-tyrosine concentration was calculated according to the standard curve, with the inactivated crude enzyme solution used as a control. The enzyme activity unit is expressed in U / mL, and the calculation formula is as follows:

[0157]

[0158] Where: C: tyrosine concentration in the reaction system, mg / mL; V: total volume of the reaction system, mL; W: dilution factor of crude enzyme solution; t: reaction time, min; V1: amount of enzyme involved in the reaction, mL.

[0159] 4. Carboxymethyl cellulose (CMC) enzyme activity assay

[0160] The assay was performed using Li Hao's method. 0.5 mL of a five-fold diluted crude enzyme solution and 1.5 mL of 0.5% sodium carboxymethyl cellulose solution (prepared with 0.1 mol / L, pH 4.6 acetate buffer) were placed in a 50°C water bath for 30 minutes, followed by the addition of 2.00 mL of DNS. The reaction was terminated by boiling in a water bath for 5 minutes and then cooled to 25 mL. The OD value at 540 nm was measured, and the glucose concentration was calculated based on the standard curve. The inactivated crude enzyme solution was used as the control group. Enzyme activity units are expressed in U / mL, and the calculation formula is as follows:

[0161]

[0162] Where: C: glucose concentration in the reaction system, mg / mL; V: total volume of the reaction system, mL; W: dilution factor of crude enzyme solution; 1000: conversion factor of mg glucose to μg; t: reaction time, min; V1: amount of enzyme involved in the reaction, mL.

[0163] 5. Hemicellulose (HC) enzyme activity determination

[0164] Huang Wanbing's method was modified with reference to the following: 0.5 mL of a five-fold diluted crude enzyme solution preheated at 50°C for 5 min was mixed with 1.0 mL of a 1% xylan solution (preheated at 50°C for 5 min using 0.1 mol / L pH 4.6 acetate buffer). The mixture was incubated in a 50°C water bath for 30 min. 2.00 mL of DNS reagent was added and the mixture was boiled in a water bath for 5 min before cooling to terminate the reaction. The volume was then adjusted to 25 mL and the OD at 540 nm was measured. The xylose concentration was calculated using the standard curve. The inactivated crude enzyme solution was used as the control group. Enzyme activity was expressed in U / mL using the following formula:

[0165]

[0166] Where: C: xylose concentration in the reaction system, mg / mL; V: total volume of the reaction system, mL; W: dilution factor of crude enzyme solution; 1000: conversion factor of mg xylose to μg; t: reaction time, min; V1: amount of enzyme involved in the reaction, mL.

[0167] 2. Changes in nutrients during fermentation

[0168] The changes in the content of nutrients and dietary fiber during the fermentation process were detected. The detection methods for total starch, reducing sugar, total protein, soluble protein, free amino acids, and dietary fiber content were the same as those in Example 2.

[0169] 3. Statistics and Analysis

[0170] Data were analyzed using IBM SPSS Statistics 23 software, and graphs were plotted using Graph Pad Prism 8 software. Each experiment was repeated three times.

[0171] IV. Results

[0172] 1. Changes in amylase activity during fermentation

[0173] 1) Glucose standard curve

[0174] A standard curve was drawn to detect amylase activity. The glucose concentration was calculated based on the standard curve regression equation: y = 19.464x -0.0168, R² = 0.9993.

[0175] 2) Changes in amylase activity

[0176] Changes in amylase activity during fermentation Figure 5 As shown in A. Figure 5 A shows that the amylase activity showed an overall trend of first increasing and then decreasing during the fermentation process. The amylase activity was significantly enhanced in the first 6 days of fermentation (P < 0.05) and reached a maximum of 189.90 U / mL on the 6th day. The amylase activity showed a downward trend from 8 to 12 days, and the lowest activity dropped to 159.69 U / mL on the 12th day.

[0177] 2. Changes in acid protease activity during fermentation

[0178] 1) L-Tyrosine standard curve

[0179] Draw the standard curve of acidic protease activity and use the regression equation according to the standard curve: y = 9.0536 x +0.0719, R ² = 0.9995, calculate the L-tyrosine concentration.

[0180] 2) Changes in acidic protease activity

[0181] The changes of acid protease activity during fermentation Figure 5 As shown in Figure B. The figure shows that the activity of acid protease showed a trend of continuous growth from 0 to 7 days during the fermentation process, and the activity growth was relatively slow in the first 4 days of fermentation, and the activity growth was most significant on the 7th day ( P<0.05), after a slight decrease on the 8th day, there was an inflection point of activity change on the 9th day and reached a peak of 149.33 U / mL on the 11th day, and then decreased to 97.81 U / mL on the 12th day.

[0182] 3. Changes in carboxymethyl cellulase activity during fermentation

[0183] 1) Glucose standard curve

[0184] Draw the standard curve of carboxymethyl cellulase activity and use the regression equation according to the standard curve: y = 14.536 x -0.0622, R ² = 0.9990, calculate the glucose concentration.

[0185] 2) Changes in carboxymethyl cellulase activity

[0186] Changes in carboxymethyl cellulose activity during fermentation Figure 5 As shown in Figure C. As can be seen from the figure, the activity of carboxymethyl cellulase showed an increasing trend in the early stage of fermentation, and the activity was significantly enhanced during the 1-4 days of fermentation ( P <0.05), the activity decreased from 4 to 6 days and reached a peak of 120.18 U / mL on the 7th day. The activity decreased from 7 to 10 days and there was an inflection point on the 11th day. The activity decreased to 95.95 U / mL on the 12th day.

[0187] 4. Changes in hemicellulase activity during fermentation

[0188] 1) Xylose standard curve

[0189] Draw the standard curve of hemicellulase activity and use the regression equation according to the standard curve: y =18.302 x -0.0003, R =0.9993, calculate the xylose concentration.

[0190] 2) Changes in hemicellulase activity

[0191] Changes in hemicellulase activity during fermentation Figure 5 As shown in Figure D, the activity of hemicellulase increased significantly in the first 4 days of fermentation and reached a peak activity of 211.04 U / mL on the 4th day. The activity decreased from 4 to 6 days and the decrease was the largest on the 6th day. The hemicellulase activity reached the lowest value of 166.30 U / mL. The activity increased significantly on the 7th day, decreased on the 8th day, and then slowly increased from 9 to 10 days, and there was an inflection point on the 11th day.

[0192] 5. Detection of changes in nutrient content during fermentation

[0193] 1) Changes in total starch and reducing sugar content during fermentation

[0194] The changes of total starch content during fermentation Figure 6 As shown in A, the reducing sugar content changes as Figure 6 As shown in B. Figure 6 As shown in A, the starch content showed an overall downward trend during the fermentation process. The starch content decreased the most during the 0-6 days of fermentation. The downward trend was relatively slow and tended to be gentle in the middle and late stages of fermentation, fluctuating around 3.27%. This may be because the Hericium erinaceus grew rapidly in the early stage of fermentation and the amylase activity was high. In the late stage of fermentation, the growth of Hericium erinaceus tended to be stable or began to age, and the amylase activity decreased. In addition, the culture medium was rich in nutrients, which reduced the utilization of starch. Figure 6 As shown in Figure 2, the reducing sugar content showed an overall downward trend during fermentation. It increased significantly between days 0 and 2, reaching a peak of 7.04 mg / g on day 2. It then decreased significantly between days 2 and 6, with a sharp drop on day 6. It then showed an initial increase followed by a decrease, reaching a minimum of 0.56 mg / g on day 10 before stabilizing. This is consistent with Yuan Shengdong's findings on changes in reducing sugar content within the substrate during Hericium erinaceus fermentation. Changes in reducing sugar content during fermentation can reflect changes in the activity of carboxymethyl cellulase and hemicellulase, as well as the conversion between IDF and SDF. In the early stages of fermentation, IDF is hydrolyzed to produce reducing sugars, and the amount of reducing sugar produced exceeds the amount consumed, leading to accumulation. In the later stages of fermentation, the consumption of reducing sugars by Hericium erinaceus fermentation growth, limited enzyme activity, and the inhibition of enzymes by enzymatic hydrolysis products lead to a gradual decrease in reducing sugar content.

[0195] 2) Changes in the content of total protein, soluble protein and free amino acids during fermentation

[0196] The changes of total protein content during fermentation Figure 6 As shown in C, the changes in soluble protein content are as follows Figure 6 As shown in D, the changes in free amino acid content are as follows Figure 6 As shown in Figure E. As can be seen from the figure, the change trends of total protein, soluble protein and free amino acids during the fermentation process are roughly the same. The total protein content reached a peak of 12.78% on the 10th day. In the early stage of fermentation, Hericium erinaceus grows and reproduces rapidly, and the mycelium fermentation growth produces bacterial protein, which leads to an increase in total protein content. In the late stage of fermentation, the culture medium is lacking in nutrients, and the mycelium may undergo autolysis, resulting in a decrease in total protein content. Figure 6 D and Figure 6E showed that both soluble protein and free amino acid contents increased significantly during fermentation (P < 0.05), with similar trends to those of acidic protease activity. Soluble protein content peaked on day 11 at 21.37 mg / g, 1.75 times the pre-fermentation level. Free amino acid content showed no significant changes during the early stages of fermentation (P < 0.05), but began to rise sharply on day 5, reaching a peak of 245.64 mg / g on day 9, 2.06 times the pre-fermentation level. During the fermentation process, the activity of the acidic protease continued to increase, along with its hydrolytic capacity, which in turn increased the content of soluble protein and free amino acids.

[0197] 3) Changes in IDF and SDF content during fermentation

[0198] The changes of IDF content during fermentation Figure 6 As shown in F, the SDF content changes as Figure 6 As shown in G. Figure 6 It can be seen from the results that the IDF content showed a gentle downward trend during the fermentation process. The IDF content remained almost unchanged in the early stage of fermentation. As the fermentation time prolonged, the IDF content gradually decreased. On the 12th day of fermentation, the IDF content in the culture medium dropped to 64.29% of the raw material content. Figure 6 As shown in Figure 1, the SDF content initially increased and then slowly decreased during fermentation. On days 6 and 7 of fermentation, the SDF content in the culture medium stabilized at approximately 9.38 g / 100 g before slowly decreasing. This suggests that IDF is converted to SDF during fermentation. Furthermore, in the early stages of fermentation, the hydrolytic action of enzymes such as amylase and protease produced by Hericium erinaceus breaks the chemical bonds connecting SDF to components such as starch and protein, thereby increasing the purity and content of SDF. As the small-molecule nutrients in the culture medium are consumed, enzymes such as carboxymethyl cellulase and hemicellulase produced during fermentation degrade IDF and SDF in the corn husk, producing small-molecule nutrients such as oligosaccharides, disaccharides, and monosaccharides that feed bacterial growth, leading to a gradual decrease in IDF and SDF content. However, due to the gradual aging of the mycelium in the later stages of fermentation and its weakened ability to absorb nutrients, the decrease in IDF and SDF was minimal.

[0199] Example 6 Effect of solid-state fermentation on the properties of corn husk soluble dietary fiber

[0200] 1. SDF Preparation

[0201] Corn husk processing: 1) Wash the corn husks with deionized water until the pH is neutral before fermentation, dry them at 50°C, and crush them for later use; 2) Solid-state ferment the corn husks according to the optimal fermentation conditions of Example 4, using a fermentation medium prepared using the medium conditions optimized in Example 4; dry and crush the fermented sample for later use;

[0202] Accurately weigh the samples before and after fermentation, pass them through a 60-mesh sieve, add distilled water at a solid-liquid ratio of 1:20 (W / V), and extract them in an 80°C water bath for 2 h. Use a vortex mixer during the extraction. After cooling, centrifuge at 4000 r / min for 20 min. Add 4 times the volume of 95% ethanol to the supernatant and let it stand at 4°C overnight. The precipitate obtained is SDF, which is freeze-dried for 48 h to obtain SDF powder.

[0203] 2. Detection of SDF structural properties before and after fermentation

[0204] (1) Scanning electron microscopy (SEM) observation

[0205] Fix the conductive glue on the sample stage, take an appropriate amount of SDF sample and shake it onto the conductive glue, evenly spray the gold layer, observe the microstructure of the sample at different magnifications under a 12-15 kV scanning electron microscope, and obtain the scanning electron microscope image.

[0206] (2) Fourier transform infrared spectroscopy (FT-IR) analysis

[0207] Using blank potassium bromide as background, SDF was detected by Fourier transform infrared spectrometer at a scanning wavelength of 400-4000 cm -1 The transmittance under different conditions was measured to detect the chemical groups and molecular structure of SDF before and after fermentation.

[0208] (III) Physical and chemical properties of SDF before and after fermentation

[0209] 1. Water holding capacity (WHC) test

[0210] Accurately weigh 0.20 g of dry SDF sample (accurate to 0.0001 g), place it in a 15 mL centrifuge tube, add 10.00 mL of distilled water, shake well, place it at room temperature for 24 h, centrifuge it at 5000 r / min for 10 min, discard the supernatant, weigh the residue, and repeat the measurement three times to take the average value.

[101] , the calculation formula of water holding capacity is as follows:

[0211]

[0212] Where: m: sample mass, g; m0: centrifuge tube and sample mass, g; m1: centrifuge tube and sample mass after water absorption, g.

[0213] 2. Expansion force (WSC) test

[0214] Accurately weigh 0.20 g of dry SDF sample (accurate to 0.0001 g) and place it in a 15 mL centrifuge tube. Record the volume of the dry sample in its natural stacking state, then add 10.00 mL of distilled water. Shake evenly and leave it at room temperature for 24 h. Record the volume of the SDF sample after it absorbs water and swells. Repeat the measurement three times and take the average value. The expansion force is calculated as follows:

[0215]

[0216] Where: m: sample mass, g; V0: sample volume, mL; V1: volume of the sample after water absorption and expansion, mL.

[0217] 3. Oil holding capacity (OHC) test

[0218] Accurately weigh 0.20 g of dry SDF sample (accurate to 0.0001 g), place it in a 15 mL centrifuge tube, add 10.00 mL of soybean oil, shake well, place at room temperature for 24 h, centrifuge at 5000 r / min for 10 min, discard the upper layer of oil, weigh the remaining residue, repeat the measurement three times and take the average value.

[103] , the calculation formula of oil holding capacity is as follows:

[0219]

[0220] Where: m: sample mass, g; m0: centrifuge tube mass, g; m1: centrifuge tube and sample mass after oil absorption, g.

[0221] 4. Detection of SDF adsorption characteristics before and after fermentation

[0222] (1) Determination of nitrite adsorption capacity (NAC)

[0223] Drawing of the standard curve of NaNO2 solution: Prepare a 50 μg / mL standard solution with NaNO2, and pipette 0.00, 1.00, 2.50, 5.00, 7.50, and 10.00 mL of the standard solution into a 100 mL volumetric flask, respectively. Add 2.00 mL of 4 g / L p-aminobenzenesulfonic acid solution, let it stand for 5 minutes, add 1.00 mL of 2 g / L naphthylethylenediamine hydrochloride solution, make up to volume with distilled water, shake well, let it stand for 15 minutes, use distilled water as the blank, measure the OD value at 538 nm, and draw the NaNO2 standard curve.

[0224] Determination of SDF's adsorption capacity for nitrite at different times: Reference Hua

[104] The pH of a 0.1 mg / mL NaNO2 standard solution was adjusted to 2 (to simulate the environment in the stomach) and 7 (to simulate the environment in the intestine) for later use. Accurately weigh two portions (0.20 g) of SDF before and after fermentation (accurate to 0.0001 g) and add them to 50.00 mL of the NaNO2 standard solution. Stir evenly and shake at 37°C for 30 min, 45 min, 60 min, 75 min, 90 min, and 120 min. Quantitatively remove the solution and centrifuge at 4000 r / min for 15 min. Pipette 1.00 mL of the supernatant into a 10 mL centrifuge tube. The determination method was the same as the standard curve. The remaining NaNO2 content in the supernatant was calculated based on the standard curve to obtain the adsorption capacity of SDF for nitrite. The formula for nitrite adsorption capacity is as follows:

[0225]

[0226] Where: m: sample mass, g; V: total reaction volume, mL; n0: NO2- concentration after adsorption, μg / mL; n1: NO2- concentration before adsorption, μg / mL.

[0227] (2) Cholesterol adsorption capacity (CAC) determination

[0228] Cholesterol standard curve drawing: Use cholesterol standard to prepare 100 mg / mL cholesterol standard solution, pipette 0.00, 0.20, 0.40, 0.60, 0.80, and 1.00 mL of cholesterol standard solution into 10 mL centrifuge tubes, add glacial acetic acid in sequence to make the total volume reach 8 mL, then add 2.00 mL of ferroallum colorimetric solution, mix well and let stand for 30 min, use 8.00 mL of glacial acetic acid as blank, measure the OD value at 570 nm, and draw the cholesterol standard curve.

[0229] Determination of the cholesterol adsorption capacity of SDF at different times: Referring to Yang Xue's method, the pH of the 0.05 mg / mL cholesterol standard solution was adjusted to 2 (simulating the environment in the stomach) and 7 (simulating the environment in the intestine) for later use. Accurately weigh two 0.50 g samples (accurate to 0.0001 g) and add them to 50.00 mL of the cholesterol standard solution respectively. After stirring evenly, oscillate at a constant temperature of 37 ° C for 30 min, 45 min, 60 min, 75 min, 90 min and 120 min. After quantitative removal, centrifuge at 4000 r / min for 20 min, take 4.00 mL of the supernatant into a 10 mL centrifuge tube, add 4.00 mL of glacial acetic acid, and use the same determination method as the standard curve. Calculate the cholesterol content in the supernatant based on the cholesterol standard curve and calculate the adsorption capacity of dietary fiber for cholesterol. The formula for cholesterol adsorption capacity is as follows:

[0230]

[0231] Where: m: sample mass, g; V: total reaction volume, mL; n0: cholesterol concentration in the supernatant before adsorption, mg / mL; n1: cholesterol concentration in the supernatant after adsorption, mg / mL.

[0232] (3) Determination of bile salt adsorption capacity

[0233] Preparation of sodium cholate standard curve: Pipette 2.00, 4.00, 6.00, 8.00, and 10.00 mL of 1.0 mg / mL sodium cholate standard solution into 10 mL volumetric flasks to make up the volume, and pipette 1.00 mL of sodium cholate standard solution of different concentrations into 10 mL centrifuge tubes. Add 6.00 mL of 45% sulfuric acid and 1.00 mL of 0.3% furfural in sequence. Mix well, place in a constant temperature water bath at 65°C and heat for 30 min. Take out and cool to room temperature. Measure the OD value at 620 nm to draw the sodium cholate standard curve.

[0234] Determination of the adsorption capacity of sodium cholate by SDF at different times: Reference Chu

[103] Adjust the pH of the bile salt standard solution to 7 (simulating the intestinal environment) using the method described above. Weigh 0.50 g of sample (accurate to 0.0001 g) and add 50.00 mL of a 1 mg / mL sodium cholate standard solution. Stir thoroughly and shake in a 37°C water bath for 30, 45, 60, 75, 90, and 120 minutes. Quantitatively remove the sample and centrifuge at 4000 rpm for 20 minutes. Determine the sodium cholate content in 1.00 mL of the supernatant using the standard solution assay method. The adsorption capacity of dietary fiber for sodium cholate is then calculated. The formula for sodium cholate adsorption capacity is as follows:

[0235]

[0236] Where: m: sample mass, g; V: total reaction volume, mL; n0: cholesterol concentration in the supernatant before adsorption, mg / mL; n1: cholesterol concentration in the supernatant after adsorption, mg / mL.

[0237] 3. Results

[0238] 1. Scanning electron microscopy analysis

[0239] Scanning electron microscopy images of corn husk SDF before and after fermentation Figure 7 As shown. Figure 7 (A1, A2) It can be seen that the corn husk SDF particles before fermentation are large and uneven in size, with irregular flakes on the particle surface and a dense structure. Figure 7 (B1, B2) It can be seen that the fermented corn husk SDF particles are smaller and uniform in size, with irregular protrusions on the surface and a honeycomb structure. The number of loose small particles is significantly greater than that of the corn husk SDF before fermentation. This may be because the hydrolysis of enzymes such as carboxymethyl cellulase and hemicellulase during the fermentation process converts IDF into SDF, and the original SDF structure in the corn husk is also changed, resulting in a decrease in molecular weight, a decrease in polymerization degree, and smaller SDF particles. The fermented modified SDF has a larger specific surface area, and the loose structure is conducive to enhancing the physical and chemical properties of SDF, such as water holding capacity, oil holding capacity, and cholesterol binding capacity.

[26] .

[0240] 2. Infrared spectroscopy analysis

[0241] Infrared spectroscopy is an effective method for analyzing the possible functional groups in compounds. Figure 8 As shown. Figure 8 It can be seen that at 3300 cm -1 The broad peak near 2930 cm is caused by OH stretching vibration, indicating the presence of free hydroxyl groups in SDF; -1 The absorption peaks appearing near 1600 cm are caused by the CH stretching vibration of the -CH2- (methine) and -CH3 methyl groups of sugars, which are typical cellulose absorption peaks. -1 The absorption peak near 1030cm is formed by the stretching vibration of C=O in esterification, which is the characteristic peak of hemicellulose. The width of this peak in corn husk SDF becomes slightly wider after fermentation; -1 The absorption peak near 1350 cm is a typical absorption peak of arabinoxylan. The absorption peak intensity is stronger after fermentation, indicating that the corn bran SDF contains more arabinoxylan after fermentation. -1 The absorption peak near 880 cm is formed by CH bending vibration; -1 The nearby absorption peak is that of the β-glycosidic bond. This peak weakened after fermentation, likely due to the cleavage of the glycosidic bond during fermentation. The types of characteristic absorption peaks in corn bran SDF did not change significantly before and after fermentation, and the functional groups remained essentially the same, indicating that the types of hydrophilic and some active groups in SDF remained unchanged. However, some changes in the width, intensity, and wavenumber of the absorption peaks occurred, likely due to hydrolysis during fermentation, which disrupted the molecular structure of the dietary fiber and exposed the functional groups.

[0242] 3. Analysis of physical and chemical properties

[0243] The results of the effect of Hericium erinaceus He-02 solid-state fermentation on the water holding capacity, swelling capacity and oil holding capacity of corn husk SDF are shown in Table 11. As can be seen from the table, the water holding capacity, swelling capacity and oil holding capacity of corn husk SDF are significantly improved after fermentation. The water holding capacity of SDF after fermentation increased by 1.57 times compared with that before fermentation, reaching 3.65±0.12 g / g, the swelling capacity of SDF after fermentation increased by 1.95 times compared with that before fermentation, reaching 4.31±0.04 mL / g, and the oil holding capacity of SDF after fermentation increased by 1.80 times compared with that before fermentation, reaching 3.93±0.09 g / g. The hydrolysis effect of microbial metabolism during the fermentation process causes the corn husk SDF particles to become smaller, increasing the contact area between water and oil and SDF. After fermentation, the structure of SDF is destroyed, exposing more groups, increasing the binding sites of SDF, and thus increasing the water holding capacity, swelling capacity and oil holding capacity of corn husk SDF. This result is consistent with Wu Liping's

[106] The test results are consistent.

[0244]

[0245] 4. Analysis of nitrite adsorption capacity

[0246] Draw a standard curve and use the regression equation of the NaNO2 standard curve as follows: y = 0.6725 x + 0.5304, R =0.9996, calculate the concentration of NaNO2.

[0247] The adsorption capacity of corn bran SDF on nitrite at different times before and after fermentation Figure 8 As shown in the figure, it can be seen that the nitrite adsorption capacity of SDF gradually increases with time. After 120 minutes of adsorption, the nitrite adsorption capacity of SDF before fermentation under pH 2 conditions reached 488.73 μg / g, and the nitrite adsorption capacity of SDF after fermentation was 493.99 μg / g. The nitrite adsorption capacity of SDF before fermentation under pH 7 conditions reached 378.73 μg / g, and the nitrite adsorption capacity of SDF after fermentation was 426.58 μg / g. This shows that the nitrite adsorption capacity of SDF in the pH 2, i.e., gastric environment, is higher than that in the pH 7, i.e., small intestine environment. The nitrite adsorption capacity of corn bran SDF is enhanced after fermentation. This may be because SDF has a loose structure after fermentation, and fermentation helps to expose more carboxyl and hydroxyl groups, thereby enhancing the adsorption capacity of corn bran SDF for nitrite. Under the conditions of pH 2 and pH 7, the adsorption capacity of corn bran SDF for nitrite is stronger than that of Yunnan olive pomace SDF.

[107] .

[0248] 5. Cholesterol adsorption capacity analysis

[0249] Draw the standard curve and use the regression equation of the standard curve: y = 10.946 x + 1.0081, R ² = 0.9991, calculate the cholesterol concentration.

[0250] Cholesterol is closely related to cardiovascular diseases such as atherosclerosis and hypertension. Figure 8 As shown in the figure, after 120 minutes of adsorption, the cholesterol adsorption capacities of fermented corn bran SDF at pH 2 and pH 7 were 3.61 mg / g and 6.92 mg / g, respectively, both exceeding those of pre-fermented corn bran SDF. Furthermore, the cholesterol adsorption capacity at pH 7 was higher than that at pH 2, indicating that SDF has a greater cholesterol adsorption capacity in the small intestine, the primary site of cholesterol digestion and absorption. This result is consistent with the actual digestive environment of the human body. Fermentation modification can enhance the cholesterol adsorption capacity of corn bran SDF, making it suitable for use as a functional ingredient for lowering cholesterol in food processing.

[0251] 6. Analysis of bile salt adsorption capacity

[0252] Draw the standard curve and use the regression equation of the standard curve: y = 0.7443 x + 0.0688, R ² = 0.9991, calculate the sodium cholate concentration.

[0253] Bile salts are secondary metabolites of bile acid, which are related to some gastrointestinal diseases and cancers. They are also necessary emulsifiers and carriers for intestinal absorption of cholesterol. Dietary fiber can bind to bile salts, promote the excretion of bile salts, reduce the body's contact with and absorption of bile salts, and further inhibit the absorption of cholesterol. The adsorption capacity of corn bran SDF on bile salts at different times before and after fermentation is shown in Figure 2. Figure 9 (pH 7). As shown in the figure, the adsorption capacity of corn bran SDF for bile salts gradually increased over time. After 120 minutes of adsorption, the bile salt adsorption capacity of fermented corn bran SDF reached 0.83 mg / g, 1.34 times that of unfermented corn bran SDF. Fermented corn bran SDF exposes more active groups, increasing its affinity for bile salts and enhancing its lipid-lowering potential.

[0254] In summary, a comparison of the structural properties of corn bran SDF before and after fermentation revealed that solid-state fermentation modified corn bran SDF from a dense, flaky structure with large, unevenly sized particles and irregularly shaped surfaces to a honeycomb-like structure with smaller, uniformly sized particles and irregularly shaped bumps. While there was no significant difference in the types of functional groups in corn bran SDF before and after fermentation, the enzyme-producing fermentation process exposed more functional groups, enhancing its functional activity.

[0255] Comparison of the physical and chemical properties of corn bran SDF before and after fermentation revealed that the water-holding capacity, swelling capacity, and oil-holding capacity of fermented corn bran SDF increased from 2.33±0.11 g / g, 2.21±0.07 mL / g, and 2.18±0.06 g / g to 3.65±0.12 g / g, 4.31±0.04 mL / g, and 3.93±0.09 g / g, respectively, representing increases of 1.57-fold, 1.95-fold, and 1.80-fold, respectively. The adsorption properties of corn bran SDF were enhanced through solid-state fermentation modification. The study found that fermented corn bran SDF exhibited a certain degree of improvement in its adsorption capacity for nitrite, cholesterol, and bile salts.

[0256] Example 7 Modification Method of Corn Husk Soluble Dietary Fiber

[0257] Corn husks and water were mixed in a 1:2 ratio. Anhydrous glucose (3.0% by weight of corn husk), potassium dihydrogen phosphate (0.1%), and magnesium sulfate heptahydrate (0.04%) were added. After mixing, the mixture was sterilized at 121°C for 20 minutes. After cooling, the mixture was inoculated with Hericium erinaceus He-02-06 at an inoculum size of 8% and cultured at 25°C for 7 days. (The optimal fermentation conditions were: inoculum size 8.205%, culture temperature 25.395°C, and culture time 7.132 days, yielding a theoretical maximum SDF content of 8.245 g / 100g. For experimental convenience, the fermentation conditions were adjusted to an inoculum size of 8%, culture temperature 25°C, and culture time of 7 days.)

Claims

1. Hericium erinaceus He-02-06, its preservation number is CCTCC NO: M 20221043.

2. A method for preparing corn husk soluble dietary fiber, comprising: 1) Corn husk processing: wash the corn husk until the pH is neutral, dry it, and crush it; 2) Solid-state fermentation: a solid-state fermentation medium was prepared with corn husks, and the Hericium erinaceus He-02-06 described in claim 1 was inoculated at an inoculum size of 8%, the culture temperature was 25°C, and the culture time was 7 days; 3) Preparation of soluble dietary fiber: Weigh the fermented sample from step 2), sieve it, add water, and extract it in a water bath at 75-85°C for 1.5-2.5 hours. Cool it, centrifuge it, and retain the supernatant. Add ethanol, let it stand at 0-4°C for 6-12 hours, and dry it to obtain corn husk soluble dietary fiber. The solid-state fermentation medium in step 2) is prepared according to the proportions of 100 g corn husk, 200 g water, 3 g anhydrous glucose, 0.1 g potassium dihydrogen phosphate, and 0.04 g magnesium sulfate heptahydrate, mixed well, and sterilized at 121° C. for 20 min.

3. The method for preparing corn husk soluble dietary fiber according to claim 2, wherein: The water bath temperature in step 3) is 80° C. and the time is 2 h.

4. The method for preparing corn husk soluble dietary fiber according to claim 3, wherein: The centrifugation in step 3) is carried out at 4000 r / min for 20 min; the ethanol concentration is 95%, and the standing temperature is 4°C.

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