Water-insoluble dietary fiber obtained by fermenting black fungus through eurotium cristatum, method and application

Abstract

The invention relates to a method for obtaining insoluble dietary fiber (IDF) by fermenting black fungus through eurotium cristatum and extracting black fungus polysaccharide, and application of the insoluble dietary fiber in reducing blood sugar and blood fat. Eurotium cristatum is used for carrying out fermentation treatment on the black fungus; changes of physicochemical properties (water-holding capacity, oil-holding capacity and expansibility) and functional characteristics (alpha-amylase inhibition rate, glucose adsorption capacity, glucose dialysis delay capacity, pancreatic lipase inhibition rate and cholesterol adsorption capacity) of the IDF obtained after treatment with different fermentation time (2-10 days) and eurotium cristatum inoculation amount (1%-9%) are systematically studied. Results show that the water-holding capacity, the oil-holding capacity and the expansive force of the IDF after fermentation are respectively increased to 30.27 g / g, 3.31 g / g and 30.17 mL / g, and compared with the IDF of the unfermented black fungus, the water-holding capacity, the oil-holding capacity and the expansive force of the IDF are obviously enhanced (Plt; when the inoculation amount of eurotium cristatum is 5% and the fermentation time is 6 days, the alpha-amylase inhibition rate and glucose adsorption capacity of the obtained IDF respectively reach 79.34% and 83.06%, which are significantly higher than those of an unfermented control group (P is less than 0.05). The cholesterol adsorption capacity of the IDF after fermentation is improved by nearly 5 times compared with that of an unfermented group. The method provides a theoretical basis for reutilization of the black fungus residues, and lays a foundation for further development and application of insoluble dietary fibers.

Description

Technical Field

[0001] The invention relates to a method for fermenting black fungus with Eurotium cristatum, and obtaining insoluble dietary fiber therefrom with soluble solid content as an index to achieve the level of reducing blood sugar and lipid. Background Art

[0002] With the changes in modern lifestyle and dietary needs, people have gradually shifted from pursuing food and clothing to healthy eating, and from taking medicine to treating diseases to mild diet therapy. Contemporary people pay more attention to whether fruits and vegetables are green and pollution-free, and whether the food they eat is rich in nutritional value. At the same time, more and more people are paying attention to gastrointestinal health and pursuing foods rich in dietary fiber. The nutritional and functional values ​​of many foods are gradually being discovered.

[0003] Black fungus is a precious medicinal and edible colloid fungus in my country, and is also a beneficial fungus recognized by the public in the world. my country is the hometown of black fungus. Its industry scale is large in Heilongjiang, Jilin and other three provinces in the northeast, with high output and good quality. In addition, my country's black fungus cultivation technology has always been in the world's leading position. Not only is the market price relatively stable, but it also has a very broad development prospect. Black fungus not only has a smooth taste and delicious taste, but also contains dietary fiber, crude protein, amino acids, polysaccharides, polyphenols, and mineral elements required by the human body such as calcium, phosphorus, and iron. Among them, dietary fiber and oligosaccharides account for a large proportion, so black fungus can be used as a high-quality source of dietary fiber.

[0004] Dietary fiber is defined as the "seventh nutrient". It is a biological macromolecule composed of different monosaccharide molecules that cannot be digested and absorbed in the small intestine but can be completely or partially fermented in the large intestine and mainly comes from various plants. It can also be obtained from fruits, vegetables and cereals in the daily diet. Because it is composed of polysaccharides, hemicellulose and lignin, it is divided into soluble dietary fiber (SDF) and insoluble dietary fiber (IDF). The two have different morphological structures and chemical properties, so they also play different physiological roles in the gastrointestinal tract. Soluble dietary fiber is soluble in water, mainly including pectin, inulin, fructan, β-glucan and other substances; insoluble dietary fiber cannot be digested and absorbed by the human gastrointestinal tract, including cellulose, hemicellulose and lignin. It mainly manifests in lowering blood lipids and cholesterol, lowering blood sugar, reducing weight, regulating autoimmunity, proliferating beneficial bacteria and anti-oxidation aging and other beneficial functions.

[0005] In recent years, more and more studies have been conducted on the beneficial functions of insoluble dietary fiber (IDF) on the human body. IDF plays a series of beneficial roles based on its unique chemical groups, such as water-holding capacity and oil-holding capacity. It also has the ability to absorb lipids and cholesterol, which helps it lower blood lipids and has a certain regulatory effect on intestinal flora imbalance. At present, due to the gradual refinement of people's diet, there is a lack of intake of substances such as insoluble dietary fiber, resulting in an increase in the incidence of constipation and colon cancer year by year. In addition, in modern society, humans have a high-fat and high-sugar diet, which can cause various sub-health diseases, such as hypertension, hyperlipidemia, and hyperglycemia. Therefore, based on dietary balance, it is of great significance to fully develop and utilize the resources of insoluble dietary fiber and conduct in-depth research on its beneficial functions.

[0006] Fermentation is a biochemical process that uses the action of microorganisms to convert raw materials into desired products. This process mainly relies on microorganisms to convert raw materials into products needed by humans through specific metabolic pathways under suitable conditions. The level of fermentation production depends on the genetic characteristics of the strain itself and the culture conditions. In recent years, studies have confirmed that many edible and medicinal plants can improve their active functions and enhance their sensory quality by fermenting with fungi or probiotics. Foods fermented by microorganisms, such as kimchi, yogurt, bacon, etc., have excellent flavor and have a series of excellent properties such as adjusting product pH, antibacterial, and producing or enriching bioactive compounds, thereby improving product quality.

[0007] Eurotium cristatum, also known as "golden flower fungus", is a potential probiotic fungus belonging to the genus Eurotium of the family Aspergillus of the order Aspergillus of the class Ascomycetes. It is a key microorganism in the "flowering" process of traditional Fuzhuan tea. It is also a unique probiotic fungus that is widely used in the fermentation of food and Chinese medicinal materials, as well as safe and environmentally friendly in food processing. Eurotium cristatum can secrete a variety of extracellular enzymes, such as cellulase and pectinase. During the fermentation process, the extracellular enzymes secreted by Eurotium cristatum promote the metabolism of compounds in the fermentation substrate, thereby affecting the quality of food. In addition, a variety of bioactive metabolites can also be produced during the fermentation process, and the body's immune function can be increased by regulating the intestinal flora and improving the intestinal barrier, and it has a good lipid-lowering and weight-loss effect. At present, the fermentation process of Eurotium cristatum is mainly solid, and liquid form is less.

[0008] In summary, the numerous medicinal values ​​and health functions of black fungus have been recognized by people. Relevant clinical studies have shown that black fungus has the functions of clearing intestines and moistening lungs, and is conducive to timely removal and excretion of toxic substances in the body to the outside of the body. However, the extraction of active ingredients from black fungus is mostly concentrated on polysaccharides and soluble components, and the utilization of the remaining substances after extraction is relatively low. This trend has also greatly improved the utilization rate of food processing by-products, and improved the drawback that many value-added products in most by-products are not utilized. The present invention studies and utilizes the remaining residues produced by extracting active ingredients from black fungus, and finds that it contains a large amount of insoluble dietary fiber. Further studies have found that the insoluble dietary fiber of black fungus after pre-treatment and fermentation by Aspergillus niger has in vitro functional characteristics such as hypoglycemic and lipid-lowering. Summary of the invention

[0009] The invention aims to improve the blood sugar and lipid lowering effects of black fungus insoluble dietary fiber through fungus fermentation treatment.

[0010] In this experiment, the research group isolated Eurotium cristatum as the fermentation strain, activated it in potato dextrose agar medium, subcultured it, and then inoculated it in black fungus residue for fermentation. Insoluble dietary fiber was extracted from it and compared with unfermented black fungus as a control. The effect of bacterial fermentation treatment on the physicochemical properties and functional characteristics of insoluble dietary fiber from black fungus was explored.

[0011] The research significance lies in:

[0012] 1. To provide a theoretical and technical basis for further exploring the quality of deep-processed products from black fungus residue and to provide a reference for the nutritional value of dietary fiber from black fungus.

[0013] 2. Study the effects of fungal fermentation on black fungus, analyze the physical, chemical and functional properties of the insoluble dietary fiber therein, and lay the foundation for the subsequent development of dietary fiber foods.

[0014] Based on the above purpose, the present invention inoculates Eurotium cristatum into black fungus after pretreatment such as high pressure sterilization for solid fermentation, sets a group of control groups - control group 1 (pre-treated, not inoculated with Eurotium cristatum, i.e. 0d / 1%), and measures the content, water holding capacity, oil holding capacity, and swelling capacity of the two groups of insoluble dietary fiber. Subsequently, the in vitro hypoglycemic function of the insoluble dietary fiber is analyzed by measuring the glucose adsorption capacity, glucose dialysis retardation index, and α-amylase inhibition rate. Finally, the cholesterol adsorption capacity and pancreatic lipase inhibition rate of the insoluble dietary fiber are measured to analyze its in vitro lipid-lowering function.

[0015] Explanation of the attached table BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1Effect of different fermentation time and inoculation amount on the yield of insoluble dietary fiber from black fungus

[0017] Figure 2 Effects of different fermentation times on the physicochemical properties of black fungus IDF

[0018] Figure 3 Effects of different inoculation doses on the physicochemical properties of black fungus IDF

[0019] Figure 4 Results of α-amylase inhibition rate of insoluble dietary fiber under different fermentation conditions

[0020] Figure 5 Results of glucose adsorption capacity of insoluble dietary fiber under different fermentation conditions

[0021] Figure 6 Results of glucose dialysis retardation index of insoluble dietary fiber under different fermentation conditions

[0022] Figure 7 Results of pancreatic lipase inhibition rate of insoluble dietary fiber under different fermentation conditions

[0023] Figure 8 Results of cholesterol adsorption capacity of insoluble dietary fiber under different fermentation conditions DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention will be described clearly and completely below.

[0025] The influence of the fermentation of black fungus by Eurotium cristatum in this embodiment on the physicochemical properties and structural properties of its insoluble dietary fiber is achieved according to the following method steps:

[0026] Experimental Example 1 Effects of different fermentation time and inoculation amount on the yield of insoluble dietary fiber from black fungus

[0027] 1. Preparation of Eurotium cristatum Seed Solution

[0028] Activation of Eurotium cristatum and preparation of spore suspension The preserved strain of Eurotium cristatum was inoculated on the prepared PDA medium using a loop, and the mycelium was scraped off after culturing at 28°C for 4 days and transferred to a triangular flask containing 100 mL of sterile water. The mixture was shaken at 28°C and 120 r / min for 30 min. The above operation was repeated twice to fully activate the strain. The cells were counted using a hemocytometer and diluted to a cell density of 1×10 6 Cfu / mL is obtained.

[0029] 2. Fermentation experiment of Eurotium cristatum inoculated into black fungus

[0030] (1) To investigate the effect of inoculation amount on the yield of insoluble dietary fiber from black fungus

[0031] The raw material black fungus was crushed and passed through an 80-mesh sieve, then put into a 250mL conical flask, with 8g in each bottle. Then distilled water was added to each bottle at a material-liquid ratio of 1:5 (v / v), and the bottle was sealed with a breathable membrane and sterilized in an autoclave at 121°C. After the sterilization, the fermented matrix was cooled to room temperature and the prepared suspension of Eurotium coronatum was inoculated at 1%, 3%, 5%, 7%, and 9% of the raw material, and then placed in a 28°C incubator for 6 days. Each level was repeated three times. After the fermentation was completed, each group of samples was fully stirred by adding 1:60 (v / v) distilled water and ultrasonically treated at 200-400W and 40-80°C for 20-40min, magnetically stirred for 0.5-2h, and the pH value was adjusted to 11. After centrifugation at 4000r / min for 20min, the samples were dried at 55°C and weighed for later use.

[0032] (2) Experiment on the effect of fermentation time on the yield of insoluble dietary fiber from black fungus

[0033] After the raw material black fungus is crushed and passed through an 80-mesh sieve, it is placed in a 250mL conical flask, with 8g of material in each bottle. After that, distilled water is added to each bottle according to a material-liquid ratio of 1:5 and sealed with a breathable membrane, and sterilized in an autoclave at 121°C. After the sterilization is completed, the crown-processed spores are inoculated into the black fungus matrix according to an inoculation amount of 5% (v / w) and cultured at 28°C. The fermentation is repeated three times at each level for 0d, 2d, 4d, 6d, 8d, and 10d. The black fungus matrix is ​​covered with crown-processed spores hyphae and grows vigorously, which means the fermentation is over. The sterilized uninoculated black fungus (0d) is used as the control. After the fermentation was completed, each group of samples was added with 1:60 distilled water and fully stirred, and ultrasonically treated at 200-400W and 40-80℃ for 20-40min. After magnetic stirring for 0.5-2h, the pH value was adjusted to 11, centrifuged at 4000r / min for 20min, dried at 55℃, and weighed for later use.

[0034] 3. Dietary fiber yield determination experiment

[0035] After grinding and dehydration, the sample was transferred to a 500mL beaker, 35mL of 50mmol / L maleic acid buffer was added, and stirred with a magnetic stirrer until the sample was completely dispersed in the buffer. Two blank samples were prepared at the same time. Then 50μL of heat-stable α-amylase solution was added to the beaker, stirred slowly, covered with aluminum foil, and placed in a constant temperature shaking water bath at 95℃~100℃. When the temperature rises to 95℃, the timing was started and the shaking reaction was carried out for 35min. The beaker was taken out, cooled to 60℃, the aluminum foil cover was opened, and the paste on the inner wall of the beaker and the colloid on the bottom of the beaker were scraped off with a spatula, and the beaker wall and spatula were rinsed with 5mL 50mmol / L maleic acid buffer. 100μL of amyloglucosidase solution was added to the high-foot beaker while stirring, covered with aluminum foil, and continued to be placed in a 60℃ water bath. When the water temperature reaches 60℃, the timing was started and the shaking reaction was carried out for 30min. After the reaction, add 100 μL of protease solution to each beaker, cover with aluminum foil, and place in a 60°C water bath and shake continuously for 30 min. Open the aluminum foil cover, add 4 mL of 2 mol / L acetic acid solution while stirring, and adjust the pH to 4.3 ± 0.2 with 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid solution. Centrifuge at 4000 r / min for 20 min, obtain the residue, rinse with distilled water, and dry at 55°C to obtain dry IDF.

[0036] The IDF yield calculation formula is as follows:

[0037] R=M1 / M2×100%

[0038] Wherein: R represents the yield of insoluble dietary fiber of fungus, %; M1 represents the mass of insoluble dietary fiber of fungus, g; M2 represents the mass of fungus sample, g.

[0039] according to Figure 1 It can be seen that according to the control of inoculation amount and fermentation time, compared with the non-fermentation (control group), the IDF yield mostly showed an upward trend, indicating that the crown-like spores may change the structure of black fungus dietary fiber through its own secreted enzymes such as cellulase during the fermentation process, making the insoluble fiber that was originally difficult to detect or separate easy to extract or measure. When the inoculation amount was 5% and the fermentation time was 6 days, the lowest IDF yield was 69.99±5.11% and 68.84±8.11%, respectively. The reason may be that under this condition, the metabolic activity of the flora is at its peak, and the microorganisms secrete a large amount of cellulase, hemicellulase, etc., which will significantly reduce the insoluble dietary fiber in black fungus. These enzymes break down insoluble dietary fiber into smaller molecules, such as oligosaccharides and monosaccharides, or convert them into soluble dietary fiber, thus resulting in a decrease in the yield of insoluble dietary fiber. In addition, if the fermentation time is further extended, the metabolic activity of the microorganisms may decrease, and the yield of insoluble dietary fiber may have a rebound trend, which is consistent with the results in the figure.

[0040] Physicochemical properties of insoluble dietary fiber under different fermentation time and inoculum amount

[0041] Water holding capacity: Weigh 0.50 g of black fungus IDF sample dried to constant mass into a 50 mL centrifuge tube (the dry mass of the sample is recorded as m0 and the mass of the tube is recorded as m1), add 20 mL of distilled water, stir well to make the sample evenly dispersed in the centrifuge tube, seal it and place it in a refrigerator overnight, centrifuge it at 4000 r / min for 25 min, then discard the supernatant, use filter paper to absorb the residual water on the tube wall, weigh the mass (the mass of the sample and centrifuge tube after absorbing water is recorded as m2), and calculate the IDF water holding capacity.

[0042] Water holding capacity / (g / g)=(m2-m1) / m0

[0043] Oil holding capacity Weigh 0.50 g of black fungus IDF sample dried to constant mass into a 50 mL centrifuge tube (the dry mass of the sample is recorded as m0, and the mass of the tube is recorded as m1), add 10 mL of edible oil, shake well, let stand at room temperature for 1 hour, and then centrifuge at 4000 r / min for 20 minutes. Immediately discard the supernatant, wipe off the grease attached to the inner and outer walls of the centrifuge tube, and weigh it (the mass of the sample and the centrifuge tube after oil absorption is recorded as m2). Calculate the IDF oil holding capacity.

[0044] Oil holding capacity / (g / g)=(m2-m1) / m0

[0045] Swelling force Weigh 0.50 g of black fungus IDF sample dried to constant mass and place it in a 20 mL graduated test tube (the dry mass of the sample is recorded as m), read the volume of the dry product (V1), and add 10 mL of distilled water to it, stir it thoroughly to remove bubbles in the solution, shake it evenly, and place it in a refrigerator overnight. Accurately read the volume of the material in the test tube after expansion (V2), and calculate the IDF swelling force.

[0046] Expansion force / (mL / g) = (V2-V1) / m

[0047] like Figure 2 The water holding capacity, oil holding capacity and swelling capacity of black fungus IDF under different inoculation amounts are shown in Figure 2. Insoluble dietary fiber has good water holding capacity. The water holding capacity, oil holding capacity and swelling capacity of black fungus IDF before and after fermentation increased from 25.68±0.24 c , 2.44±0.06 b , 22.57±2.53 c Increased to 28.97±0.62 b , 3.33±0.02 a , 23.99±0.22 bcCompared with that before fermentation, it was significantly improved (P<0.05), and during the entire fermentation stage, IDF was between 3% and 9%, showing stronger water holding capacity, oil holding capacity, and swelling capacity.

[0048] like Figure 3 The water holding capacity, oil holding capacity and swelling capacity of black fungus IDF increased to 28.78±0.26 before and after fermentation. ab , 3.48±0.12 a , 26.70±0.73 bc Compared with the level before fermentation, it was significantly improved (P < 0.05), and during the entire fermentation stage, IDF showed stronger water holding capacity, oil holding capacity, and swelling capacity from 4d to 10d.

[0049] Experimental Example 2 Experiment on determination of α-amylase inhibition rate of insoluble dietary fiber under different fermentation conditions

[0050] Add α-amylase (2u / mL) to black fungus IDF under different fermentation conditions, place in a 37°C water bath for 10 minutes, then add 500μL of 1% soluble starch solution, and keep warm in a 37°C water bath for 10 minutes. Then add 1mL of DNS reagent, heat in a boiling water bath for 5 minutes to terminate the reaction, and cool quickly. Add 10mL of distilled water. Use a phosphate buffer solution system instead of α-amylase as the control group, and distilled water instead of IDF as the blank group. Measure the OD value of each reaction system at 540nm. Perform 3 parallel experiments to ensure the accuracy of the test. The inhibition rate of IDF on α-amylase is calculated as follows:

[0051]

[0052] Wherein: A0 represents the absorbance of phosphate buffer; A1 represents the absorbance of the entire reaction system; A2 represents the absorbance of the α-amylase-catalyzed reaction system.

[0053] Figure 4It can be seen that with the change of inoculation amount and fermentation time, the α-amylase activity of IDF also showed a change of first rising and then falling. When the fermentation time was 6 days and the inoculation amount was 5%, the inhibitory activity reached 79.34±9.96% and 83.06±0.57%, respectively, which was 48.92±1.59% higher than that of the non-fermented control group, with significant differences (P<0.05). This shows that when the inoculation amount is 5%, the appropriate inoculation amount makes the bacteria sufficient but not too dense, so that the bacteria can be fully active and the enzymes and metabolites produced are rich. After the growth and metabolism of the bacteria in the first few days of fermentation, on the 6th day, the cellulase and hemicellulase produced by the crown-like spores can fully act on the IDF of black fungus, resulting in partial degradation of the fiber structure, thereby enhancing the inhibitory effect. The later period showed a downward trend, which may be because after a long period of fermentation, the activity of the bacteria decreased, and the excessive decomposition of the fiber led to a decrease in the inhibitory effect.

[0054] Experimental Example 3: Determination of glucose adsorption capacity of insoluble dietary fiber under different fermentation conditions

[0055] (1) Drawing of glucose standard curve

[0056] Take 6 20mL graduated test tubes, add 0, 0.2, 0.4, 0.6, 0.8, 1.0mL of 1mg / mL glucose standard solution, add distilled water to make the volume reach 1mL, add 2mL DNS reagent to the test tube, mix well and heat in a boiling water bath for 5 minutes, cool with cold water immediately after taking out, make up to 15mL with distilled water, and shake well. Measure the absorbance A at OD of 520nm, and draw a standard curve with absorbance as the ordinate and glucose content as the abscissa. Obtain the standard curve and regression equation of glucose concentration (X) and absorbance (Y), where the glucose standard curve is y=0.8127x+0.0768, R 2 =0.9962.

[0057] (2) Determination of glucose adsorption capacity of insoluble dietary fiber

[0058] Take 0.25g (m0) of the sample under different fermentation conditions and add it to 25mL (V) of 1mg / mL (m1) glucose solution, stir evenly, and incubate in a 37℃ water bath oscillator for 6h. After adsorption equilibrium, centrifuge the mixture at 4000r / min for 20min. Determine the glucose content (m2) in the supernatant according to the above method. The glucose adsorption capacity is calculated as follows:

[0059]

[0060] Where: m1 is the glucose content in the supernatant before adsorption; m2 is the glucose content in the supernatant after adsorption; m0 is the dry mass of the sample.

[0061] like Figure 5 As shown in the figure, when the fermentation time is 0 days (unfermented group), the fiber structure is relatively stable, the porosity and specific surface area are relatively small, and the glucose adsorption is low at 2.67±0.06 mg / g. When the fermentation time reaches 6 days and the inoculation amount is 5%, the glucose adsorption reaches a peak of 3.34±0.05 mg / g. The glucose adsorption is mainly affected by the structural changes, porosity, specific surface area and surface chemical properties of insoluble dietary fiber. Fermentation with Aspergillus crownus can make the dietary fiber structure looser, increase the surface area, increase or expose the binding sites, and help the adsorption capacity of glucose.

[0062] Experimental Example 4: Experiment on the Delaying Ability of Glucose Dialysis of Insoluble Dietary Fiber under Different Fermentation Conditions

[0063] Accurately weigh 0.2g of sample and mix with 10mL of 0.1% glucose solution and put it into a dialysis bag (Mw=14000). Place the dialysis bag in a 500mL beaker filled with 200mL of distilled water, oscillate at 37℃ and 160r·min-1 for 1.5h, take 2mL of dialysate every 15min, and measure the glucose mass concentration.

[0064] Distilled water was used to replace glucose as a blank group, and a glucose solution without a sample was used as a control group. The glucose dialysis delay capacity (%) of the sample was calculated according to the following formula:

[0065]

[0066] Where: A1 is the glucose concentration of the sample group, A2 is the glucose concentration of the blank group, and A3 is the glucose concentration of the control group, in mg·mL -1

[0067] Figure 6 a is the corresponding change of glucose dialysis delay capacity of different insoluble dietary fiber samples obtained at different fermentation times. Overall, with the passage of time, the glucose dialysis delay index (GDRI) of most fermentation groups showed a trend of first decreasing, then increasing, and then decreasing. The downward trend may be due to the fact that the fiber structure was in a state of partial degradation or excessive degradation in the early and late fermentation, respectively, resulting in a rapid decrease in dialysis delay capacity. Or physical factors such as glucose concentration gradient and fiber water saturation effect accelerated diffusion in a short period of time, further reducing GDRI. The IDF of fermentation for 6 days reached its maximum value at 60 minutes, while most of the other samples reached their maximum values ​​at 30 minutes and 45 minutes, respectively. In addition, the GDRI of insoluble dietary fiber fermented for 6 days reached 34.51±2.77% at 60 minutes, which was significantly higher than the IDF samples of other time periods (P<0.05), such as 20.86±1.32% for 10 days of fermentation (30 minutes).

[0068] Figure 6 b shows the corresponding changes in the glucose dialysis delaying ability of different insoluble dietary fiber samples obtained with different inoculation amounts. The figure shows that with the increase in the inoculation amount, GDRI increases in the early time period (15min and 30min). At 5% inoculation amount, GDRI is significantly higher than other inoculation amounts at each time point. The remaining samples show a good delay effect at 15-30min, and some samples tend to stabilize after a slight increase after 60min. The performance of too high inoculation amount is relatively mediocre, which may be because the bacterial community with a high inoculation amount needs to consume more nutrients, while the available nutrients in the fermentation matrix are limited. When the inoculation amount is too high, there will be nutritional competition between bacterial species, resulting in a reduction in the nutrients that each bacterial community can obtain, affecting its metabolic efficiency. When the bacterial population density is too high, the bacterial species may secrete some inhibitory substances to inhibit the growth and metabolism of itself or other bacterial communities. This self-inhibition effect may be one of the reasons why too high inoculation amounts (7% and 9%) did not bring significant improvements.

[0069] In summary, each microorganism has an optimal inoculum size during the fermentation process. If the inoculum size exceeds this optimal range, the fermentation efficiency will decrease. For E. coronariae, a fermentation time of 6 days and a 5% inoculum size may be the optimal fermentation time and bacterial concentration under these conditions, making the fermentation product the most ideal in terms of improving the ability to delay glucose dialysis.

[0070] Experimental Example 5: Determination of pancreatic lipase inhibition rate of insoluble dietary fiber under different fermentation conditions

[0071] (1) Determination of pancreatic lipase activity

[0072] Take three 50mL conical flasks, add 5mL 0.025moI / L PBS buffer (pH=7.4) and 4mL polyvinyl triolein substrate emulsion to each flask, preheat in a 37°C incubator for 10 minutes, then add 1mL 2mg / mL pancreatic lipase solution (prepared with pH=7.4 0.025mol / L PBS buffer) (except the blank), start timing from the addition of the pancreatic lipase solution, and immediately add 15mL 95% ethanol solution after keeping warm for 15 minutes to stop the enzyme reaction. Add three drops of phenolphthalein indicator to each conical flask, and use 0.025mol / L sodium hydroxide standard solution until it turns slightly red, and do a blank control at the same time. In the blank control experiment, no pancreatic lipase solution is added first. After adding PBS and substrate emulsion, keep warm for 15 minutes, and then immediately add 15mL 95% ethanol solution.

[0073] Then add 1 mL of pancreatic lipase solution and phenolphthalein indicator. Calculate pancreatic lipase activity according to the following formula:

[0074]

[0075] (2) Determination of pancreatic lipase inhibition rate

[0076] Add PBS buffer and a certain amount of IDF sample to a 50mL conical flask, keep warm at 37℃ for 10min, then add 1mL pancreatic lipase solution, start timing for 10min from the addition of enzyme solution, add substrate emulsion after the end, keep warm at 37℃ for 15min, immediately add 15mL 95% ethanol solution and phenolphthalein indicator, and measure the residual enzyme activity. For the blank test, first add PBS and substrate to keep warm, then add IDF, and finally add ethanol and enzyme solution.

[0077] Definition of inhibition activity unit: Under the conditions of pH 7.4 and temperature of 37°C, one unit of enzyme activity is inactivated to form one inhibitor activity unit.

[0078] Inhibition rate of pancreatic lipase / % = (inhibition of pancreatic lipase activity before - inhibition of pancreatic lipase activity after) / inhibition of pancreatic lipase activity before

[0079] Figure 7 a is the correlation between the inhibition rate of pancreatic lipase by insoluble dietary fiber at different fermentation times. As the fermentation time increases, the inhibition rate of pancreatic lipase shows a trend of first increasing and then decreasing. Compared with the inhibition rate of 36.39±3.28% when the fermentation time is 0d, the inhibition rate is relatively high when the fermentation days are 6d, reaching 60.08±2.88%, which is significantly different from 0d and 2d (P<0.05). Microbial metabolic activity can improve the surface activity of some insoluble dietary fiber and further inhibit pancreatic lipase. It can be proved that insoluble dietary fiber can be used as a pancreatic lipase inhibitor to reduce the digestion and absorption of fat.

[0080] Figure 7 b is the results of the pancreatic lipase inhibition rate obtained when the inoculation amount was 1%, 3%, 5%, 7%, and 9%, with no inoculation (0%) as the control. As can be seen from the figure, with the increase of the inoculation amount, the pancreatic lipase inhibition rate showed a continuous upward trend. The inhibition rates of the three groups of 1%, 3%, and 5% were 51.07±2.52%, 56.22±2.56%, and 56.99±2.22%, respectively. There was no significant difference among the three, but it was significantly higher than the non-fermented group (P<0.05). The inhibition rate was the best when the inoculation amount was 9%, reaching a peak value (67.82±1.64%), which was significantly higher than other inoculation groups (P<0.05). This shows that the inoculation amount is low, the metabolic activity of the strain is weak, and the dietary fiber structure has no obvious change. With the increase of the inoculation amount, the metabolic activity and metabolites of the crown-like cysts continue to increase, showing a more obvious inhibitory activity.

[0081] Experimental Example 6 Experiment on the Determination of Cholesterol Adsorption Capacity of Insoluble Dietary Fiber under Different Fermentation Conditions

[0082] (1) Cholesterol standard curve drawing

[0083] The o-phthalaldehyde method was used to determine the cholesterol content in the sample solution. Accurately weigh 250 mg of o-phthalaldehyde, dilute to a 250 mL volumetric flask and refrigerate in a brown reagent bottle. To prepare a 1.0 mg / mL standard cholesterol stock solution, weigh 250 mg of the cholesterol standard and dilute to 250 mL with glacial acetic acid; to prepare a 0.1 mg / mL standard cholesterol working solution, accurately draw 10 mL from the standard cholesterol stock solution and dilute to 100 mL with anhydrous ethanol.

[0084] Transfer 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, and 0.40 mL of 0.1 mg / mL cholesterol standard working solution to colorimetric tubes, add glacial acetic acid to each tube to a volume of 0.4 mL, and add 0.1 mL of 1 mg / mL o-phthalaldehyde reagent and 2.0 mL of mixed acid (glacial acetic acid and concentrated sulfuric acid mixed in a ratio of 1:1) to each tube. After fully and evenly mixing, place in a water bath at 25°C for 10 minutes. Measure the OD value at a wavelength of 550 nm, and calculate the cholesterol content according to the standard curve. Repeat each test 3 times.

[0085] (2) Cholesterol adsorption capacity determination

[0086] Add distilled water to fresh raw egg yolks and mix thoroughly to obtain 10% egg yolk emulsion. Weigh IDF powder with different fermentation time and inoculation amount respectively, and add 25g egg yolk liquid into a conical flask. Adjust the solution system to pH = 2.0 (simulated gastric fluid environment) and pH = 7.0 (simulated small intestine environment), shake at 37℃ for 2h, then centrifuge at 4000r / min for 20min, take 0.1mL supernatant, and detect it at 550nm wavelength using o-phthalaldehyde method.

[0087] Cholesterol adsorption capacity = (cholesterol content of egg yolk before adsorption - cholesterol content of supernatant after adsorption) / sample mass

[0088] like Figure 8 a is the cholesterol standard curve, and the standard curve and regression equation of cholesterol concentration (X) and absorbance (Y) are obtained. The cholesterol standard curve is y = 0.4471x ± 0.0551, R 2 =0.9943.

[0089] like Figure 8As shown in b, the adsorption capacity of IDF for cholesterol at different fermentation times was better than that of unfermented IDF, and the maximum cholesterol adsorption capacity of fermented wood ear IDF at pH = 2 (simulated gastric juice) and pH = 7 (simulated intestinal juice) was 8.82±1.17 mg / kg and 24.26±0.51 mg / kg, respectively; the adsorption capacity of IDF for cholesterol at different inoculum amounts was 8.33±1.17 mg / kg and 18.06±0.36 mg / kg, respectively, which was significantly enhanced compared with the unfermented 5.22±0.02 mg / kg and 4.20±0.11 mg / kg (P<0.05) because fermentation made the surface structure of IDF loose, the pores increased, and the particle size decreased, thereby enhancing the adsorption of cholesterol.

Claims

1. A method and application of fermenting black fungus with Eurotium cristatum and extracting the residue of black fungus soluble polysaccharide to prepare insoluble dietary fiber (IDF), characterized in that It is implemented as follows: (1) Prepare the seed solution of Eurotium cristatum, activate and subculture the preserved strain of Eurotium cristatum, count the cells using a hemocytometer, and dilute to a cell density of 1×10 5 ~1×10 9 CFU / mL, that’s it.

2. (2) After the raw material black fungus is crushed and sieved (40-200 mesh), distilled water is added according to the mass volume ratio of material to liquid of 1:4~1:20 and sealed with a breathable membrane for sterilization. After sterilization, the prepared suspension of Eurotium cristatum is inoculated at 1%~9% of the raw material amount and cultured, and each level is repeated three times. Each inoculated amount of black fungus is fermented for 0 d~10 d, and each level is repeated three times. After the fermentation is completed, each group of samples is subjected to ultrasound and magnetic stirring to adjust the pH value, centrifuged, dried, and weighed for later use.

3. (3) Grind the dried black fungus residue and determine the IDF yield according to the National Food Safety Standard Determination of Dietary Fiber in Food GB 5009.88-2023.

4. (4) Obtain IDF under different fermentation conditions and measure its water holding capacity, oil holding capacity and swelling capacity. Three parallel samples are collected in each group, and the unfermented sample is used as the control.

5. (5) Add α-amylase to IDF at different fermentation times and inoculation amounts, place in a 37°C water bath for 10 min, and then perform an α-amylase inhibition test.

6. (6) Draw a standard curve of 1 mg / mL glucose standard solution, take samples under different fermentation conditions, add 1 mg / mL glucose solution, stir evenly, incubate in a 37 ℃ water bath oscillator for 6 h, and conduct a glucose adsorption capacity determination experiment.

7. (7) Accurately weigh the sample and mix it with 0.1% glucose solution and put it into a dialysis bag. Then put it into a beaker with distilled water and conduct a glucose dialysis delay test.

8. (8) Prepare a sufficient number of conical flasks and add PBS buffer and different IDF samples into them. Set up blank test and blank control test to carry out the experiment of determining the inhibition rate of pancreatic lipase.

9. (9) Determine the cholesterol content in the sample solution using the o-phthalaldehyde method. Prepare o-phthalaldehyde solution, standard cholesterol storage solution and standard cholesterol working solution, and draw a 0.1 mg / mL cholesterol standard curve. Repeat each test 3 times. Use fresh raw egg yolk to prepare egg yolk emulsion. Weigh the IDF of different fermentation times and inoculation amounts respectively. Adjust the solution system to pH=2.0 (simulated gastric fluid environment) and pH=7.0 (simulated small intestine environment) to conduct cholesterol adsorption test experiments.

10. The method according to claim 1, characterized in that The described Eurotium cristatum has the ability to secrete cellulase, pectinase and protease. Therefore, the strain used in this scheme is not limited to Eurotium cristatum, and other strains that can secrete corresponding enzymes (including one or two enzymes) can be used.

11. The method according to claim 1, characterized in that The insoluble black fungus dietary fiber is prepared by extracting the residue after extracting the soluble black fungus polysaccharide, which further increases the utilization efficiency of the black fungus.

12. The method according to claim 1, characterized in that The black fungus fermentation process is to crush the raw black fungus and pass it through an 80-mesh sieve, then put it into a 250 mL conical flask, with 8 g of material in each bottle, and then add distilled water to each bottle at a material-liquid ratio of 1:5 (v / v), seal it with a breathable membrane, and put it in a high-pressure sterilizer for sterilization at 121 °C. After the sterilization is completed, the fermentation matrix is ​​cooled to room temperature and inoculated at 1%~9% (v / w) of the raw material to obtain a cell density of 1×10 6 CFU / mL suspension of Eurotium cristatum was prepared and then cultured in a constant temperature incubator at 28 ℃ for 6 days.

13. The method according to claim 1, characterized in that The density of Eurotium cristatum was adjusted to 1×10 5 ~ 1×10 9 CFU / mL.

14. The method according to claim 1, characterized in that After the fermentation was completed, 1:60 (v / v) distilled water was added to each group of samples, and they were fully stirred and ultrasonically treated at 200-400 W and 40-80 °C for 20-40 min. After magnetic stirring for 0.5-2 h, the pH value was adjusted to 11, and the samples were centrifuged at 4000 r / min for 20 min, dried at 55 °C, and weighed for later use.

15. The method for determining the yield of insoluble dietary fiber according to claim 1, characterized in that After the sample is ground, crushed and dehydrated, it is further hydrolyzed with α-amylase and amyloglucosidase solution to remove starch and low molecular weight sugars before calculating the yield.

16. The method for measuring IDF water holding capacity, oil holding capacity and swelling capacity according to claim 1, characterized in that The above indicators of IDF were significantly improved after 6 days of fermentation.

17. The IDF method for determining the α-amylase inhibition rate and glucose adsorption capacity according to claim 1, characterized in that After 6 days of fermentation, the α-amylase inhibition rate, glucose adsorption capacity and glucose dialysis delaying ability of IDF were significantly improved.

18. The method for determining the pancreatic lipase inhibition and cholesterol adsorption capacity of IDF according to claim 1, characterized in that After 6 days of fermentation, the pancreatic lipase inhibition and cholesterol adsorption capacity of IDF were significantly improved.

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