Application of Armillaria mellea Am-07-22 in Modification of Ginseng Insoluble Dietary Fiber

Abstract

The invention discloses an application of Armillaria mellea in modifying ginseng insoluble dietary fiber; a preparation method of the modified ginseng insoluble dietary fiber comprises the following steps: 1) preparing a liquid culture medium for bacterial strain activation; 2) preparing a ginseng fermentation culture medium: adding ginseng residue to the liquid culture medium for bacterial strain activation, sterilizing, and cooling; 3) inoculating: inoculating Armillaria mellea into the ginseng fermentation culture medium at an inoculum rate of 6-10%, and culturing for 4-8 days; 4) extracting the modified ginseng insoluble dietary fiber: extracting the fermented ginseng insoluble dietary fiber by an enzymatic method; the results show that the ginseng insoluble dietary fiber obtained by fermentation and modification of Armillaria mellea Am-07-22 has good functional properties, and its water holding capacity, oil holding capacity, water swelling capacity, glucose adsorption capacity, glucose dialysis delay capacity, cholesterol adsorption capacity, sodium cholate adsorption capacity, and nitrite adsorption capacity are significantly improved compared with unfermented samples, and the cation exchange capacity is also significantly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to the application of Armillaria mellea Am-07-22 in the modification of ginseng insoluble dietary fiber. Background Art

[0002] Ginseng is a traditional and precious Chinese medicinal herb and a nutritious food that serves both medicinal and edible purposes. Ginseng residue, a byproduct of the extraction process for ginseng's active ingredients, contains a large amount of dietary fiber (DF). However, it is often discarded during production and processing, resulting in resource waste and environmental pollution. Insoluble dietary fiber (IDF), a major component of dietary fiber, contains numerous active groups such as hydroxyl, carboxyl, and amide groups, which impart excellent water- and oil-retention, adsorption, and cation exchange capacities. These groups can regulate blood sugar, lower cholesterol, and prevent obesity. However, the compact structure of naturally occurring insoluble dietary fiber prevents the full exposure of these active groups, limiting its functional properties. Furthermore, due to its water insolubility and rough texture, its addition to foods can negatively impact color, texture, flavor, and taste. Therefore, the use of appropriate modification techniques to fully improve the structure and physicochemical properties of insoluble dietary fiber and, thereby, enhance its functional properties has become a research hotspot in recent years.

[0003] Currently, the main methods for modifying insoluble dietary fiber include physical, chemical, and biological methods, such as extrusion, steam heat treatment, ultrasonication, cellulase, xylanase, and microbial fermentation. Microbial fermentation is a gentle and efficient method for modifying dietary fiber. Insoluble dietary fiber prepared by microbial fermentation generally has better structural and functional properties. Chu et al. treated millet bran insoluble dietary fiber with Bacillus natto. The modified sample had a looser and more porous structure and significantly improved adsorption capacity for glucose, cholesterol, and bile salts. Wang et al. modified bean dregs insoluble dietary fiber with Kluyveromyces marxii. The modified sample had a looser and more porous microstructure, improved thermal stability, and improved water and oil holding capacity, cholesterol adsorption capacity, bile acid adsorption capacity, and glucose adsorption capacity, making it suitable for use as a functional ingredient in food. Morels, Hericium erinaceus, and Armillaria mellea are well-known edible and medicinal fungi in my country and are recognized as safe strains. Their fruiting bodies contain a large number of nutrients and active ingredients and are widely used in the development and utilization of functional foods. However, the cultivation of edible fungi fruiting bodies requires a long time and high environmental requirements. Therefore, obtaining edible fungi fermentation products through liquid fermentation technology has become a research hotspot in recent years. Research shows that edible fungi fermentation products (microorganisms and fermentation liquids), like fruiting bodies, are not only rich in nutrients but also contain active ingredients such as polysaccharides, polyphenols, and enzymes, which can be used in the development and manufacture of functional foods. Summary of the Invention

[0004] The present invention aims to provide application of Armillaria mellea Am-07-22 in modification of ginseng insoluble dietary fiber.

[0005] Application of Armillaria mellea in modification of ginseng insoluble dietary fiber;

[0006] The modification is to improve the water holding capacity, oil holding capacity, water swelling capacity, glucose adsorption capacity, glucose dialysis delaying capacity, cholesterol adsorption capacity, sodium cholate adsorption capacity and nitrite adsorption capacity of the ginseng insoluble dietary fiber.

[0007] The honey fungus is Armillaria mellea Am-07-22, with a deposit number of CCTCC NO: M 20221044;

[0008] The ginseng is ginseng residue.

[0009] A method for preparing modified ginseng insoluble dietary fiber, comprising:

[0010] 1) Prepare liquid activation culture medium for bacterial strains;

[0011] 2) Preparing Armillaria fermentation seed liquid: Activate the Armillaria, inoculate the activated Armillaria into liquid activation culture medium, and culture with shaking at 25-30°C and 150-200 rpm for 5-7 days to obtain the Armillaria fermentation seed liquid;

[0012] 3) Prepare ginseng fermentation medium: Add 1g of ginseng residue to 10-20mL of liquid culture medium for bacterial activation, sterilize, and cool.

[0013] 4) Inoculation: Inoculate 6-10% of the Armillaria fermentation seed solution into the ginseng fermentation medium and incubate at 25-30°C and 150-200 rpm for 4-8 days.

[0014] 5) Extraction of modified ginseng insoluble dietary fiber: Extract the fermented ginseng insoluble dietary fiber by enzymatic hydrolysis.

[0015] The honey fungus is Armillaria mellea Am-07-22, with a deposit number of CCTCC NO: M 20221044;

[0016] The liquid culture medium for bacterial activation comprises 20% potato, 0.5% silkworm pupa powder, 1% glucose, 1% sucrose, 2% yeast extract powder, 0.15% potassium dihydrogen phosphate, 0.075% magnesium sulfate heptahydrate, 0.001% vitamin B1, and the balance is water;

[0017] The ginseng residue is the solid ginseng residue remaining after extracting total ginsenosides, which is obtained by drying and crushing;

[0018] The Armillaria inoculation amount in step 3) is 8%;

[0019] The culture conditions in step 3) are 27°C and 160 rpm for 6 days.

[0020] The present invention provides an application of Armillaria mellea in modifying ginseng insoluble dietary fiber; the Armillaria mellea is Armillaria mellea Am-07-22, with a deposit number of CCTCC NO: M 20221044; a method for preparing modified ginseng insoluble dietary fiber, comprising: 1) preparing a liquid culture medium for bacterial strain activation; 2) preparing a ginseng fermentation medium: taking 1 g of ginseng residue, adding it to 10-20 mL of the liquid culture medium for bacterial strain activation, sterilizing it, and cooling it; 3) inoculating: inoculating the Armillaria mellea into the ginseng fermentation medium at an inoculum rate of 6-10%, and incubating the culture medium at 25-30°C and 100-200°C. The modified ginseng insoluble dietary fiber was cultured at rpm for 4-8 days; 4) Extraction of modified ginseng insoluble dietary fiber: the fermented ginseng insoluble dietary fiber was extracted by enzymatic hydrolysis; the results showed that the ginseng insoluble dietary fiber modified by Armillaria mellea Am-07-22 fermentation had the best functional properties, and its water holding capacity, oil holding capacity, water swelling capacity, glucose adsorption capacity, glucose dialysis delay capacity, cholesterol adsorption capacity, sodium bile adsorption capacity, and nitrite adsorption capacity were increased by 74.2%, 93.6%, 124.38%, 82.17%, 20.24%, 14.04%, 49.41%, and 24.49% respectively compared with those of the unfermented sample, and the cation exchange capacity was also significantly improved compared with that of the unfermented modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Effects of strain mutation on the soluble dietary fiber content of ginseng residue;

[0022] Figure 2 Effects of strain mutation on the water-holding capacity, oil-holding capacity and water-swelling capacity of insoluble dietary fiber in ginseng residue;

[0023] Figure 3 Tree diagram of strains;

[0024] Figure 4 SEM images of ginseng IDF-C (A, a), IDF-M (B, b), IDF-H (C, c), IDF-A (D, d);

[0025] Figure 5 Particle size distribution of ginseng IDF;

[0026] Figure 6 FT-IR image of ginseng IDF;

[0027] Figure 7 Ginseng IDF XRD pattern;

[0028] Figure 8 DSC thermogram of ginseng IDF;

[0029] Figure 9 Glucose adsorption capacity of ginseng IDF;

[0030] Figure 10 Glucose Dialysis Retarding Rating (GDRI) of Ginseng IDF;

[0031] Figure 11 Cholesterol adsorption capacity of ginseng IDF;

[0032] Figure 12 Sodium bile acid adsorption capacity of ginseng IDF;

[0033] Figure 13 Nitrite adsorption capacity of ginseng IDF;

[0034] Figure 14 Cation exchange capacity of ginseng IDF.

[0035] In the figure, IDF-C is an insoluble dietary fiber derived from unfermented ginseng; IDF-M is an insoluble dietary fiber derived from fermented ginseng by Morchella oleracea; IDF-H is an insoluble dietary fiber derived from fermented ginseng by Hericium erinaceus; and IDF-A is an insoluble dietary fiber derived from fermented ginseng by Armillaria mellea. DETAILED DESCRIPTION

[0036] Experimental materials: 1) Ginseng residue, selected from garden ginseng (4-5 years old); Armillaria mellea Am-07-22 ( Armillaria mellea ), the deposit number is CCTCC NO: M 20221044; 2) Based on the results of the previous experiments of the research team, the strains with better (cellulose biodegradation) indicators were selected, and Morchella Me-01 ( Morchella esculenta )、Hericium erinaceus He-02-06( Hericiumerinaceus ) and Armillaria Am-07-22 ( Armillaria mellea ), provided by the National Engineering Center for Deep Processing of Wheat and Corn; all three fungi were isolated from the seed bodies of fungi collected in Changbai Mountain, Morchella Me-01 was isolated from the fruiting bodies of Morchella, Hericium erinaceus He-02-06 was isolated and induced from the fruiting bodies of Hericium erinaceus, and Armillaria mellea Am-07-22 was isolated from the fruiting bodies of Armillaria mellea; 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 mellea Am-07-22 was deposited in the China Center for Type Culture Collection on July 6, 2022, with the strain collection number CCTCC NO: M20221044.

[0037] Example 1 Screening and Identification of Armillaria Am-07-22

[0038] 1. Mutation method of bacterial strains

[0039] The laboratory-preserved Armillaria Am-07 ( Armillaria mellea ) was used as the starting strain. The protoplast suspension of the strain to be mutated was placed on a plate, a rotor was placed in the plate for stirring, and the plate was 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 a regeneration medium and incubated in a 27°C constant temperature incubator in the dark for 10 days. The colonies grown were counted, and a lethality curve was plotted. Strains with good growth were selected as mutants and cultured under appropriate conditions. Mutant strains with a soluble dietary fiber content that was more than 15% higher than that of the starting strain were selected as positive mutants. They were serially passaged 10 times, and fermentation tests were performed 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.

[0040] 2. Comparison of mutagenic effects

[0041] The Armillaria Am-07-22 strain obtained after UV mutagenesis was subjected to solid-state fermentation, and its effect on the soluble dietary fiber content of ginseng residue was as follows: Figure 1 As shown in the figure, after solid-state fermentation of ginseng residue using its mutant strain, the content of soluble dietary fiber in the ginseng residue was increased, and the hydration properties of the insoluble dietary fiber in the ginseng residue, such as water holding capacity, oil holding capacity and water swelling capacity, were also improved. Figure 2 shown.

[0042] 3. Sequencing results of Am-07-22 strain

[0043] 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.

[0044]

[0045] 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 result is shown in the sequence listing SEQ ID NO.1. The sequence was determined and the tree diagram of the strain was drawn using MEGA7.1 software, as shown in the figure. Figure 3The figure shows that strain Am-07-22 has high sequence similarity to Armillaria sp., and the strain was identified as Armillaria mellea and named Armillaria mellea Am-07-22. Armillaria mellea Am-07-22 was deposited with the China Center for Type Culture Collection on July 6, 2022, with the strain accession number CCTCC NO: M20221044.

[0046] Example 2 Bacteria culture

[0047] 1. Bacteria Activation

[0048] The strains Me-01, He-02-06, and Am-07-22 were transferred to slant culture medium, cultured at 27°C for 12 days;

[0049] Prepare liquid activation culture medium: 20% potato, 0.5% silkworm pupa powder, 1% glucose, 1% sucrose, 2% yeast extract powder, 0.15% potassium dihydrogen phosphate, 0.075% magnesium sulfate heptahydrate, 0.001% vitamin B1, natural pH, sterilize at 121°C for 20 minutes;

[0050] Preparation of primary fermentation seed: In a sterile environment, take 8 bacterial cells from the activated slant culture medium of the three strains and inoculate them into a 100 mL Erlenmeyer flask containing 30 mL of liquid activation medium. Incubate at 27°C in a shaker at 160 rpm for 6 days to obtain the primary fermentation seed.

[0051] Preparation of secondary fermentation seed liquid: crush the primary fermentation seed, inoculate the strain into a 500 mL Erlenmeyer flask containing 200 mL of liquid activation culture medium at an addition rate of 8%, and culture on a shaker at a constant temperature of 27°C and 160 r / min for 6 days to obtain the secondary fermentation seed liquid.

[0052] 2. Preparation of Ginseng Fermentation Medium

[0053] Ginseng fermentation medium was prepared at a material-liquid ratio of ginseng residue to liquid culture medium of 1:15 (g / mL), sterilized at 121°C for 20 min, and cooled for later use.

[0054] The ginseng residue is obtained by washing and cutting garden ginseng (4-5 years old), boiling it with water to extract total ginsenosides, filtering out the remaining ginseng segments (solid matter), and then drying and crushing them.

[0055] 3. Inoculation and Fermentation

[0056] According to preliminary laboratory research, the three fermentation seed liquids were inoculated into ginseng fermentation medium at an 8% inoculation rate (equivalent to 8% of the mass fraction of the strain in the ginseng fermentation medium), and the culture conditions were 27°C, 160 rpm, and cultured for 6 days.

[0057] Example 3 Extraction of Ginseng Insoluble Dietary Fiber (IDF)

[0058] IDF was extracted according to the enzymatic hydrolysis method in the national standard GB 5009.88-2014: the raw material sample was used as a control and was directly extracted according to the national standard method, and the fermented ginseng residue prepared in Example 2 was extracted according to the national standard method after ginseng fermentation and cultivation.

[0059] Example 4 Determination of the structure of ginseng insoluble dietary fiber

[0060] 1. Scanning electron microscopy (SEM)

[0061] The microstructure of ginseng IDF was characterized by SEM. The accelerating voltage was set to 5 kV, and the freeze-dried sample was gold-plated and then observed on the machine.

[0062] 2. Particle size analysis

[0063] The particle size distribution of the sample was determined using a laser particle size analyzer, and the refractive index of the particles and the refractive index of the dispersant were 1.470 and 1.330, respectively.

[0064] 3. Fourier transform infrared spectroscopy (FT-IR)

[0065] The sample was mixed with potassium bromide at a ratio of 1:100, ground into powder and pressed into tablets. The whole band scanning (4000-500 cm -1 ), and measure the FT-IR spectrum curve.

[0066] 4. X-ray diffraction (XRD)

[0067] The crystal structure of the sample was analyzed using an X-ray diffractometer. Key parameters: Cu target, tube voltage 20 kV, scan speed 4° / min, measurement range 5°–50° (2θ angle), angular step size 0.02°.

[0068] 5. Differential Scanning Calorimetry (DSC)

[0069] The thermal properties of IDF were measured using a DSC instrument, following the method of Niu et al. A 3 mg sample was placed in an aluminum container and pressed into a pellet. An empty aluminum container was used as a control. The temperature was then increased from 30°C to 300°C at a rate of 10°C / min. Thermal curves were obtained using Universal Analysis software.

[0070] 6. Results

[0071] SEM analysis: Figure 4 As shown in the results, the samples fermented with the three edible fungi showed a large number of flaky structures and honeycomb-like holes compared to those before fermentation, the specific surface area increased, and the overall structure became loose. This indicates that the three edible fungi can produce active substances during the fermentation process to destroy the structure of the fiber, making the originally dense structure loose and porous. This loose and porous structure can lead to the exposure of more polar and non-polar groups, thereby improving the water-soluble properties and adsorption capacity of dietary fiber. Chu et al. reported that the structure of millet bran dietary fiber after fermentation with Bacillus natto was looser and more porous, which enhanced its adsorption properties. Wang et al. reported that the structure of insoluble dietary fiber in ginger residue treated with cellulase changed from dense to loose, and a sponge-like structure appeared on the surface and a large number of pores were generated. This study also showed similar changes and characteristics.

[0072] Particle size analysis: Figure 5 As shown, the main peak of the ginseng IDF particle size distribution after fermentation with the three edible fungi exhibited a leftward shift. The unfermented sample had a single particle size distribution peak, while the fermented sample exhibited a smaller peak between 3 and 26 μm. This indicates that active substances such as cellulase produced by fermentation have a certain degrading effect on ginseng IDF, breaking down large particles into smaller molecules. This is consistent with the SEM results mentioned above. The reduction in particle size exposes more functional groups in ginseng IDF, contributing to its enhanced physical, chemical, and functional properties.

[0073] Fourier transform infrared spectroscopy (FT-IR): Figure 6 As shown in the figure, both ginseng IDF before and after fermentation have the typical characteristics of dietary fiber, with basically the same absorption peaks and some changes in absorption intensity. -1 Corresponding to the hydroxyl stretching vibration peaks of cellulose and hemicellulose, 2800-3000 cm -1 The peaks between the two locations correspond to the vibration peaks of the -CH and -CH2 groups of cellulose. The peak intensities of the two locations of ginseng IDF weakened after fermentation, indicating that the active substances produced by fermentation had a certain degradation effect on the cellulose and hemicellulose in the sample, destroying the hydrogen bonds within the groups and promoting the exposure of the hydroxyl groups. -1 The characteristic peaks near this point are related to the carboxyl groups in uronic acid and polyphenols. The peak intensity of the fermented ginseng IDF is enhanced to a certain extent. -1 The peaks at 1050cm correspond to the vibration of OH or CO groups of cellulose and hemicellulose, and the peak intensity does not change significantly. -1The absorption peak at is attributed to the COO stretching vibration of lignin or hemicellulose. The intensity of this peak in ginseng IDF decreases significantly after fermentation, indicating that the active substances produced by fermentation also have a certain degrading effect on the lignin in the sample. In summary, edible fungus fermentation does not destroy the basic chemical structure of the fiber. However, the difference in peak intensity indicates that fermentation effectively removes the amorphous components in the ginseng IDF structure.

[0074] X-ray diffraction (XRD) analysis: Figure 7 As shown, the samples before and after fermentation exhibited a strong absorption peak at 2q of 21.72° and a weak absorption peak at 2q of 14.81°, reflecting typical cellulose type I structure. This indicates that fermentation did not alter the crystalline configuration of ginseng IDF, but only differences in peak intensity were observed. Origin software calculations revealed that the crystallinity of the unfermented sample was 13.78%, while that of the samples fermented with Morchella, Hericium erinaceus, and Armillaria mellea reached 15.37%, 18.57%, and 19.36%, respectively. This increase in crystallinity suggests that the active substances produced by the fermentation of these three edible fungi degraded the amorphous components of ginseng IDF, promoting the exposure of crystalline regions and leading to changes in the physicochemical properties of ginseng IDF, such as water-holding capacity, oil-holding capacity, and swelling capacity.

[0075] Thermal properties analysis (DSC): Figure 8 As shown, all samples exhibit two endothermic peaks. The first endothermic peak for IDF-C, IDF-M, IDF-H, and IDF-A occurs at 110.82°C, 137.84°C, 139.80°C, and 145.84°C, respectively. This is inferred to be the endothermic evaporation of water in the samples at these temperatures. The second endothermic peak for IDF-C, IDF-M, IDF-H, and IDF-A, at 203.02°C, 248.41°C, 250.15°C, and 257.56°C, respectively, likely represents the thermal decomposition of soluble substances and hemicellulose polysaccharides in the samples, or the pre-carbonization of cellulose. Compared to unfermented samples, both endothermic peaks of ginseng IDF fermented with the three edible fungi exhibit a significant rightward shift, indicating that the fermented ginseng IDF exhibits enhanced thermal stability. This may be due to the removal of some cellulose and lignin during the fermentation process, resulting in a higher crystallinity in the IDF.

[0076] Example 5 Determination of functional properties of ginseng insoluble dietary fiber

[0077] 1. Water holding capacity

[0078] Following the method of Zhou Hexia et al. with slight modifications, a 50 mL centrifuge tube was dried to constant weight. 0.1 g (accurate to 0.001 g) of sample powder was accurately weighed into the centrifuge tube. 10 mL of distilled water was added, shaken, and allowed to soak at room temperature for 1 h. Subsequently, the tube was centrifuged at 4500 rpm for 15 min, the supernatant was discarded, and the sample was weighed. This was repeated three times. The water holding capacity was calculated using the formula:

[0079] WHC (g / g) =

[0080] Where: M0 is the mass of the sample (g); M1 is the mass of the sample after water absorption (g).

[0081] 2. Oil holding capacity

[0082] Following the method of Marcin et al. with slight modifications, dry a 50 mL centrifuge tube to constant weight. Accurately weigh 0.1 g (accurate to 0.001 g) of sample powder into the tube, add 10 mL of cooking oil, and mix at room temperature for 1 hour. Centrifuge at 4500 rpm for 15 minutes, discard the supernatant, and weigh the sample. Repeat three times. The oil retention capacity is calculated as follows:

[0083] OHC (g / g) =

[0084] Where: M0 is the mass of the sample (g); M1 is the mass of the sample after oil absorption (g).

[0085] 3. Water expansion force

[0086] Following the method of Zhang et al. with slight modifications, accurately weigh 0.3 g of sample into a dry 10 mL graduated cylinder, smooth the surface, and record the volume reading (V2). Pour 5 mL of distilled water into the cylinder and mix at room temperature for 24 h. Record the volume reading (V1) after hydration. Repeat three times. The water expansion force is calculated as follows:

[0087] SC (mL / g) =

[0088] Where: M0 is the sample mass (g); V2 is the sample volume before expansion (mL); V1 is the liquid volume after standing for 24 h (mL).

[0089] 4. Glucose adsorption capacity

[0090] Using the method of Peerajit et al. with slight modifications, 0.5 g of sample was added to 50 mL of glucose solutions at concentrations of 50 mmol / L, 100 mmol / L, 150 mmol / L, and 200 mmol / L, respectively, and the mixture was shaken thoroughly. After incubation at 37°C in a water bath for 6 hours, the mixture was centrifuged at 4000 rpm for 20 minutes. The supernatant was collected and the glucose concentration was determined using the DNS method, with a glucose solution without sample added serving as a control. The glucose concentrations before and after adsorption were determined, and the GAC of the sample was calculated according to the following formula:

[0091] GAC (mg / g) = ×V

[0092] Where: M0 is the sample mass (g); C1 is the glucose concentration in the supernatant before adsorption (mg / mL); C2 is the glucose concentration in the supernatant after adsorption (mg / mL); V is the volume of the glucose solution (mL).

[0093] 5. Glucose dialysis delay ability

[0094] Using the method of Zheng et al. with slight modifications, 0.5 g of sample was mixed with 15 mL (0.2 g / 100 mL) of glucose solution and placed in a dialysis bag (Mw = 80,000-14,000 Da). The bag was placed in a conical flask containing 200 mL of distilled water and shaken at 37°C for 1.5 hours. Every 15 minutes, 2 mL of dialysate was sampled and the glucose concentration was determined using the DNS method. A blank control was prepared by replacing the glucose with distilled water to remove the sugar from the sample. A control control was prepared by using a glucose solution without sample. The glucose dialysis delay capacity of the sample was calculated according to the following formula:

[0095] GDRI(%) = ×100%

[0096] Where A1 is the glucose concentration in the sample group (mg / mL); A2 is the glucose concentration in the sample blank group (mg / mL); and A3 is the glucose concentration in the control group (mg / mL).

[0097] 6. Cholesterol adsorption capacity

[0098] Refer to the method of Cheng Mingming with slight modifications. Take egg yolk and thoroughly beat it with 9 times distilled water to form an emulsion. Adjust the pH of the system to 2 (simulating the gastric environment) and pH = 7 (simulating the intestinal environment). Accurately weigh 1.0 g of sample into a 100 mL Erlenmeyer flask, add 25 mL of egg yolk emulsion, stir thoroughly, place in a shaker, and shake thoroughly at 37°C for 2 h. Then, centrifuge at 4000 rpm for 20 min. The cholesterol content is determined by the o-phthalaldehyde method, and the cholesterol adsorption capacity of the sample is calculated according to the following formula:

[0099] Cholesterol adsorption capacity (mg / g) =

[0100] Where M1 is the cholesterol content in the supernatant before adsorption (mg); M2 is the cholesterol content in the supernatant after adsorption (mg); and M0 is the sample mass (g).

[0101] 7. Sodium bile acid adsorption capacity

[0102] Weigh 0.1 g of sample into a 100 mL Erlenmeyer flask, add 20 mL of sodium cholate solution (0.2 mg / mL), adjust the system pH to 7 (bile acid is only metabolized in the intestine, so it is only necessary to simulate the intestinal environment), shake at 37°C for 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min, respectively, and centrifuge at 4000 r / min for 20 min. Determine the sodium cholate content by furfural colorimetry, and calculate the sodium cholate adsorption capacity of the sample according to the following formula.

[0103] Sodium cholate adsorption capacity (mg / g) =

[0104] Wherein, M1 is the sodium cholate content in the supernatant before adsorption (mg); M2 is the sodium cholate content in the supernatant after adsorption (mg); M0 is the sample mass (g).

[0105] 8. Sodium nitrite adsorption capacity

[0106] With slight modifications, the method of Yang Xiaokuan et al. was used. 0.1 g of sample was accurately weighed into a 250 mL Erlenmeyer flask, and 50 mL of 20.0 μg / mL NaNO2 solution was added. The pH of the system was adjusted to 2 (simulating the gastric environment) and pH = 7 (simulating the intestinal environment). After shaking at 37°C for 15 min, 30 min, 60 min, 120 min, 150 min, and 180 min, 5 mL of each sample solution was diluted to 100 mL. 25.0 mL of the dilution was then added to 2 mL of p-aminobenzenesulfonic acid solution (4 g / L), mixed and allowed to stand for 3 min, and then 1 mL of 2 g / L naphthylethylenediamine hydrochloride solution was added. Distilled water was added to the mark and mixed. After standing for 15 min, the absorbance was measured at 538 nm. A blank control was performed using an equal-ratio diluted solution without sample. The nitrite adsorption capacity of the sample was calculated according to the following formula:

[0107] Nitrite adsorption capacity (μg / g) =

[0108] Where M1 is the nitrite content in the system before adsorption (μg); M2 is the nitrite content in the system after adsorption (μg); M0 is the sample mass (g).

[0109] 9. Cation exchange capacity

[0110] Refer to the method of Zhang et al. with slight modifications. Accurately weigh 1.0 g of sample, thoroughly mix with 50 mL of 0.1 mol / L hydrochloric acid solution, and acidify at 37°C for 24 h. After acidification, centrifuge at 4000 rpm for 15 min, collect the precipitate, and thoroughly wash with distilled water until it is free of Cl. - The sample was then dried in a 60°C oven to obtain the acidified sample. Weigh 0.1 g of the sample, add 50 mL of 5% NaCl solution, mix thoroughly, and titrate with 0.01 mol / L NaOH solution. Record the initial pH and the pH of the mixture after each 50 μL of NaOH solution is added.

[0111] 10. Results:

[0112] (1) Water and characteristics

[0113]

[0114] Note: Different letters in the same column indicate significant differences ( P<0.05). Insoluble dietary fiber from unfermented ginseng IDF-C; insoluble dietary fiber from ginseng fermented with Morchella oleracea IDF-M; insoluble dietary fiber from ginseng fermented with Hericium erinaceus IDF-H; insoluble dietary fiber from ginseng fermented with Armillaria mellea IDF-A.

[0115] As shown in Table 2, fermentation with the three edible fungi significantly increased the water-holding capacity, oil-holding capacity, and water-swelling capacity of ginseng IDF. This is due to structural changes caused by fermentation, which create numerous pores on the surface, increase the specific surface area, and expose hydrophilic and lipophilic functional groups, thereby increasing the sample's permeability and absorption capacity for water and oil molecules. SEM and particle size analysis results also confirm this finding. Comparing the three fungi, ginseng IDF fermented with Armillaria mellea exhibited the greatest improvement compared to unfermented ginseng, with increases in water-holding capacity, oil-holding capacity, and water-swelling capacity by 74.2%, 93.6%, and 124.38%, respectively. In summary, edible fungi fermentation imparts enhanced water-holding properties to ginseng IDF, contributing to its enhanced functional properties.

[0116] (2) Glucose adsorption capacity

[0117] like Figure 9 As shown in the figure, compared with the unfermented treatment, the glucose adsorption capacity of ginseng IDF after fermentation with the three edible fungi was significantly improved. Among them, the glucose adsorption capacity of the sample fermented with Armillaria mellea was significantly stronger than that of the other two fungi, and the adsorption capacity of the sample increased significantly with the gradient of glucose concentration. This may be because the reduction in particle size exposes the internal functional groups, thereby increasing the contact area with glucose, and the loose and porous structure of the fermented sample is also conducive to promoting glucose adsorption. The in vitro hypoglycemic function of dietary fiber is mainly due to its ability to effectively adsorb glucose. It can produce an obstructive effect on the diffusion and absorption of glucose, thereby reducing the glucose concentration in the intestine, reducing intestinal wall absorption, and maintaining postprandial blood sugar levels. The ability of ginseng IDF to effectively adsorb glucose after fermentation provides ideas for the development of functional foods.

[0118] (3) Glucose dialysis delay ability

[0119] like Figure 10As shown, the GDRI of ginseng IDF fermented with the three edible fungi was significantly higher than that of the unfermented samples, with the GDRI of the sample fermented with Armillaria mellea showing the greatest increase compared to the unfermented sample. From 15 to 45 minutes, the GDRI of all samples increased continuously with dialysis time, reaching a maximum at 45 minutes before rapidly decreasing. These results indicate that ginseng IDF effectively adsorbs glucose, trapping it within its fiber network. The surface of the fermented ginseng IDF becomes loose, with numerous honeycomb-like pores, enhancing glucose adsorption and trapping more glucose molecules. This glucose trapping can reduce the effective glucose concentration in the small intestine, thereby lowering postprandial blood glucose levels. However, this adsorption and trapping effect is time-limited and quickly reaches saturation. Diffusion accelerates after 45 minutes because the insoluble dietary fiber absorbs water and expands to saturation, reducing its viscosity and thus its binding capacity for glucose.

[0120] (4) Cholesterol adsorption capacity

[0121] like Figure 11 As shown in the results, compared with the unfermented ginseng IDF, the ginseng IDF fermented by the three edible fungi has a better ability to adsorb cholesterol. Among them, the cholesterol adsorption capacity of the ginseng IDF fermented by Armillaria mellea at pH=2 and pH=7 is 1.28 times and 1.14 times that of the unfermented ginseng IDF, respectively. This may be because the fermentation makes the surface structure of the ginseng IDF loose, the pores increase, and the particle size decreases, thereby enhancing the adsorption of cholesterol. In addition, the acidity and alkalinity of the system will also affect the adsorption capacity of ginseng IDF for cholesterol. The adsorption capacity under neutral conditions is greater than that in an acidic environment, which indicates that the intestinal environment is more conducive to the adsorption of cholesterol by dietary fiber. This is because there are a large number of hydrogen ions in the system under acidic conditions. With the increase of pH value, the carboxyl groups in the dietary fiber molecules dissociate and are converted into carboxyl anions with strong binding ability to cholesterol molecules, thereby enhancing the adsorption capacity of dietary fiber for cholesterol, which is consistent with Si

[52] et al., and the results of Wang et al. are consistent.

[0122] (5) Sodium bile acid adsorption capacity

[0123] like Figure 12As shown in the results, the adsorption capacity of fermented ginseng IDF for sodium cholate increased to varying degrees compared to unfermented ginseng IDF. This capacity initially increased and then decreased with adsorption time, reaching a maximum value between 45 and 60 minutes, consistent with Tian Hailong's research. Studies have shown that the adsorption capacity of dietary fiber for bile salts is primarily due to the adsorption and entrainment of insoluble dietary fiber. Substances such as cellulase are produced during the fermentation of edible fungi, which have a certain degrading effect on insoluble dietary fiber. The structural analysis above revealed that a large number of pores appeared on the surface of fermented ginseng IDF, which effectively bound and entrained bile salt molecules, thereby enhancing the sample's binding capacity for sodium cholate.

[0124] (6) Nitrite adsorption capacity

[0125] like Figure 13 As shown in the results, compared with unfermented ginseng IDF, the adsorption capacity of nitrite by ginseng IDF fermented with the three edible fungi was significantly enhanced, and the adsorption capacity continued to increase with adsorption time. The sample fermented with Armillaria mellea exhibited the best adsorption efficiency, with adsorption capacities reaching 1642.37 μg / g and 1249.13 μg / g at pH = 2 and pH = 7, respectively, after 150 min of adsorption. IDF adsorbs nitrite primarily through physical adsorption via its fluffy structure and chemical adsorption via its active molecular groups, particularly phenolic acid groups. SEM results showed that fermentation loosened the surface structure of ginseng IDF, while FT-IR results indicated an increase in phenolic acid groups in the fermented sample, both of which promoted the physical and chemical adsorption of nitrite by ginseng IDF. Furthermore, the adsorption efficiency of IDF at pH 2 was greater than that at pH 7, indicating that pH significantly affects nitrite adsorption by IDF. This may be because an increase in pH causes the dissociation of carboxyl groups in IDF, increasing the negative charge on the surface and generating a repulsive effect, thereby hindering the sample's absorption of NO. 2- This also shows that ginseng IDF can absorb more nitrite ions in the stomach than in the intestine.

[0126] (7) Cation exchange capacity

[0127] like Figure 14As shown, with the addition of NaOH solution, the pH of the solutions containing several samples continued to increase. The pH of the unfermented IDF-C solution increased from 3.12 to 9.45. The pH increase, as well as the initial and final pH values, of the solutions containing IDF-M (2.91-9.30), IDF-H (2.69-9.16), and IDF-A (2.63-9.12), fermented with the three edible fungi, were significantly lower than those of IDF-C. IDF-A, fermented with Armillaria mellea, maintained a lower pH for the longest period. Studies have shown that IDF contains numerous specialized functional groups, such as hydroxyl and carboxyl groups, which impart weakly acidic cation exchange capacity. Structural analysis suggests that fermentation disrupts the structure of ginseng IDF, loosening it and exposing more functional groups, such as carboxyl and hydroxyl groups. FT-IR analysis of the functional groups confirms this finding, showing that the fermented samples possess more uronic acid groups, including numerous hydroxyl and carboxyl groups, which enhances their cation exchange properties.

[0128] Conclusion: In summary, fermentation with all three edible fungi can degrade ginseng insoluble dietary fiber to some extent, resulting in a reduced particle size, a loose and porous surface structure, the exposure of more active groups, an increased crystallinity, and higher thermal stability. These structural changes, in turn, affect the functional properties of ginseng insoluble dietary fiber. Comparing the three strains, the ginseng insoluble dietary fiber fermented with Armillaria mellea Am-07-22 exhibited the best functional properties. Its water-holding capacity, oil-holding capacity, water-swelling capacity, glucose adsorption capacity, glucose dialysis retardation capacity, cholesterol adsorption capacity, sodium cholate adsorption capacity, and nitrite adsorption capacity increased by 74.2%, 93.6%, 124.38%, 82.17%, 20.24%, 14.04%, 49.41%, and 24.49%, respectively, compared to the unfermented sample. The cation exchange capacity was also significantly improved compared to the unfermented sample. Therefore, fermentation with large edible fungi can be used as a method for preparing highly active ginseng insoluble dietary fiber, providing insights into the high-quality utilization of the large amount of insoluble dietary fiber in ginseng residue.

Claims

1. Application of Armillaria mellea in the modification of ginseng insoluble dietary fiber; The modification is to improve the water holding capacity, oil holding capacity, water swelling capacity, glucose adsorption capacity, glucose dialysis delay capacity, cholesterol adsorption capacity, sodium cholate adsorption capacity and nitrite adsorption capacity of the ginseng insoluble dietary fiber; The Armillaria mellea is Armillaria Am-07-22, and its deposit number is CCTCC NO: M 20221044; The ginseng is ginseng residue, which is obtained by drying and crushing the ginseng solids remaining after extracting total ginsenosides.

2. A method for preparing modified ginseng insoluble dietary fiber, comprising: 1) Prepare liquid activation culture medium for bacterial strains; 2) Preparing a fermentation seed solution of Armillaria mellea: activating the Armillaria mellea, inoculating the activated Armillaria mellea into a liquid activation culture medium, and shaking culturing the culture at 25-30°C and 150-200 rpm for 5-7 days to obtain a fermentation seed solution of Armillaria mellea; the Armillaria mellea Am-07-22, with a deposit number of CCTCC NO: M 20221044; 3) Prepare ginseng fermentation medium: Add 1g of ginseng residue to 10-20mL of liquid culture medium for inoculum activation, sterilize, and cool. The ginseng residue is the solid ginseng residue remaining after extracting total ginsenosides, which is dried and crushed. 4) Inoculation: Inoculate 6-10% of the Armillaria fermentation seed solution into the ginseng fermentation medium and incubate at 25-30°C and 150-200 rpm for 4-8 days. 5) Extraction of modified ginseng insoluble dietary fiber: Extract the fermented ginseng insoluble dietary fiber by enzymatic hydrolysis.

3. The method for preparing a modified ginseng insoluble dietary fiber according to claim 2, wherein: The liquid culture medium for activating the bacterial strain comprises 20% potato, 0.5% silkworm pupa powder, 1% glucose, 1% sucrose, 2% yeast extract powder, 0.15% potassium dihydrogen phosphate, 0.075% magnesium sulfate heptahydrate, 0.001% vitamin B1, and the balance is water.

4. The method for preparing a modified ginseng insoluble dietary fiber according to claim 3, wherein: The inoculation amount of Armillaria mellea in step 4) is 8%.

5. The method for preparing a modified ginseng insoluble dietary fiber according to claim 4, characterized in that: The culture conditions in step 4) are 27° C. and 160 rpm for 6 days.

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