Modified insoluble dietary fiber as well as preparation method and application thereof

Abstract

The invention provides modified insoluble dietary fibers as well as a preparation method and application thereof, and relates to the technical field of modified dietary fibers. The preparation method of the modified insoluble dietary fiber comprises the following steps: preparing Chinese cabbage insoluble dietary fiber, adding the Chinese cabbage insoluble dietary fiber into sterile water according to a material-liquid ratio of 1g: (10-30) mL, fully stirring, and inoculating lactobacillus plantarum for fermentation culture; and after fermentation culture is finished, adding an ethanol aqueous solution, carrying out alcohol precipitation, carrying out suction filtration, collecting precipitates, and carrying out freeze drying to obtain the lactobacillus plantarum fermentation modified Chinese cabbage outer leaf insoluble dietary fibers. Test results show that the insoluble dietary fibers of the Chinese cabbages fermented and modified by the lactobacillus plantarum have the best improvement effect on the oil holding capacity, the glucose adsorption capacity, the alpha-amylase activity inhibition capacity and other properties, have better performance in the aspects of blood sugar and blood fat regulation and antioxidant activity, and can be used as a good functional food raw material.

Description

Technical Field

[0001] The invention belongs to the technical field of modified dietary fiber, and specifically relates to a modified insoluble dietary fiber and a preparation method and application thereof. Background Art

[0002] Dietary fiber (DF), as one of the seven major nutrients, is a type of carbohydrate that is beneficial to human health. It exists in grains, fruits and vegetables, beans, seaweed and other plants. It cannot be digested and absorbed by the human gastrointestinal tract, nor does it provide energy for the human body, but it can be fermented in the large intestine to promote intestinal function. Dietary fiber can be divided into soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) according to its solubility. Insoluble dietary fiber mainly acts on the intestine to produce mechanical peristalsis, while soluble dietary fiber plays a more metabolic role, such as affecting the metabolism of available carbohydrates and lipids.

[0003] The outer leaves of Chinese cabbage are byproducts produced during the harvesting, transportation and sales of Chinese cabbage. Currently, they are mainly used as animal feed or waste and have not been fully utilized. However, the outer leaves of Chinese cabbage contain a large amount of dietary fiber and have great development value. However, there are few studies on their preparation, modification and application. Therefore, it is of great research significance to extract insoluble dietary fiber from Chinese cabbage and modify it to enhance its functional properties and improve its utilization rate. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a modified insoluble dietary fiber and a preparation method and application thereof, wherein the modified insoluble dietary fiber has more stable properties and has better blood sugar and blood lipid regulation and antioxidant activity.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a method for preparing modified insoluble dietary fiber, comprising the following steps: preparing Chinese cabbage insoluble dietary fiber, adding sterile water to the Chinese cabbage insoluble dietary fiber according to a solid-liquid ratio of 1g:10-30mL, fully stirring, inoculating plant lactobacillus for fermentation and culturing; after the fermentation and culturing is completed, adding ethanol aqueous solution, precipitating by alcohol, collecting precipitates by suction filtration, and freeze-drying to obtain the modified insoluble dietary fiber.

[0007] Preferably, the inoculation amount of the Lactobacillus plantarum is 5-10%.

[0008] Preferably, the fermentation culture is carried out at 35-37° C. for 40-50 hours.

[0009] Preferably, the alcohol precipitation time is 20 to 30 hours.

[0010] Preferably, the preparation method of the insoluble dietary fiber of Chinese cabbage comprises the following steps: taking cabbage residue, adding distilled water according to a solid-liquid ratio of 1g:30-40mL, fully stirring, adjusting the pH value, and adding alkaline protease for enzymolysis; after the enzymolysis is completed, heating in a boiling water bath for 8-15min and then cooling to room temperature, centrifuging and discarding the supernatant to obtain a precipitate; washing the precipitate and collecting the precipitate by suction filtration, freeze-drying, crushing and sieving, and obtaining the insoluble dietary fiber of Chinese cabbage.

[0011] Preferably, the enzymatic hydrolysis condition is 35-45° C. water bath enzymatic hydrolysis for 35-45 min.

[0012] Preferably, the precipitate is washed with 65-75°C hot water, 75-80% ethanol aqueous solution, and 90-98% ethanol aqueous solution in sequence.

[0013] The invention also provides modified insoluble dietary fiber prepared by the preparation method.

[0014] The present invention also provides an application of the modified insoluble dietary fiber in preparing an antioxidant activity product.

[0015] The present invention also provides an application of the modified insoluble dietary fiber in preparing a product for regulating blood sugar and blood lipids.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a modified insoluble dietary fiber and a preparation method thereof. The present invention extracts insoluble dietary fiber (CIF) by enzymatic hydrolysis, and then ferments and modifies CIF by using plant lactobacillus. The surface structure of the modified CIF is rough and porous, the particle size is reduced, the specific surface area is increased, the crystallinity is increased, and the CIF has better thermal stability. The test results show that the three properties of the fermented modified CIF (L-CIF) are improved best, namely, oil holding capacity, glucose adsorption capacity and α-amylase activity inhibition capacity, which are increased by 33.13%, 118.18% and 31.00% respectively. At the same time, the modified CIFs have good performance in regulating blood sugar and blood lipids and antioxidant activity, and can be used as a good functional food raw material. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The scanning electron microscope images of unmodified cabbage insoluble dietary fiber (CIF) and fermented modified cabbage insoluble dietary fiber (L-CIF) of the present invention, in which A and B are the observation results of CIF and L-CIF at 200×, respectively, and a and b are the observation results of CIF and L-CIF at 2000×, respectively;

[0019] Figure 2 is the particle size distribution diagram of CIFs;

[0020] Figure 3 is the infrared spectrum of CIFs;

[0021] Figure 4 is the X-ray diffraction pattern of CIFs;

[0022] Figure 5 The thermogravimetric analysis diagram of CIFs, in which A is unmodified cabbage insoluble dietary fiber (CIF), and B is the fermented and modified cabbage insoluble dietary fiber (L-CIF) of the present invention;

[0023] Figure 6 is the water holding capacity of CIFs;

[0024] Figure 7 The oil holding capacity of CIFs;

[0025] Figure 8 is the expansion force of CIFs;

[0026] Fig. 9 is the glucose adsorption capacity of CIFs, and different letters in the figure indicate significant differences at the p<0.05 level;

[0027] Fig.10 is the α-amylase activity inhibition ability of CIFs, and different letters in the figure indicate significant differences at the p<0.05 level;

[0028] Fig.11 is the inhibitory ability of CIFs on α-glucosidase activity, and different letters in the figure indicate significant differences at the p<0.05 level;

[0029] Fig.12 is the cholesterol adsorption capacity of CIFs. Different letters in the figure indicate significant differences at the p<0.05 level;

[0030] Fig.13 is the sodium cholate adsorption capacity of CIFs, and different letters in the figure indicate significant differences at the p<0.05 level;

[0031] Fig.14 is the free radical scavenging ability of CIFs, in which A is the DPPH free radical scavenging ability and B is the ABTS free radical scavenging ability;

[0032] Fig.15The effect of L-CIF intervention on the body weight, food intake and energy intake of mice with glucose and lipid metabolism disorders. In the figure, A is the body weight of mice; B is the food intake of mice; C is the energy intake of mice; n=10, mean±SEM, compared with the CON group: *P<0.05, **P<0.01; compared with the HFD group: #P<0.05, ##P<0.01;

[0033] Fig.16 The effect of L-CIF intervention on the levels of serum inflammatory factors in mice with glucose and lipid metabolism disorders. In the figure, A is the level of lipopolysaccharide, B is the level of tumor necrosis factor-α (TNF-α); C is the level of interleukin-6 (IL-6); D is the level of interleukin-1β (IL-1β); n=10, mean±SEM, compared with the CON group: *P<0.05, **P<0.01; compared with the HFD group: #P<0.05, ##P<0.01;

[0034] Fig.17 The effect of L-CIF intervention on insulin resistance in mice with glucose and lipid metabolism disorders. Figure A is fasting blood glucose (FGB); B is insulin (INS); C is insulin resistance index (HOMA-IR); n=10, mean±SEM, compared with CON group: *P<0.05, **P<0.01; compared with HFD group: #P<0.05, ##P<0.01;

[0035] Fig.18 The effect of L-CIF intervention on glucose tolerance in mice with glucose and lipid metabolism disorders. A is glucose tolerance; B is the area under the glucose tolerance curve; C is the pancreatic tissue pathological section; n = 10, mean ± SEM, compared with the CON group: *P < 0.05, **P < 0.01; compared with the HFD group: #P < 0.05, ##P < 0.01;

[0036] Fig.19 The effect of L-CIF intervention on serum lipid levels in mice with glucose and lipid metabolism disorders, A is the total cholesterol (TC) level; B is the triglyceride (TG) level; C is the low-density lipoprotein cholesterol (LDL-C) level; D is the high-density lipoprotein cholesterol (HDL-C) level; n = 10, mean ± SEM, compared with the CON group: *P < 0.05, **P < 0.01; compared with the HFD group: #P < 0.05, ##P < 0.01;

[0037] Fig. 20The effect of L-CIF intervention on the organ index of mice with glucose and lipid metabolism disorders, A is the fat index; B is the adipose tissue section; C is the organ index; n = 10, mean ± SEM, compared with the CON group: *P < 0.05, **P < 0.01; compared with the HFD group: #P < 0.05, ##P < 0.01;

[0038] Fig.21 The effect of L-CIF intervention on the liver oxidative stress level of mice with glucose and lipid metabolism disorders. In the figure, A is the level of superoxide dismutase; B is the level of glutathione; C is the level of malondialdehyde; n=10, mean±SEM, compared with the CON group: *P<0.05, **P<0.01; compared with the HFD group: #P<0.05, ##P<0.01;

[0039] Fig. 22 The effect of L-CIF intervention on the level of liver inflammation in mice with glucose and lipid metabolism disorders. In the figure, A is the level of tumor necrosis factor-α (TNF-α); B is the level of interleukin-6 (IL-6); C is the level of interleukin-1β (IL-1β); n=10, mean±SEM, compared with the CON group: *P<0.05, **P<0.01; compared with the HFD group: #P<0.05, ##P<0.01;

[0040] Fig.23 The effect of L-CIF intervention on liver glucose metabolism in mice with glucose and lipid metabolism disorders. In the figure, A is liver glycogen; B is the expression level of liver glucose metabolism-related proteins; C is the relative expression of liver glucose metabolism-related proteins; n = 10, mean ± SEM, compared with the CON group: *P < 0.05, **P < 0.01; compared with the HFD group: #P < 0.05, ##P < 0.01;

[0041] Fig.24 Effects of L-CIF intervention on lipid accumulation in the liver of mice with glucose and lipid metabolism disorders. A is the total cholesterol (TC) level; B is the triglyceride (TG) level; C is the liver pathological section; D is the expression level and relative expression of liver lipid-related proteins; n=10, mean±SEM, compared with the CON group: *P<0.05, **P<0.01; compared with the HFD group: #P<0.05, ##P<0.01. DETAILED DESCRIPTION

[0042] The invention provides a method for preparing modified insoluble dietary fiber, comprising the following steps: preparing Chinese cabbage insoluble dietary fiber, adding sterile water to the Chinese cabbage insoluble dietary fiber according to a solid-liquid ratio of 1g:10-30mL, fully stirring, inoculating plant lactobacillus for fermentation and culturing; after the fermentation and culturing is completed, adding ethanol aqueous solution, precipitating by alcohol, collecting precipitates by suction filtration, and freeze-drying to obtain the modified insoluble dietary fiber.

[0043] In the present invention, the preparation method of the insoluble dietary fiber of Chinese cabbage preferably comprises the following steps: taking cabbage residue, adding distilled water according to the solid-liquid ratio of 1g: 30-40mL, stirring thoroughly, adjusting the pH value, and adding alkaline protease for enzymolysis; after the enzymolysis is completed, heating in a boiling water bath for 8-15min and then cooling to room temperature, centrifuging and discarding the supernatant to obtain a precipitate; washing the precipitate and collecting the precipitate by suction filtration, freeze-drying, crushing and sieving, and obtaining the insoluble dietary fiber of Chinese cabbage. The present invention does not specifically limit the source of the cabbage residue. In the present invention, the cabbage residue is preferably the residue after the soluble dietary fiber of the outer leaves of Chinese cabbage is extracted. In the present invention, the solid-liquid ratio is more preferably 1g: 35mL. In the present invention, the pH value is preferably adjusted to 7-8, and the pH value is more preferably adjusted to 7.5. The present invention does not specifically limit the solvent for adjusting the pH value, and the adjusting solvent can be selected according to actual needs. In the present invention, the solvent for adjusting the pH value is preferably an alkaline solution, and more preferably a 1mol / LNaOH solution. In the present invention, the enzymatic activity of the alkaline protease is preferably 2800-3500U, more preferably 3000U. In the present invention, the conditions of the enzymolysis are preferably 35-45°C water bath enzymolysis for 35-45min, more preferably 40°C water bath enzymolysis for 40min. In the present invention, the centrifugal conditions are preferably 4000r / min centrifugation for 5min. In the present invention, the precipitate obtained after centrifugation is preferably washed with 65-75°C hot water, 75-80% ethanol aqueous solution, 90-98% ethanol aqueous solution, more preferably with 70°C hot water, 78% ethanol aqueous solution, 95% ethanol aqueous solution. After washing, the precipitate is filtered and collected, freeze-dried at -50°C, crushed through an 80-120 mesh sieve, and Chinese cabbage insoluble dietary fiber (CIF) is obtained, and stored at -20°C for use.

[0044] In the present invention, the fermented and modified Chinese cabbage insoluble dietary fiber obtained by fermenting and modifying the above-obtained Chinese cabbage insoluble dietary fiber using plant lactobacillus preferably comprises the following steps: CIF adds sterile water according to a solid-liquid ratio of 1g: 10-30mL, stirs thoroughly, inoculates plant lactobacillus, the inoculation amount is 5-10%, and the culture condition is 35-37°C for 40-50h. After the fermentation is completed, 4 times the volume of 95% ethanol aqueous solution is added, the alcohol precipitation is 20-30h, the precipitate is collected by suction filtration, and freeze-dried at -50°C to obtain plant lactobacillus fermented and modified Chinese cabbage outer leaf insoluble dietary fiber. In the present invention, the solid-liquid ratio is more preferably 1g: 15-25mL, and further 1g: 20mL. In the present invention, the fermentation and culture conditions are preferably cultured at 37°C for 48h. In the present invention, the alcohol precipitation time is more preferably 24h. The present invention does not specifically limit the purchase source of the lactobacillus. The plant lactobacillus of the present invention is preferably purchased from Beina Biotechnology Co., Ltd., and its name is Lactobacillus acidophilus and its number is BNCC-185343.

[0045] The invention also provides modified insoluble dietary fiber prepared by the preparation method.

[0046] The present invention also provides an application of the modified insoluble dietary fiber in preparing an antioxidant activity product.

[0047] The present invention also provides an application of the modified insoluble dietary fiber in preparing a product for regulating blood sugar and blood lipids.

[0048] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0049] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0050] Example 1

[0051] A preparation method of modified insoluble dietary fiber, the specific steps are as follows: weigh cabbage residue, add distilled water according to the material-liquid ratio of 1g:35mL, stir thoroughly, adjust the pH value to 7.5 with 1mol / LNaOH solution; add 3000U alkaline protease, and perform enzymolysis in a 40℃ water bath for 40min. After completion, heat in a boiling water bath for 10min and cool to room temperature. Centrifuge at 4000r / min for 5min, and discard the supernatant. The precipitate is washed with 70℃ hot water, 78% ethanol aqueous solution, and 95% ethanol aqueous solution in sequence, and the precipitate is collected by suction filtration, freeze-dried at -50℃, and crushed through an 80-mesh sieve to obtain Chinese cabbage insoluble dietary fiber. Add sterile water to the Chinese cabbage insoluble dietary fiber according to the material-liquid ratio of 1g:20mL, stir thoroughly, inoculate plant lactobacillus, the inoculation amount is 9%, and the culture conditions are 37℃ and 48h. After the fermentation, 4 times the volume of 95% ethanol aqueous solution was added, and the precipitate was collected by suction filtration and freeze-dried at -50°C to obtain the insoluble dietary fiber of Chinese cabbage fermented by Lactobacillus plantarum. The Lactobacillus plantarum was purchased from Beina Biotechnology Co., Ltd., named Lactobacillus acidophilus, and numbered BNCC-185343.

[0052] Example 2

[0053] A preparation method of modified insoluble dietary fiber, the specific steps are as follows: weigh cabbage residue, add distilled water according to the material-liquid ratio of 1g:30mL, stir thoroughly, adjust the pH value to 7.5 with 1mol / LNaOH solution; add 3000U alkaline protease, and perform enzymolysis in a 35°C water bath for 45min. After completion, heat in a boiling water bath for 10min and cool to room temperature. Centrifuge at 4000r / min for 5min, and discard the supernatant. The precipitate is washed with 75°C hot water, 75% ethanol aqueous solution, and 90% ethanol aqueous solution in sequence, and the precipitate is collected by suction filtration, freeze-dried at -50°C, and crushed through an 80-mesh sieve to obtain Chinese cabbage insoluble dietary fiber. Add sterile water to the Chinese cabbage insoluble dietary fiber according to the material-liquid ratio of 1g:30mL, stir thoroughly, inoculate plant lactobacillus, the inoculation amount is 5%, and the culture conditions are 35°C for 50h. After the fermentation, 4 times the volume of 95% ethanol aqueous solution was added, and the mixture was precipitated for 20 hours. The precipitate was collected by suction filtration and freeze-dried at -50°C to obtain insoluble dietary fiber fermented by Lactobacillus plantarum and modified Chinese cabbage. The Lactobacillus plantarum was purchased from Beina Biotechnology Co., Ltd., named Lactobacillus acidophilus, and numbered BNCC-185343.

[0054] Example 3

[0055] A preparation method of modified insoluble dietary fiber, the specific steps are as follows: weigh cabbage residue, add distilled water according to the material-liquid ratio of 1g:40mL, stir thoroughly, adjust the pH value to 7.5 with 1mol / LNaOH solution; add 3000U alkaline protease, and perform enzymolysis in a 45℃ water bath for 35min. After completion, heat in a boiling water bath for 10min and cool to room temperature. Centrifuge at 4000r / min for 5min, and discard the supernatant. The precipitate is washed with 65℃ hot water, 80% ethanol aqueous solution, and 98% ethanol aqueous solution in sequence, and the precipitate is collected by suction filtration, freeze-dried at -50℃, and crushed through an 80-mesh sieve to obtain Chinese cabbage insoluble dietary fiber. Add sterile water to the Chinese cabbage insoluble dietary fiber according to the material-liquid ratio of 1g:20mL, stir thoroughly, inoculate plant lactobacillus, the inoculation amount is 10%, and the culture conditions are 36℃ for 40h. After the fermentation, 4 times the volume of 95% ethanol aqueous solution was added, and the mixture was precipitated for 30 hours. The precipitate was collected by suction filtration and freeze-dried at -50°C to obtain the insoluble dietary fiber of Chinese cabbage fermented by Lactobacillus plantarum. The Lactobacillus plantarum was purchased from Beina Biotechnology Co., Ltd., named Lactobacillus acidophilus, and numbered BNCC-185343.

[0056] Control group

[0057] The Chinese cabbage insoluble dietary fiber was obtained according to the steps of Example 1.

[0058] Example 4

[0059] In this example, the basic components and physicochemical properties of the insoluble dietary fiber (L-CIF) of Chinese cabbage modified by fermentation of Lactobacillus plantarum obtained in Example 1 and the insoluble dietary fiber (CIF) of Chinese cabbage obtained in the control group were determined. The cabbage residues used to prepare the insoluble dietary fiber of Chinese cabbage in the example and the control group were all from the residues after the soluble dietary fiber of Chinese cabbage outer leaves was extracted.

[0060] 1. Test method:

[0061] (1) Determination of basic components: The moisture content is determined by the direct drying method in GB 5009.3-2016 "Determination of moisture in foods". The protein content is determined by the Kjeldahl method in GB 5009.5-2016 "Determination of protein in foods". The fat content is determined by the Soxhlet extraction method in GB 5009.6-2016 "Determination of fat in foods". The ash content is determined by the muffle furnace method in GB 5009.4-2016 "Determination of ash in foods". The IDF and SDF contents are determined by the enzymatic weight method in GB5009.88-2016 "Determination of dietary fiber in foods". The cellulose, hemicellulose and lignin contents are determined by the method of the National Renewable Energy Laboratory (NREL). The polyphenol content is determined by the Folin-Ciocalteu method.

[0062] (2) Determination of monosaccharide composition: The monosaccharide composition was determined by HPLC. 10 mg of sample was accurately weighed into a 10 mL ampoule, 3 mL of 2 mol / L trifluoroacetic acid solution was added, nitrogen was filled, the tube was sealed, and acid hydrolysis was carried out at 120 °C for 4 h. After the temperature dropped to room temperature, 1.0 mL was aspirated and added to 1.0 mL of methanol, the mixture was evaporated to dryness, and then the sample was redissolved with 1.0 mL of distilled water. The sample was derivatized with PMP (1-phenyl-3-methyl-5-pyrazolone), and the sample hydrolyzate was accurately aspirated and mixed with 0.6 M NaOH and 0.4 M PMP-methanol solution in a ratio of 1:1:2, and reacted at 70 °C for 1 h. After the temperature dropped to 25 °C, 500 μL of 0.3 M HCl solution was added to neutralize it, and the solution was extracted with 1 mL of chloroform three times. After standing, the supernatant was aspirated and filtered with a 0.45 μm filter membrane for loading.

[0063] Each monosaccharide standard was dissolved in distilled water to prepare a standard solution of a certain concentration, and PMP derivatization and chloroform extraction were performed according to the above method, and the membrane was filtered and put on the machine. The HPLC determination conditions were as follows: using an Xtimate C18 (4.6×200mm, 5μm) chromatographic column, acetonitrile and potassium dihydrogen phosphate solution (0.05M, pH=6.85) in a ratio of 17:83 as the mobile phase, the flow rate was 1.0mL / min, the column temperature was 30℃, the detection wavelength was 250nm, and the injection volume was 20μL.

[0064] (3) Scanning electron microscopy measurement

[0065] The surface microstructure of CIFs was observed using a scanning electron microscope. A small amount of CIFs sample was attached to the conductive tape and fixed on the sample stage of the vacuum sprayer. The CIFs sample was sprayed with a gold layer. The surface microstructure of CIFs was observed at 200x and 2000x magnifications, and photographed and recorded.

[0066] (4) Particle size measurement

[0067] The particle size distribution of CIFs samples was measured by a laser particle size analyzer. Water was used as the dispersant, and the refractive index of the particles and the refractive index of the dispersant were 1.50 and 1.33, respectively.

[0068] (5) Fourier transform infrared spectroscopy

[0069] Fourier transform infrared spectrometer was used to analyze the functional group types of CIFs samples. CIFs and potassium bromide were mixed evenly at a mass ratio of 1:100, ground into powder, and pressed into tablets. The resolution was set to 4 cm -1 , scanning times 32 times, scanning wavelength 400~4000cm -1 Read the spectrum within the range.

[0070] (6) X-ray diffraction measurement

[0071] The crystal structure of CIFs samples was analyzed by X-ray diffractometer. The main parameters were Cu target, tube voltage 20 kV, scanning speed 5° / min, test range 5°~50° (2θ angle), and angle step size of 0.02°.

[0072] (7) Thermogravimetric measurement

[0073] The thermal stability of CIFs samples was analyzed by thermogravimetric analyzer. 5 mg of CIFs sample was accurately weighed and placed in a crucible, and the temperature was raised at a rate of 10 °C / min in the temperature range of 25 °C to 600 °C, with nitrogen as the protective gas and the flow rate controlled at 50 mL / min.

[0074] (8) Water holding capacity

[0075] Dry a 50mL centrifuge tube to constant weight (M0), accurately weigh 0.5g CIFs sample (M1) into the centrifuge tube, add 10mL distilled water, shake well, and let stand at room temperature for 1h. Centrifuge at 4000r / min for 15min, discard the supernatant, carefully wipe off the supernatant remaining on the tube wall with filter paper, and weigh the mass of the centrifuge tube containing the CIFs sample (M2). Calculate WHC according to the following formula:

[0076] WHC(g / g)=(M2-M0) / M1;

[0077] (9) Oil holding capacity

[0078] Dry a 50mL centrifuge tube to constant weight (M0), accurately weigh 0.5g CIFs sample (M1) into the centrifuge tube, add 10mL peanut oil, mix well, and let stand at room temperature for 1h. Centrifuge at 4000r / min for 15min, discard the supernatant, carefully wipe off the supernatant remaining on the tube wall with filter paper, and weigh the mass of the centrifuge tube containing the CIFs sample (M2). Calculate OHC according to the following formula:

[0079] OHC (g / g) = (M2 - M0) / M1.

[0080] (10) Expansion force

[0081] Accurately weigh 0.25g CIFs sample (M) and place it in a 10mL measuring cylinder, record the volume (V1), add 5mL distilled water, stir thoroughly, shake to remove bubbles, let stand at room temperature for 24h, and record the volume of the CIFs sample after water absorption and expansion (V2). Calculate WSC according to the following formula:

[0082] WSC(g / g)=(V2-V1) / M.

[0083] (11) Glucose adsorption capacity

[0084] Accurately weigh 0.5g of CIFs sample and place it in 50mL of 50, 100, 150 and 200mmol / L glucose solution respectively, and shake it thoroughly. Incubate in a 37℃ water bath for 6h, centrifuge at 4000r / min for 20min, and aspirate the supernatant. Measure the glucose concentration in the supernatant with a glucose kit, and use the glucose solution without CIFs sample as a control. Calculate GAC according to the following formula:

[0085] GAC(mmol / g)=(C1-C2) / M0×V;

[0086] Where: M0 is the sample mass (g); C1 is the glucose concentration in the original solution before adsorption (mmol / L); C2 is the glucose concentration in the supernatant after adsorption (mmol / L); V is the volume of glucose solution (L).

[0087] (12) α-amylase activity inhibition ability

[0088] Accurately weigh 10 mg of CIFs sample and mix with 75 μL of amylase solution (2U / mL), shake at 37°C for 20 min. Add 75 μL of starch solution (1%, w / w) as substrate, continue shaking for 30 min, then add 125 μL of 3,5-dinitrosalicylic acid and boil for 10 min. After cooling, add distilled water to 5 mL, centrifuge at 8000 r / min for 10 min, and absorb the supernatant. Determine its absorbance at 540 nm. Calculate α-AAIR according to the following formula:

[0089] α-AAIR(%)=[1-(A1-A2) / (A3-A4)]×100%;

[0090] Wherein: A1 is the absorbance value of CIFs sample; A2 is the absorbance value of phosphate buffered saline (PBS) instead of amylase solution; A3 is the absorbance value without adding sample; A4 is the absorbance value without adding sample and PBS instead of amylase solution.

[0091] (13) α-glucosidase activity inhibition ability

[0092] Accurately weigh 1 mg of CIFs sample and mix it with 40 μL of α-glucosidase solution (1U / mL), and shake it at 37°C for 15 minutes. Add 20 μL of 4-nitrophenyl-α-D-pyranoglucoside solution (2.5mmol / L), continue shaking for 10 minutes, then add 150 μL of Na2CO3 solution (1mol / L), centrifuge at 8000r / min for 5 minutes, and aspirate the supernatant. Determine its absorbance value at 405nm. Calculate α-AGIR according to the following formula:

[0093] α-AGIR(%)=[1-(A1-A2) / (A3-A4)]×100%;

[0094] Wherein: A1 is the absorbance value of the sample; A2 is the absorbance value of PBS instead of glucosidase solution; A3 is the absorbance value without adding sample; A4 is the absorbance value without adding sample and PBS instead of glucosidase solution.

[0095] (14) Cholesterol adsorption capacity

[0096] First, add 9 times distilled water to the egg yolk and stir thoroughly until it is in an emulsion to prepare the egg yolk emulsion. Then, accurately weigh 0.1g of CIFs sample and place it in a 50mL centrifuge tube, add 2.5mL of egg yolk emulsion, stir thoroughly, oscillate at 37℃ for 2h, centrifuge at 5000r / min for 15min at 4℃, and aspirate the supernatant. No sample was added to the control group, and the other conditions were the same. The cholesterol content of the supernatant was determined by the o-phthalaldehyde method. CAC was calculated according to the following formula:

[0097] CAC (mg / g) = (M1-M2) / M0;

[0098] Where: M1 is the cholesterol content in the control group (mg); M2 is the cholesterol content in the sample group (mg); M0 is the sample mass (g).

[0099] (15) Sodium bile acid adsorption capacity

[0100] Accurately weigh 0.1g CIFs sample and 0.2g sodium cholate in a 50mL centrifuge tube, add 20mL 0.15M NaCl solution, and place in a 37℃ water bath for 3h. Centrifuge at 5500r / min for 10min, aspirate 1mL supernatant, add 6mL 45% H2SO4 and 1mL 0.3% furfural, and place in a 65℃ water bath for 30min, and measure its absorbance at 620nm. No sample was added to the control group, and the other conditions were the same. Calculate SCAC according to the following formula:

[0101] SCAC (mg / g) = (M0-M1) / M;

[0102] Wherein: M0 is the mass of sodium cholate in the control group (mg); M1 is the mass of sodium cholate in the sample group (mg); M is the mass of the sample (g).

[0103] (16) DPPH free radical scavenging ability

[0104] Accurately weigh 3, 6, 12, 18, 24 and 30 mg of CIFs samples, add 3 mL of ddH2O and 3 mL of 0.2 mM DPPH-ethanol solution respectively, mix well, and keep it in the dark for 30 min. Centrifuge at 4000 r / min for 10 min, aspirate the supernatant, and measure its absorbance at 517 nm. Ascorbic acid solution (1 mg / mL) is a positive control, and the DPPH free radical scavenging rate is calculated according to the following formula:

[0105] DPPH free radical scavenging rate (%) = [1-(Ai-Aj) / A0] × 100%;

[0106] Where: Ai is the absorbance value of the sample and DPPH-ethanol solution; Aj is the absorbance value of the sample and ethanol solution; A0 is the absorbance value of the DPPH-ethanol solution.

[0107] (17) ABTS free radical scavenging ability

[0108] K2S2O8 solution (2.5 mM) and ABTS solution (7 mM) were mixed in equal proportions and protected from light for 12 to 16 hours to prepare ABTS mother solution. Then, PBS solution (0.1 M, pH = 7) was slowly added to the ABTS mother solution until its absorbance at 734 nm was 0.7 ± 0.02 to obtain ABTS working solution.

[0109] Accurately weigh 3, 6, 12, 18, 24 and 30 mg of CIFs samples, add 1.5 mL of ddH2O and 6 mL of ABTS working solution, mix well, and keep it in the dark for 6 minutes. Centrifuge at 4000 r / min for 10 minutes, aspirate the supernatant, and measure its absorbance at 734 nm. Ascorbic acid solution (1 mg / mL) is a positive control, and the ABTS free radical scavenging rate is calculated according to the following formula:

[0110] ABTS free radical scavenging rate (%) = [1-(Ai-Aj) / A0] × 100%;

[0111] Where: Ai is the absorbance value of the sample and ABTS solution; Aj is the absorbance value of the sample; A0 is the absorbance value of the ABTS solution.

[0112] 2. Results Analysis

[0113] (1) Basic ingredients

[0114] According to the results in Table 1, compared with CIF, the SDF content of L-CIF is significantly increased, which may be due to the cohesion generated by ultrafine grinding and the organic acid produced by fermentation, which can destroy the glycosidic bonds in cellulose and convert IDF into small molecular SDF. Compared with CIF, the polyphenol content after modification is significantly increased, which may be due to the modification treatment that destroys the covalent bond between phenolic substances and IDF, which may give L-CIF a higher antioxidant capacity.

[0115] Table 1 Basic components of CIFs

[0116] sample CIF L-CIF Moisture <![CDATA[8.68±0.49 a ]]> <![CDATA[9.13±1.25 a ]]> protein <![CDATA[2.17±0.09 a ]]> <![CDATA[2.14±0.10 a ]]> Ash <![CDATA[6.46±0.04 a ]]> <![CDATA[6.37±0.05 a ]]> Fat <![CDATA[0.27±0.01 b ]]> <![CDATA[0.20±0.01 c ]]> IDF <![CDATA[79.34±0.93 a ]]> <![CDATA[74.02±0.52 b ]]> SDF <![CDATA[2.93±0.35 b ]]> <![CDATA[8.11±0.20 a ]]> Polyphenols (mgGAE / g) <![CDATA[0.60±0.01 d ]]> <![CDATA[0.86±0.01 b ]]> Cellulose <![CDATA[48.97±0.37 b ]]> <![CDATA[48.91±0.54 b ]]> Hemicellulose <![CDATA[14.25±0.25 a ]]> <![CDATA[12.90±0.14 b ]]> Lignin <![CDATA[3.81±0.42 b ]]> <![CDATA[3.26±0.74 b ]]>

[0117] Note: Different letters in the table indicate significant differences at the p<0.05 level

[0118] (2) Monosaccharide composition

[0119] According to the results in Table 2, both CIF and modified CIF are composed of 10 monosaccharides, including mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose, but the proportion of each monosaccharide changes after modification. Studies have shown that mannose, rhamnose, arabinose and galactose are typical monosaccharides of hemicellulose, while glucose is a typical monosaccharide of cellulose, which indicates that cellulose and hemicellulose are the main components of CIF.

[0120] Table 2 Monosaccharide composition of CIFs

[0121]

[0122]

[0123] (3) Scanning electron microscopy

[0124] according to Figure 1 The results showed that the contents of galacturonic acid, glucose, galactose and xylose in L-CIF increased compared with CIF, which may be due to the degradation and rearrangement of cellulose and hemicellulose in CIF by fermentation treatment, increasing the cellulose content and reducing the hemicellulose content. The change in monosaccharide composition may lead to changes in the microstructure and physicochemical functional properties of CIF samples.

[0125] (4) Particle size

[0126] according to Figure 2 The results showed that compared with CIF, the main peak of L-CIF was basically the same, but a small peak appeared on the left side of the main peak of L-CIF, indicating that fermentation modification can decompose some CIF large particles into small particles, which has a certain degradation effect on CIF. Studies have shown that particle size can reflect its dispersibility and solubility in the food system, and affect the exposure of its internal active groups and the release of nutrients. The smaller the particle size of dietary fiber, the larger the specific surface area, which is conducive to the exposure of its internal active groups and improves its hydration capacity and adsorption capacity.

[0127] (5) Fourier transform infrared spectroscopy

[0128] according to Figure 3 The results show that compared with CIF, the modified CIF sample has basically the same characteristic peaks, but the absorption intensity changes slightly. -1 Corresponding to the associative vibration of hydrogen bonds and free -OH groups, CIFs before and after modification have broad and strong absorption peaks here, indicating that there are many associative hydrogen bonds in their molecular structure. -1 Corresponding to the stretching vibration of -CH in the methyl or methylene groups on cellulose, CIFs all have a typical cellulose structure. -1 The absorption peaks near 1060cm are related to the carboxyl groups in uronic acid and polyphenols; -1 Corresponding to the stretching vibration of the ether bonds in lignin or hemicellulose. In summary, the modification did not destroy the basic chemical structure of CIF, and its typical characteristics of dietary fiber were retained.

[0129] (6) X-ray diffraction

[0130] according to Figure 4The results showed that the peak diffraction in the XRD spectrum reflects the crystalline structure of the sample, and the diffuse diffraction reflects the non-crystalline structure of the sample. CIF showed strong diffraction peaks near 15° and 21°, indicating that it has a typical type I cellulose structure, that is, a coexistence state of crystals and non-crystals. Compared with CIF, the peak position of the modified sample did not change, indicating that the fermentation modification method did not change the type of crystal. However, the diffraction peak of the fermentation modified sample became broader near 15°, which may be caused by cellulose denaturation during the modification process.

[0131] (7) Thermogravimetry

[0132] according to Figure 5 The results showed that the weight loss rates of CIF and fermentation-modified CIF samples were CIF (77.45%) > L-CIF (73.39%). Specifically, when the temperature was 50-200°C, the mass of the fermentation-modified CIF sample decreased by about 10%, which may be due to the evaporation of bound water; when the temperature was 200-400°C, the sample weight loss reached about 50%, and a peak appeared on the DTG curve, indicating that the sample mass decreased rapidly at this stage, and the weight loss rate was fast, which may be caused by the thermal decomposition of cellulose and hemicellulose; when the temperature was 400-600°C, the sample weight loss reached about 11%, and the weight loss rate slowed down, which may be due to the partial thermal cracking of lignin and other components with strong chemical bonds. In summary, the thermogravimetric indicators of CIF and fermentation-modified CIF samples showed a significant downward trend in the three temperature stages.

[0133] (8) Water holding capacity

[0134] Water holding capacity (WHC) is the ability of a sample to retain water under external centrifugal force conditions, including physically trapped water, hydrodynamic water, and bound water. It can reflect the hydration capacity of dietary fiber and is an important indicator for evaluating its water retention capacity. Figure 6 The results showed that the WHC of L-CIF was significantly increased by 9.34% (P < 0.05) compared with CIF ((6.64 ± 0.09) g / g). This may be due to the fact that fermentation modification breaks the β-glycosidic bonds between cellulose and hemicellulose, increases the pores, and exposes more hydrophilic groups.

[0135] (9) Oil holding capacity

[0136] Oil holding capacity (OHC) reflects the ability of dietary fiber to bind oil, which mainly depends on the total charge density, hydrophobicity and surface properties. Figure 7The results showed that the oil holding capacity of L-CIF was significantly increased by 33.13% (P < 0.05) compared with CIF ((3.29 ± 0.03) g / g). This may be because the fermentation process made the originally smooth and dense CIF structure become rough and porous, increasing its oil storage capacity.

[0137] (10) Expansion force

[0138] The WSC is the ability of a sample to swell or form a gel-like substance after adding water. The hydrogen bonds and dipole hydrophilic groups in dietary fiber can bind to water molecules and preserve them in the capillary structure inside the structure. Figure 8 The results showed that the WSC of L-CIF was significantly increased by 116.40% (P < 0.05) compared with CIF ((3.74 ± 0.44) g / g). This may be because the fermentation process formed a loose and rough structure, which was conducive to the exposure of hydrogen bonds and hydrophilic active groups, increasing the penetration and absorption of water molecules.

[0139] (11) Glucose adsorption capacity

[0140] according to Fig. 9 The results showed that, consistent with CIF, the GAC of L-CIF showed an increasing trend with the increase of glucose concentration. However, at a low glucose concentration of 50 mmol / L, the GAC of L-CIF decreased significantly, while at high concentrations, the GAC was the highest; when the glucose concentration was 200 mmol / L, the GAC of L-CIF was 118.18% higher than that of CIF ((6.68±0.47) mmol / g) (P<0.05). This phenomenon may be due to the exposure of polar and non-polar groups of CIF, including free hydroxyl, ether, phenol and carboxyl groups, which are conducive to improving the interaction between CIF and glucose molecules. The concentration difference generated at a high glucose concentration promotes the entry of glucose molecules into the dietary fiber, thereby better utilizing the pores inside the dietary fiber. The above in vitro experiments show that L-CIF has a good glucose adsorption capacity, suggesting that it may have certain application prospects in regulating blood sugar.

[0141] (12) α-amylase activity inhibition ability

[0142] according to Fig.10 The results showed that the α-AAIR of L-CIF was significantly increased by 31.00% (P < 0.05) compared with CIF ((37.14 ± 2.14)%). Studies have shown that the surface structure of dietary fiber is an important factor affecting the activity of α-amylase. IDF significantly inhibits the activity of amylase by encapsulating the enzyme to reduce the binding of the enzyme to the substrate. This is consistent with the results of SEM. The rough and porous surface structure promotes the binding of active functional groups to α-amylase or its substrate.

[0143] (13) α-glucosidase activity inhibition ability

[0144] according to Fig.11 The results showed that CIFs had a certain inhibitory effect on α-glucosidase. Compared with CIF ((35.27±5.92)%), L-CIF increased by 11.16%.

[0145] (14) Cholesterol adsorption capacity

[0146] according to Fig.12 The results showed that the CAC of L-CIF increased by 253.85% (P < 0.05) compared with CIF ((5.98 ± 0.91) mg / g). This may be due to the changes in the surface and structure of CIF caused by the fermentation modification method, including an increase in specific surface area, an increase in surface voids, and a loose internal structure, which exposed more active groups, reduced steric hindrance, and strengthened the chemical chelation and physical adsorption interactions between insoluble dietary fiber and cholesterol. The above in vitro experiments showed that L-CIF has a good cholesterol adsorption capacity, suggesting that it may have certain application prospects in regulating blood lipids.

[0147] (15) Sodium bile acid adsorption capacity

[0148] according to Fig.13 The results showed that the SCAC of L-CIF increased by 55.10% (P < 0.05) compared with CIF ((7.15 ± 0.08) mg / g). This may be due to the fact that the SCAC of dietary fiber is related to its physicochemical properties, such as particle size distribution, charge density and the interaction of hydrophobic groups. The modified porous and uneven surface structure increases its specific surface area and the number of lipophilic groups, which is conducive to binding sodium cholate.

[0149] (16) Free radical scavenging ability

[0150] according to Fig.14 The results showed that CIFs had a certain scavenging effect on both DPPH and ABTS free radicals, and the scavenging rate increased with the increase of CIF concentration. Compared with CIF, the scavenging ability of L-CIF on the two free radicals was significantly increased at all concentrations (P < 0.05). The study showed that the modification process can release active components such as polyphenols and flavonoids in insoluble dietary fiber. At the same time, the hydroxyl and carboxyl groups in dietary fiber are fully exposed, resulting in an increase in the free radical scavenging ability of the modified IDF.

[0151] Example 5 Application of fermented modified Chinese cabbage insoluble dietary fiber in regulating blood sugar and blood lipids

[0152] 1. Test method:

[0153] Forty SPF-grade 5-week-old male C57BL / 6J mice were selected and raised in the animal laboratory of Hebei Agricultural University at a temperature of (23±2)℃, a relative humidity of 50%-60%, and a light-dark cycle of 12h. Throughout the experiment, standard feed and high-fat feed were provided by Sibeifu Biotechnology Co., Ltd., and water was provided by the laboratory. This experiment was carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council and was approved by the Research Ethics Committee of the College of Food Science and Technology of Hebei Agricultural University.

[0154] After one week of adaptive feeding, mice were randomly divided into negative control group (CON), high-fat model group (HFD), low-dose L-CIF group and high-dose L-CIF group according to body weight, with 10 mice in each group. The negative control group was fed with a low-fat diet (D12450J), while the other three groups were fed with a high-fat diet (D12492), which provided 3.85Kcal energy density per gram and 5.24Kcal energy density per gram of high-fat diet. Among them, the low-dose group was gavaged with 250mg / kg·bw of the plant lactobacillus fermented modified Chinese cabbage insoluble dietary fiber (L-CIF) suspension obtained in Example 1, and the high-dose group was gavaged with 500mg / kg·bw of the plant lactobacillus fermented modified Chinese cabbage insoluble dietary fiber (L-CIF) suspension obtained in Example 1. The negative control group and the high-fat model were gavaged with an equal amount of distilled water for 12 weeks.

[0155] The body weight of mice was recorded weekly and the food intake was recorded every 3 weeks. The fasting blood glucose of mice was measured one week before the end of the experiment, and the glucose tolerance of mice was measured three days before the end. The mice fasted for 12 hours. The next day, the eyeballs were removed to collect blood, and the mice were killed by dislocation of the neck. The organs and tissues were washed with normal saline, and the epididymal adipose tissue, subcutaneous adipose tissue, brown adipose tissue in the shoulder, as well as liver, pancreas and other organs were collected and weighed. The liver and pancreas were partially fixed in a general-purpose paraformaldehyde solution, and the epididymal adipose tissue, subcutaneous adipose tissue, and brown adipose tissue were partially fixed in a special fat fixation solution. The mouse blood was centrifuged at 3000rpm for 20 minutes at 4°C to obtain serum, and other tissues and organs were stored at -80°C for future use.

[0156] 2. Measurement indicators

[0157] (1) Determination of fasting blood glucose in mice

[0158] One week before the end of the experiment, that is, the 11th week, the mice were fasted but not watered for 12 hours the night before. The next day, blood was collected from the tail vein to measure the fasting blood glucose of the mice. The first drop of blood was discarded, and the second drop of blood was collected to measure the blood glucose of the mice using a blood glucose meter, and the blood glucose levels of the mice were recorded.

[0159] (2) Determination of glucose tolerance in mice

[0160] Three days before the end of the experiment, mice were fasted but not watered for 12 h. The next day, 0.1 mL of 20% glucose solution was gavaged per 10 g of mice. Blood was collected from the tail at 0, 15, 30, 60, 90, and 120 min, and blood glucose was measured with a blood glucose meter and recorded. A blood glucose curve was drawn and the area under the curve was recorded.

[0161] (3) Determination of organ index and fat index

[0162] The mice were killed by cervical dislocation and dissected. The subcutaneous adipose tissue, epididymal adipose tissue, brown adipose tissue, liver, spleen, kidney, lung and heart tissues of the mice were taken, washed with physiological saline, wiped clean with filter paper and weighed, and various fat indexes and organ indexes were calculated.

[0163]

[0164] (4) Determination of serum biochemical indicators

[0165] Serum lipid levels were measured by common biochemical kits including total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C), and serum INS and inflammatory factors lipopolysaccharide, tumor necrosis factor-α, interleukin-6, and interleukin-1β were measured by ELISA kits. The Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated based on the results.

[0166]

[0167] (5) Determination of biochemical indices in mouse liver

[0168] Mouse liver was taken from -80℃, liver: physiological saline was mixed and minced at a ratio of 1:9, and the temperature was 4℃, centrifuged at 3500rpm for 15min to obtain 10% liver homogenate for further use. Liver SOD, GSH, and MDA levels were determined strictly according to the instructions of the general biochemical kit, TNF-α, IL-6, and IL-1β levels in the liver were determined using ELISA kits, liver glycogen content was determined strictly according to the general biochemical kit, and liver TC and TG contents were determined using general biochemical kits.

[0169] (6) Pathological sections of organs and adipose tissue

[0170] The pancreas and liver of mice were fixed with universal paraformaldehyde solution, while the epididymal adipose tissue, subcutaneous adipose tissue and brown adipose tissue were fixed with a special fixative for adipose tissue.

[0171] Preparation of paraffin sections: Remove tissue from fixative and dehydrate through graded alcohols.

[0172] Dewaxing: Dewax twice with environmentally friendly dewaxing liquid, 20 minutes each time; soak twice with anhydrous ethanol, 5 minutes each time, then soak with 75% alcohol for 5 minutes, and finally wash with tap water.

[0173] Hematoxylin-eosin staining: The dewaxed sections were treated in a high-temperature constant stain pretreatment solution for 1 min, then placed in a hematoxylin stain solution for 5 min, washed with running water, and the washed sections were dehydrated in 95% alcohol for 1 min. Finally, the sections were placed in an eosin stain solution for 15 s.

[0174] Dehydration and sealing: The stained sections were dehydrated in anhydrous ethanol for 3 times, 2 min each time, dehydrated in n-butanol for 2 times, 2 min each time, then immersed in xylene for 2 times, 2 min each time, until transparent, and finally sealed with neutral gum.

[0175] Microscope scanning was used to collect images, and CaseViewer software was used to observe and analyze tissue pathology.

[0176] (7) Expression of proteins related to hepatic glucose and lipid metabolism

[0177] Protein extraction: Take out the mouse liver at -80℃, weigh the appropriate mouse liver tissue, add RIPA lysis (strong), 1% protease inhibitor and 1% phosphatase inhibitor to fully grind, place at 4℃ for 2h, centrifuge at 4℃, 12000rpm for 15min, and collect the supernatant. Use BCA protein concentration determination kit to determine the protein concentration, add SDS-PAGE protein loading buffer to denature the protein, add protein sample and protein loading buffer in a ratio of 1:4, heat at 100℃ or boiling water bath for 3-5min to fully denature the protein. After cooling, place at -20℃ for use.

[0178] Determination of protein loading amount: First, determine the protein loading amount according to the measured protein concentration and 25 μg protein content per well, and then estimate the protein loading amount according to the first gray value.

[0179] Prepare SDS-PAGE gel: prepare separation gel according to the common formula of the distributor or laboratory. Isopropanol can be used to eliminate bubbles. Place it in a 37℃ constant temperature incubator for 30 minutes until the separation gel solidifies. Pour out the isopropanol and add concentrated gel (the same as the preparation method of separation gel). Quickly insert the comb teeth and place it in a 37℃ constant temperature incubator for 30 minutes until the concentrated gel solidifies.

[0180] Electrophoresis: Make an electrophoresis device, add electrophoresis fluid between the plates, the electrophoresis fluid in the electrophoresis tank must cover the resistance wire, then carefully and slowly pull out the comb, add the marker and each group of protein samples to the corresponding sample wells, fast gel making voltage 150V for 70min, traditional gel making concentration gel 80V for about 30min, separation gel 120V for 70min, until the protein samples are separated and the electrophoresis is completed.

[0181] Electrotransfer: After electrophoresis, cut the separation gel according to the required protein molecular weight, and put the cut gel into the electrotransfer solution to prevent the separation gel from shrinking. Soak the PVDF membrane in methanol solution for 3 minutes until there are no white spots to fully activate it. After complete activation, put it into the electrotransfer solution to wash off the surface methanol. The electrotransfer device is assembled in the order of black plate-sponge pad-filter paper-protein gel-PVDF membrane-filter paper-sponge pad-red plate, and electrotransfer is performed at a constant voltage of 110V.

[0182] Blocking: After electroporation, take out the PVDF membrane and place it in an incubation box containing BSA blocking solution, and shake on a shaker for 90 minutes.

[0183] Incubation with primary antibody: After blocking, recover the blocking solution, then add the diluted primary antibody according to the instructions, and incubate at 4°C overnight or at room temperature for 90 minutes.

[0184] Elution: The next day, remove the primary antibody and wash with TBST solution, 5 minutes each time, for 3 times.

[0185] Incubation with secondary antibody: After washing, add the corresponding secondary antibody according to the instructions and incubate at room temperature for 1 hour while shaking on a shaker.

[0186] Elution: After the secondary antibody incubation, wash with TBST 7 times, 5 min each time.

[0187] Development: After washing, pour off TBST, prepare ECL luminescent solution in a 1:1 ratio, prepare it as needed, add ECL evenly to the PVDF membrane and place it in a dark room, expose and take pictures with a machine, use Imagepro software to analyze the gray value of the protein band for quantification, and the relative expression of the protein is expressed by the ratio of the gray value of the target protein band to the gray value of the β-actin protein band.

[0188] 3. Results Analysis

[0189] (1) Effects of L-CIF intervention on body weight, food intake, and energy intake in mice with glucose and lipid metabolism disorders

[0190] Fig.15 The results show the effects of L-CIF intervention on the basic parameters (body weight, food intake, and energy intake) of C57BL / 6J mice after 12 weeks of intervention. Fig.15As shown in A, compared with the CON group, the weight of mice in the HFD group increased significantly in the 11th and 12th weeks (P < 0.05), indicating that the high-fat model of mice was successfully established; after 12 weeks of L-CIF intervention, the weight of mice was significantly reduced in the early stage (P < 0.05), and from the 6th week to the end of the experiment, the weight of mice hardly changed, indicating that L-CIF did not significantly reduce the weight of mice on a high-fat diet. Fig.15 B and Fig.15 As shown in C, the daily food intake of each mouse was measured during the intervention period, and it was found that the food intake did not change during the intervention period. The energy intake of mice in the HFD group was significantly higher than that in the CON group at weeks 3 and 7 (P < 0.05), and the energy intake was almost unchanged after L-CIF intervention. These results indicate that the energy intake of mice on a high-fat diet increased, the weight of mice increased, and the improvement of mouse weight by L-CIF was not related to diet and energy intake.

[0191] (2) Effect of L-CIF intervention on serum inflammatory levels in mice with glucose and lipid metabolism disorders

[0192] To investigate the effect of L-CIF on serum inflammatory levels in mice with glucose and lipid metabolism disorders, the levels of serum inflammatory factors in mice were measured. Fig.16 Compared with the negative control group, there was no significant change in the serum lipopolysaccharide, interleukin-6, and interleukin-1β levels in the HFD group mice ( Fig.16 A, 16C and 16D), tumor necrosis factor-α increased significantly ( Fig.16 B). After L-CIF intervention, serum lipopolysaccharide levels were significantly reduced (P<0.05) ( Fig.16 A) There was no effect on the levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β. It may be that L-CIF is utilized by intestinal microorganisms, thereby changing the structure of intestinal flora and affecting LPS levels.

[0193] (3) Effect of L-CIF intervention on insulin resistance in mice with glucose and lipid metabolism disorders

[0194] Insulin resistance is a prerequisite for T2DM and is associated with many metabolic diseases. It can be used to evaluate glucose metabolism in mice. Fig.17 A. Fig.17 B and Fig.17 As shown in C, the fasting blood glucose and fasting serum insulin of mice were measured and the insulin resistance index was calculated. It was found that the fasting blood glucose, fasting insulin and insulin resistance index of mice fed with high fat diet were significantly increased (P < 0.05), indicating that the mice had disordered glucose metabolism and insulin resistance. After L-CIF intervention, the fasting blood glucose, fasting insulin and insulin resistance index of mice were significantly decreased (P < 0.05, P < 0.01), indicating that L-CIF can improve the insulin resistance of mice.

[0195] To further explore the glucose metabolism of mice, oral glucose tolerance and pancreatic tissue pathological sections were tested. Fig.18 As shown in A, the glucose levels of mice in each group reached the highest level 15 minutes after intragastric administration, and during the entire experiment, the glucose levels of mice in the HFD group were significantly higher than those in the control group and the L-CIF intervention group at each time period, indicating that the glucose tolerance of mice was reduced, which once again proved that the glucose metabolism of mice was disordered; the glucose tolerance of mice increased significantly after L-CIF intervention (P < 0.05), indicating that mice had a stronger tolerance to glucose after L-CIF intervention. Fig.18 As shown in B, the area under the glucose tolerance curve of mice was calculated and it can be seen that the AUC of mice in the HFD group increased significantly (P < 0.05), and the AUC decreased significantly after L-CIF intervention (P < 0.05), indicating that L-CIF improved the glucose tolerance of mice. Fig.18 As shown in C, the H&E staining results of pancreatic tissue showed that the islets were normal in all groups.

[0196] The results of sugar metabolism indicators and pancreatic tissue jointly indicated that a high-fat diet caused glucose intolerance in mice. Further observation of the pancreatic islet tissue of mice revealed that the high-fat diet did not damage the pancreatic islets of mice. The mice could secrete insulin normally, but the sensitivity of tissues and organs to insulin was reduced, there was higher insulin in the circulating blood, and the mice developed insulin resistance. L-CIF intervention can significantly improve the imbalance of blood glucose homeostasis caused by sugar and lipid metabolism disorders.

[0197] (4) Effect of L-CIF intervention on serum lipid levels in mice with glucose and lipid metabolism disorders

[0198] Serum lipid levels can be used to evaluate lipid metabolism in mice. To further explore the effect of L-CIF on lipid metabolism in mice, serum lipid levels were detected in mice. Fig.19 A~ Fig.19 As shown in C, compared with the CON group, the high-fat diet caused a significant increase in serum TC, TG, and LDL-C in mice (P < 0.05), indicating that the serum lipid metabolism of mice was disordered and the mice developed hyperlipidemia; compared with the HFD group, the serum TC, TG, and LDL-C of mice were significantly reduced after L-CIF intervention (P < 0.05, P < 0.01), especially the serum TC level was extremely significantly reduced (P < 0.01). There was no difference in HDL-C during the entire experimental period ( Fig.19 D). The above results indicate that L-CIF can reduce serum lipid profile and improve lipid metabolism disorders in mice.

[0199] (5) Effects of L-CIF intervention on organs, fat index and slices of mice with glucose and lipid metabolism disorders

[0200] Excessive fat intake will cause fat to accumulate in tissues and organs, leading to organ damage. Testing the fat index and organ index of mice revealed that Fig. 20 A and Fig. 20 As shown in B), compared with the CON group, the subcutaneous fat of the HFD group mice increased significantly (P < 0.05), and the H&E sections showed an increase in the volume of subcutaneous fat droplets, indicating that the high-fat diet led to the accumulation of subcutaneous fat in mice, which in turn caused the body's insulin sensitivity to decrease; although there was no significant difference in the subcutaneous fat index after L-CIF intervention, there was a downward trend, and the subcutaneous fat H&E staining results showed that insoluble dietary fiber can reduce the size of subcutaneous fat droplets and increase the number of fat droplets, indicating that L-CIF may improve lipid metabolism disorders by increasing the number of subcutaneous fat droplets. Although there was a downward trend in epididymal fat after L-CIF intervention, it was not significant, and the same was true for epididymal fat H&E sections. The brown fat index of mice fed a high-fat diet was reduced (P < 0.05). The results of H&E staining of brown fat again showed that lipids accumulated in brown adipose tissue and the relative reduction of brown fat content were not conducive to the health of the body. The brown fat index tended to increase after L-CIF intervention. The results of H&E staining showed that fat accumulation tended to slow down, but it was not significant. This shows that L-CIF's improvement of glucose and lipid metabolism may be related to reducing brown fat lipid accumulation and increasing the relative content of brown fat. Fig. 20 As shown in C, compared with the CON group mice, the liver index of the HFD group mice increased significantly (P < 0.05), indicating that the high-fat diet caused lipid accumulation in the liver of mice and liver damage. After L-CIF intervention, the liver showed a downward trend but was not significant; the spleen, pancreas, kidney, heart, and lung indexes did not change, indicating that L-CIF was not toxic to them. These results show that L-CIF intervention in glucose and lipid metabolism may be related to increasing the number of subcutaneous fat droplets or slowing down the whitening of brown fat and improving liver metabolism, and L-CIF has no damage to organs such as the spleen, pancreas, and kidney.

[0201] From the above results, it can be seen that L-CIF mainly acts on the liver. As the main site of glucose and lipid metabolism, high-fat diet intake can lead to fatty degeneration of the liver, accompanied by chronic low-grade inflammation and oxidative damage. In order to evaluate the effect of L-CIF on liver glucose and lipid metabolism disorders, the present invention studied the biochemical indicators of mouse liver and the expression of glucose-lowering and lipid-lowering related proteins.

[0202] (6) Effect of L-CIF on oxidative stress levels in mouse liver

[0203] The oxidative stress level in mouse liver was detected. Fig.21 As shown in A, there was no difference in superoxide dismutase SOD activity among the groups; Fig.21As shown in B, compared with the CON group, the glutathione GSH activity in the HFD group was significantly decreased (P < 0.05), and the GSH content was significantly increased after L-CIF intervention (P < 0.05); Fig.21 As shown in C, the high-fat diet significantly increased the content of peroxidation product MDA (P < 0.05), and the L-CIF group had a tendency to reduce MDA, but it was not significant. The results showed that L-CIF intervention alleviated the damage of the antioxidant defense system of mice caused by high-fat diet intake by increasing GSH activity.

[0204] (7) Effect of L-CIF on liver inflammation in mice

[0205] To further evaluate the effect of L-CIF intervention on the level of liver inflammatory factors, the liver inflammation level was measured. Compared with the CON group, although the inflammatory factor tumor necrosis factor-α ( Fig. 22 A), interleukin-6 ( Fig. 22 B) and interleukin-1β ( Fig. 22 C), but L-CIF intervention significantly reduced the level of interleukin-6 (P < 0.05). Studies have shown that interleukin-6 produced by adipose tissue can reduce GLUT4 expression, thereby reducing the uptake of glucose by muscle or adipose tissue, causing insulin resistance in the body. Therefore, the results of liver inflammation indicate that L-CIF increases insulin sensitivity by reducing the level of interleukin-6 in the liver, reduces insulin resistance and improves glucose metabolism.

[0206] (8) Effects of L-CIF on Glucose Metabolism in Mouse Liver

[0207] Detection of mouse liver glycogen revealed that Fig.23As shown in A, compared with the CON group, the liver glycogen content in the HFD group increased significantly (P < 0.05). The reason is speculated to be that insulin resistance occurs preferentially in adipose tissue. When lipid synthesis increases, adipose tissue will form huge lipid droplets, which interfere with actin movement, thereby affecting the transport of GLUT4 storage vesicles, and then affecting the uptake of glucose by adipocytes, accelerating the entry of glucose into the liver to promote liver glycogen synthesis, and in the long run, damaging the liver. Consistent with the experiment of xiuqing Han et al. using OA to induce non-alcoholic fatty liver to explore the sequence of NAFLD and IR, the liver glycogen synthesis in the model group increased and NAFLD preceded IR; after supplementing L-CIF, the liver glycogen content increased significantly (P < 0.05), indicating that L-CIF promotes liver glycogen synthesis and improves glucose metabolism. In addition, the experiment found that the amount of liver glycogen synthesis in the high-dose L-CIF group was slightly lower than that in the low-dose L-CIF group. The reason is speculated to be that the liver insulin sensitivity of this group increased, causing most carbohydrates to enter the liver for metabolism, exceeding the liver's metabolic capacity. When the carbohydrate intake exceeds storage and oxidation, it will be converted into fat through the DNL process, aggravating the development of NAFLD.

[0208] In order to further explore the effect of liver in regulating glucose metabolism, we analyzed the expression levels of liver proteins PPARγ, PI3K, and AKT. Fig.23 B and Fig.23 As shown in C, compared with the CON group, the HFD group significantly reduced the levels of PPARγ, PI3K, and AKT (P < 0.05, P < 0.01), indicating that insulin resistance has reached the liver tissue and the liver has insulin resistance; L-CIF intervention significantly increased their expression levels (P < 0.05, P < 0.01), indicating that L-CIF can increase liver insulin sensitivity and improve liver insulin resistance. These results show that L-CIF improves liver glucose metabolism by enhancing the expression of liver insulin-sensitive proteins PPARγ, PI3K, and AKT, thereby promoting the synthesis of liver glycogen.

[0209] (9) Effects of L-CIF on lipid metabolism in mouse liver

[0210] To evaluate the body's lipid metabolism, the liver TC and TG levels were detected. Fig.24 A and Fig.24 As shown in B, compared with the CON group, the TC and TG contents in the HFD group were significantly increased (P < 0.01), indicating that lipid accumulation occurred in the liver; after supplementing with L-CIF, the TC content was significantly decreased (P < 0.05), and the liver TG content showed a downward trend, but not significantly, indicating that L-CIF can improve liver lipid metabolism. Fig.24C Observation of liver tissue H&E staining revealed that there were no pathological changes in the liver tissue of the CON group, while the liver cells of the mice in the HFD group underwent obvious fatty degeneration, severe cavitation, and a large amount of fat deposition, which once again confirmed that the liver of the mice was damaged; after L-CIF intervention, the degree of adipocyte cavitation was significantly reduced, lipid accumulation was reduced, and liver cells tended to normalize, which once again showed that L-CIF can improve liver lipid metabolism and protect liver damage. In addition, it was found that the liver TG content in the low-dose L-CIF group was higher than that in the high-dose L-CIF group. The possible reason is that high-dose L-CIF increases the insulin sensitivity of the liver, resulting in an increase in the content of carbohydrates entering the liver, causing part of the carbohydrates to be converted into fat for storage, which is consistent with the above-mentioned liver glycogen content.

[0211] To further explore the role of the liver in regulating lipid metabolism, we analyzed the liver proteins PPARα, LXRα, and CYP7A1. Fig.24 As shown in D, compared with the CON group, the HFD group significantly reduced the levels of PPARα, LXRα, and CYP7A1 (P < 0.01), indicating that the liver cholesterol metabolism capacity was reduced and the liver lipid metabolism was disordered; the L-CIF group intervention significantly increased the expression levels of PPARα, LXRα, and CYP7A1 (P < 0.05, P < 0.01, P > 0.05), indicating that L-CIF can increase the liver cholesterol metabolism capacity. In summary, L-CIF reduces the liver TC level by accelerating the expression of PPARα, LXRα, and CYP7A1 proteins in the liver cholesterol-to-bile acid signaling pathway, thereby reducing serum TC content and improving the body's lipid metabolism disorder.

[0212] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing modified insoluble dietary fiber, characterized in that: The method comprises the following steps: preparing Chinese cabbage insoluble dietary fiber, adding sterile water according to a solid-liquid ratio of 1g:10-30mL to the Chinese cabbage insoluble dietary fiber, fully stirring, inoculating plant lactobacillus for fermentation and culturing; after the fermentation and culturing, adding ethanol aqueous solution, precipitating with alcohol, collecting precipitates by suction filtration, and freeze-drying to obtain modified insoluble dietary fiber.

2. The preparation method according to claim 1, characterized in that: The inoculation amount of the plant lactobacillus is 5-10%.

3. The preparation method according to claim 1, characterized in that: The fermentation culture is carried out at 35-37° C. for 40-50 hours.

4. The preparation method according to claim 1, characterized in that: The alcohol precipitation time is 20 to 30 hours.

5. The preparation method according to claim 1, characterized in that: The preparation method of the Chinese cabbage insoluble dietary fiber comprises the following steps: taking cabbage residues, adding distilled water according to a solid-liquid ratio of 1g:30-40mL, fully stirring, adjusting the pH value, and adding alkaline protease for enzymolysis; after the enzymolysis is completed, heating in a boiling water bath for 8-15min, cooling to room temperature, centrifuging and discarding the supernatant to obtain a precipitate; washing the precipitate, collecting the precipitate by suction filtration, freeze-drying, crushing and sieving, and obtaining the Chinese cabbage insoluble dietary fiber.

6. The preparation method according to claim 5, characterized in that: The enzymolysis conditions are as follows: enzymolysis in a water bath at 35-45° C. for 35-45 min.

7. The preparation method according to claim 5, characterized in that: The precipitate is washed with 65-75°C hot water, 75-80% ethanol aqueous solution, and 90-98% ethanol aqueous solution in sequence.

8. The modified insoluble dietary fiber prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the modified insoluble dietary fiber according to claim 8 in the preparation of products with antioxidant activity.

10. Use of the modified insoluble dietary fiber according to claim 8 in preparing products for regulating blood sugar and blood lipids.

Images